<%BANNER%>

Skeletal Muscle Adaptations following Incomplete Spinal Cord Injury and Exercise Training

Permanent Link: http://ufdc.ufl.edu/UFE0022057/00001

Material Information

Title: Skeletal Muscle Adaptations following Incomplete Spinal Cord Injury and Exercise Training
Physical Description: 1 online resource (182 p.)
Language: english
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: adaptations, locomotor, muscle, spinalcord, training
Rehabilitation Science -- Dissertations, Academic -- UF
Genre: Rehabilitation Science thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Recovery of function after incomplete spinal cord injury (incomplete-SCI) is in an exciting phase of research. Paralysis and paresis of lower extremity muscles following incomplete-SCI result in persistent motor dysfunction and impaired walking. Advances in research have led to promising exercise-training strategies in both humans and animals following SCI. However, the mechanisms that explain the functional improvements reported following incomplete- SCI and exercise training are not clearly understood and could possibly result from musculoskeletal changes, neural adaptations, or a combination thereof. The primary purpose of this dissertation was to explore the adaptations in lower extremity skeletal muscle following incomplete-SCI and exercise training in both humans and animals. Ours findings indicate a significant loss of both peak isometric and explosive strength in lower extremities after incomplete-SCI in humans. Additionally, this loss in strength was attributed to a severe loss in voluntary activation of the paretic muscles. Locomotor training and resistance training were two exercise interventions that were tested in our study, and our findings suggest that both locomotor training and resistance training helped in significantly improving both voluntary and explosive strength, and voluntary activation in the lower extremity muscles of persons with incomplete-SCI. In the rat model, incomplete-SCI resulted in significant atrophy in all four lower extremity muscles. In addition, SCI resulted in a shift in fiber type composition measured using myosin heavy chain (MHC) composition towards faster isoforms in all four lower extremity muscles. Locomotor training in the rats resulted in significantly reducing the atrophy in all lower extremity muscles. In addition, there was also a significant shift in fiber types in all hind limb muscles towards slower isoforms. In addition, our results indicate that recovery in muscle size following SCI and locomotor training was due to the activation of satellite cells which went to form multinucleated myotubes which repaired or replaced damaged or lost muscle fibers. The overall findings from the present work will provide essential feedback on deficits in muscle function following SCI and also effects of exercise training interventions towards reducing the musculoskeletal deficits and promoting muscle plasticity following incomplete- SCI. These findings might provide feedback for the development and integration of these exercise interventions into the community.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Thesis: Thesis (Ph.D.)--University of Florida, 2008.
Local: Adviser: Vandenborne, Krista.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0022057:00001

Permanent Link: http://ufdc.ufl.edu/UFE0022057/00001

Material Information

Title: Skeletal Muscle Adaptations following Incomplete Spinal Cord Injury and Exercise Training
Physical Description: 1 online resource (182 p.)
Language: english
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: adaptations, locomotor, muscle, spinalcord, training
Rehabilitation Science -- Dissertations, Academic -- UF
Genre: Rehabilitation Science thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Recovery of function after incomplete spinal cord injury (incomplete-SCI) is in an exciting phase of research. Paralysis and paresis of lower extremity muscles following incomplete-SCI result in persistent motor dysfunction and impaired walking. Advances in research have led to promising exercise-training strategies in both humans and animals following SCI. However, the mechanisms that explain the functional improvements reported following incomplete- SCI and exercise training are not clearly understood and could possibly result from musculoskeletal changes, neural adaptations, or a combination thereof. The primary purpose of this dissertation was to explore the adaptations in lower extremity skeletal muscle following incomplete-SCI and exercise training in both humans and animals. Ours findings indicate a significant loss of both peak isometric and explosive strength in lower extremities after incomplete-SCI in humans. Additionally, this loss in strength was attributed to a severe loss in voluntary activation of the paretic muscles. Locomotor training and resistance training were two exercise interventions that were tested in our study, and our findings suggest that both locomotor training and resistance training helped in significantly improving both voluntary and explosive strength, and voluntary activation in the lower extremity muscles of persons with incomplete-SCI. In the rat model, incomplete-SCI resulted in significant atrophy in all four lower extremity muscles. In addition, SCI resulted in a shift in fiber type composition measured using myosin heavy chain (MHC) composition towards faster isoforms in all four lower extremity muscles. Locomotor training in the rats resulted in significantly reducing the atrophy in all lower extremity muscles. In addition, there was also a significant shift in fiber types in all hind limb muscles towards slower isoforms. In addition, our results indicate that recovery in muscle size following SCI and locomotor training was due to the activation of satellite cells which went to form multinucleated myotubes which repaired or replaced damaged or lost muscle fibers. The overall findings from the present work will provide essential feedback on deficits in muscle function following SCI and also effects of exercise training interventions towards reducing the musculoskeletal deficits and promoting muscle plasticity following incomplete- SCI. These findings might provide feedback for the development and integration of these exercise interventions into the community.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Thesis: Thesis (Ph.D.)--University of Florida, 2008.
Local: Adviser: Vandenborne, Krista.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0022057:00001


This item has the following downloads:


Full Text
xml version 1.0 encoding UTF-8
REPORT xmlns http:www.fcla.edudlsmddaitss xmlns:xsi http:www.w3.org2001XMLSchema-instance xsi:schemaLocation http:www.fcla.edudlsmddaitssdaitssReport.xsd
INGEST IEID E20101107_AAAAAQ INGEST_TIME 2010-11-07T09:11:10Z PACKAGE UFE0022057_00001
AGREEMENT_INFO ACCOUNT UF PROJECT UFDC
FILES
FILE SIZE 47645 DFID F20101107_AAAKFL ORIGIN DEPOSITOR PATH jayaraman_a_Page_016.pro GLOBAL false PRESERVATION BIT MESSAGE_DIGEST ALGORITHM MD5
290bc19b607127a3af764c880ce34d61
SHA-1
43363b4485c9125a309ef9499468a1c3d71dc6ad
8724 F20101107_AAAKEX jayaraman_a_Page_054thm.jpg
e9c3ef141563495ee20dbb9424d41ad7
5fe0caa4f208e8d819fb162dfe4620649a1e19cd
49572 F20101107_AAAJZR jayaraman_a_Page_130.pro
43f2daf5b4410fa006d1aea291c2e8a5
d9306029b4693e0c204bc25a01d76c27adfc44e3
25271604 F20101107_AAAKGA jayaraman_a_Page_131.tif
1e92ed8f36084fd66a0d21f1e036b4e0
0dbcc0b66c0a48fe202879a046808c5ff408b2ae
52401 F20101107_AAAKFM jayaraman_a_Page_139.jpg
1a1d6103292e64d4dc7df6e94213e76b
ea47ae6b4a590134fb4b469039eac3b5f7f902c8
9396 F20101107_AAAKEY jayaraman_a_Page_169thm.jpg
f08b2b836cba7b9ba2b1c77b5cb76d15
7b4634cf41f9ec00b69a4cc1318b765c60f683ab
113791 F20101107_AAAJZS jayaraman_a_Page_031.jpg
1b29e3ee212ad4fe00249459f95977cf
b1d865124dc865a13f9ce257454cf730b4a5defa
F20101107_AAAKGB jayaraman_a_Page_016.tif
332e0380a2b6159d06ade4e0fc969149
85ad57b1ec661d139043f94c56d03a8c860cfaca
F20101107_AAAKFN jayaraman_a_Page_061.tif
30e5c33cf0026e107074ded62cdd7774
4aac265e76356985a961ae9cafbe24bdd56f894e
1051986 F20101107_AAAKEZ jayaraman_a_Page_145.jp2
35eb89d5f916c8e75a32072f2ac2144c
cb2f8e03adbf0d95efa39026da9101d74090d2a8
605777 F20101107_AAAJZT jayaraman_a_Page_106.jp2
dfaa613a6c9d81eeac731da086d94185
9e4f069c90532eb554a9cedd24c0424a53615eaa
46700 F20101107_AAAKGC jayaraman_a_Page_067.pro
b9d9584e03d554f38b0a9d23158c65d4
2ecf60261aa6ca4a0187bfc56dc49b685ba14b2f
F20101107_AAAKFO jayaraman_a_Page_182.tif
35be70a6f870200f9335671005797ca2
7fae70efb3cebcdeec54ebedcfe391318cca9e79
34696 F20101107_AAAJZU jayaraman_a_Page_126.QC.jpg
630dd2c522aa1a06e0eb81e8a94d270a
4aab6f12cd0412429bf2306414901ddba41d2bf4
7761 F20101107_AAAKGD jayaraman_a_Page_098thm.jpg
969e43293055be19c4a587e7c2b683af
e9315c4771229ff2ed4c3650da75822b6de84c00
2146 F20101107_AAAKFP jayaraman_a_Page_026.txt
75ce2af9b5f57a2f5d305ed4cb80683e
10411137a9fcd722f5582d157002c8e4c6056ca2
F20101107_AAAJZV jayaraman_a_Page_019.tif
485e1be17081b48058a9775f587876a0
47b97344d99c4eeb1b9e126da6673e8baaf3248d
6997 F20101107_AAAKGE jayaraman_a_Page_075.pro
48df4c8336dd2f60fd4484c42b9cfde4
854ae237efa582d9d8cdc8b46d23336b7c2275c3
34906 F20101107_AAAKFQ jayaraman_a_Page_119.QC.jpg
4dc341f466d28ef35aac436e5fb516f0
c0162270d10655fb7a2d05d1796aa5e0ab52cb38
114108 F20101107_AAAJZW jayaraman_a_Page_085.jpg
487c6355634e7efda320272d2ac324b9
e1d640ae1794da98147bec7f316162d1fbfa82ea
2202 F20101107_AAAKGF jayaraman_a_Page_037.txt
a15244b4939d44159afb68a7daf00cae
b26bade4b42fd5577e258798325c9c276a44bde6
2277 F20101107_AAAKFR jayaraman_a_Page_046.txt
e278a88192cf89712d57ea33f8b98594
6b76323557bc2afe939a5a99cfa17cfaaeed799e
2102 F20101107_AAAJZX jayaraman_a_Page_020.txt
c70d5825b2acd1fc373f4f8ac5d25277
da8f9ade93f91090423a62a0e11d776bf637d12b
8271 F20101107_AAAKGG jayaraman_a_Page_126thm.jpg
de9c678ceed068b566d7ad7886eeca88
9505ba7cfaa221f1c1abb5dea700b77a3e16e866
27393 F20101107_AAAKFS jayaraman_a_Page_059.QC.jpg
8545166158512b3e787ae6efeb8ea2ed
2a99ec74b9c3ae99d3e1e85d50c0b0bceb98becb
30694 F20101107_AAAJZY jayaraman_a_Page_082.QC.jpg
0c06f1b1ae3a0b5903703c6e4392770e
a69b70ce032d8f53ce324314ff49e1442445e829
132681 F20101107_AAAKFT jayaraman_a_Page_006.jpg
52bfd1a8e69d917ff10b8ee43a291547
18fe00c1609955557bf6f7ed1f73c81ba3bb5315
8726 F20101107_AAAJZZ jayaraman_a_Page_150thm.jpg
b784882847f7352f1a29166e0e29fde4
e5c224910f6945f21259c92c53f460575bc42227
1051918 F20101107_AAAKGH jayaraman_a_Page_033.jp2
3c2b7f5f2b70a81e4ec90a34fd720f44
a71b34166edb84e702c01799879c9333c1553f84
34677 F20101107_AAAKFU jayaraman_a_Page_113.QC.jpg
6b604031dcb44946a47d26ac04c8f808
d2c311978521307616d93b96a8bf5c88661644e4
1051926 F20101107_AAAKGI jayaraman_a_Page_048.jp2
501dbf613abbbd3002e90b9e53d4529e
6a63a3b7b9a83e3d73efb3e476bd783c33400da4
99 F20101107_AAAKFV jayaraman_a_Page_076.txt
f419b5fc8b30b88ae2b144da8373e557
8e07a27bb15845141166dee411daf0a63bf4a074
2094 F20101107_AAAKGJ jayaraman_a_Page_144.txt
ed34369aeaea0709390a7f9f4867608a
ba6a03d7b6122149b7d03f7f598c793cbce72eb4
34158 F20101107_AAAKFW jayaraman_a_Page_015.QC.jpg
6e4d8a6a95e5227a269fbfaf0c54595c
d4523d609c56993c9dfb4fa1274b289f66af36d2
551 F20101107_AAAKGK jayaraman_a_Page_108.txt
29024747d2b0e7b102b99264f5b68644
3f692b6af6432a31364a8e25fc6e1908e5a30362
604 F20101107_AAAKFX jayaraman_a_Page_002thm.jpg
abb8578444e130d1b03febd3cb9ac0bf
31e60257ca553713c4227f64f47bb0e237d0f0e9
55047 F20101107_AAAKHA jayaraman_a_Page_043.pro
8a114bc9dcc3835d79e3076caf2d1bde
1f1970f530d22e4935bee8518a5cc6b9eb57c113
36958 F20101107_AAAKGL jayaraman_a_Page_027.QC.jpg
e4323d5fef3b0f02f8d199b0770b987a
8c5d4daec8a06e4318e6774849b1bfa7c3ca55a1
3672 F20101107_AAAKFY jayaraman_a_Page_006.txt
a0b56f3ea80bf6665c8b4693a4f879eb
f7aecc31b28a9b4084720e23d332a524d3ce42eb
36259 F20101107_AAAKHB jayaraman_a_Page_026.QC.jpg
48085857f7ec1e2a407ebc6f2615f9f0
e0d2e070dc29530903f242cf273c87c5aa9cf0b2
F20101107_AAAKGM jayaraman_a_Page_134.tif
072600ab243eb89827dfb22b2063ade4
20d2515d1d2dff450f8c128d0e2a637435cda1fb
46751 F20101107_AAAKFZ jayaraman_a_Page_117.pro
b93ae552555bf59e96d5b85c690301b7
8288000387631ef61cae466700ced118e1772d4a
2053 F20101107_AAAKHC jayaraman_a_Page_039.txt
14c7113da255d577df6ebfe92af12307
456fef34c24d586fdab89a018383d4fe4124aadf
1051976 F20101107_AAAKGN jayaraman_a_Page_007.jp2
b77aa879134d887b731b11e01f4a9b38
9d84c2287ba495728d899812751f18b876ac87a3
54826 F20101107_AAAKHD jayaraman_a_Page_132.pro
80bdf38d75fa6e081f2ac357bc450a5f
c3aa019fd8f85981238b0e53c462db8d4a158912
61954 F20101107_AAAKGO jayaraman_a_Page_012.jpg
1b0ab11cbc03c27064af4bd1ddfff90d
f3281e396e50c19f51704655f99b153173cbacc1
2152 F20101107_AAAKHE jayaraman_a_Page_034.txt
6ddfcaa80acb8d9c829a10d5ec38430c
74884ee1a3c24505028c6fda8f1a152864d9a335
1051983 F20101107_AAAKGP jayaraman_a_Page_080.jp2
b60046cc40a67c9794d6f339b098fd9a
eb26b3734e7ffc282cada67816a4745c1eacfefc
57010 F20101107_AAAKHF jayaraman_a_Page_086.pro
9a3e6da389cdad5bded3e1f594e7f926
1b1b85c25f59246363defbb8f579213398859617
2071 F20101107_AAAKGQ jayaraman_a_Page_113.txt
0923b0e906a96d0cbef25dd2380e83ef
123336cdefdd2bbe6973e098f0b7ea2381d737f6
35863 F20101107_AAAKHG jayaraman_a_Page_050.QC.jpg
981071e208376471b76e409bac4a2b75
5dc9316261011dddb2659829e56ca2d21259b4c6
F20101107_AAAKGR jayaraman_a_Page_062.tif
c93c364e4e1c4adf6b491b9dae34216f
2bef87e9688fbbf88e6ecf0213c3c241f2c59dcb
2230 F20101107_AAAKHH jayaraman_a_Page_153.txt
4c7594fb97755780efa2ef4fb82c1f04
8dae43925b2c29ce90c1aeb6d62a3691aea2855e
2187 F20101107_AAAKGS jayaraman_a_Page_035.txt
bdb0873b4ccffe69b937740f47facb04
69e161fca4e90f62b971b4a1866a1688f03b7a2f
56131 F20101107_AAAKGT jayaraman_a_Page_037.pro
1f7fd03c8375ac229d91752b3f081c0c
43b0d48517f12e7bf95c9c9184d415339be62571
2157 F20101107_AAAKHI jayaraman_a_Page_155.txt
625e306a129931c306b8983a7cc899d9
e15b307a5048a79fa3181977afd5134dd7ef24bd
8423998 F20101107_AAAKGU jayaraman_a_Page_032.tif
f70a847f4e7f4afa2fc82fa3bcca2370
bb48b9e866babb27f3fa1f5bb72b654ff6515e5b
9078 F20101107_AAAKHJ jayaraman_a_Page_041thm.jpg
386f70f4a18f42a6c28577887e7606cd
c2dac39d1a594bb36d1aa89bb6147051f0bc5e27
1051970 F20101107_AAAKGV jayaraman_a_Page_096.jp2
3925fa4bc441b62f190a190bdfdc77b9
7b75c16cc69f6ae327e2ff00212585c27452224b
105771 F20101107_AAAKHK jayaraman_a_Page_004.jpg
58a193b56b9f75ce20922c2a41600db9
0a9ce5bce73f58f12af638b479c981aa4143c898
110845 F20101107_AAAKGW jayaraman_a_Page_163.jpg
d2abfce455037986b382681912348e50
f7b880befa5957216974d80ddcc2585978325568
1051974 F20101107_AAAKIA jayaraman_a_Page_095.jp2
8f224d3b642a944a98f1482ed859f1ae
502f6cad1119203d479e6903357dc5a161afc400
8768 F20101107_AAAKHL jayaraman_a_Page_110.pro
1f417f0d5344193b348d548b596f5c17
7a0018b6427efb67133d904b30c54c9e6784db18
20647 F20101107_AAAKGX jayaraman_a_Page_014.QC.jpg
373d72e79268a993a8552bcac18514d1
43edfd9d5c706ed49d71c8d800fa717d9102ee75
8846 F20101107_AAAKIB jayaraman_a_Page_134thm.jpg
430e0ffb5b0d4e0063e124ec2d78acc0
3331222aba4c076054e407c53d2b1c9896bc2fe1
34958 F20101107_AAAKHM jayaraman_a_Page_150.QC.jpg
6bb003d86edc14b46866594601158fcb
8f8d5e6f5e043965e47fbd3f1516560b04e14f89
45336 F20101107_AAAKGY jayaraman_a_Page_146.pro
0cf68e313eb88eef54dcb87468a7aab5
eeaed4d16fd4e8120c72b841f021c1e29410a1dd
F20101107_AAAKHN jayaraman_a_Page_169.tif
ce5a6f4f577cc9f5a4a07446e1d2ae6f
66faeeec39f4576b8723fcb54b740e32b671efc0
111726 F20101107_AAAKGZ jayaraman_a_Page_029.jpg
4aad08bcd9d20b46bbdd3724b251dbd1
82361931a23b847f8de4dcc15e0b3406bf2700be
2387 F20101107_AAAKIC jayaraman_a_Page_011.txt
18435f8626fd67bbd4f5fb48e11a2dbf
052ee0530c4f0d1839236ec2c243729214cbbb90
1051961 F20101107_AAAKHO jayaraman_a_Page_026.jp2
75bdd545151829e3f7b8989a28da9307
4dd57294c14207fa80ea0b1719d0157d8bb60900
9164 F20101107_AAAKID jayaraman_a_Page_181.QC.jpg
41f96c8749e5967978d9a99fc6909aca
dbd116be83779952583cf1b6609cb134c9cef0cf
2127 F20101107_AAAKHP jayaraman_a_Page_120.txt
a554e8a7648d4ddb3290eec5313c7117
e060b69f504960795d843762c19767f3519e613c
1051972 F20101107_AAAKIE jayaraman_a_Page_073.jp2
6ff0e9af1a5d204e02badd635311d107
58b03335917aa84f8ab1655e7ba346fd72960c76
100829 F20101107_AAAKHQ jayaraman_a_Page_078.jpg
b3d402f11ffb1a805e0589ad4e71b657
3e58e78056427f7e2d3398e29db96e92075a5217
102362 F20101107_AAAKIF jayaraman_a_Page_130.jpg
5f063b5487be93c7ea3aa3d1ccb24815
f4b100da90dc0d0d6b860c7087acc01287971820
879749 F20101107_AAAKHR jayaraman_a_Page_063.jp2
ff49a222f6630bced01bf2d09c9f5d95
dae950eb8af6fea836ad32dfffa13bf6c58d12fb
F20101107_AAAKIG jayaraman_a_Page_135.tif
b6623382538634f9e579167d12cd2270
991687a49bb004580a6003303ade83efd2193983
57863 F20101107_AAAKHS jayaraman_a_Page_046.pro
76d70dceda558df4722a547638219ebe
6cb996c8013331e9b8aaf71bc817d274d5d6d031
68816 F20101107_AAAKIH jayaraman_a_Page_176.pro
dc978026bca6bfe23cccb709efae4699
0ab015e5ee4218c5eac131bd26efcd15d30ec9af
2169 F20101107_AAAKHT jayaraman_a_Page_045.txt
11002135a517a9e13c4e5f203dab55f0
fcd5e39c3941191ba52459d1b39d0eb35a9f8301
8807 F20101107_AAAKII jayaraman_a_Page_021thm.jpg
5f8100b55ebfec6d78a58396a2bd336d
338c01ab1d0e4a36c721c21ef857db8328115b7b
413572 F20101107_AAAKHU jayaraman_a_Page_005.jp2
a909d8285292cc800e58b829fb2a26f9
ecb3ba1363880e58932aa5442f162d16df25e192
12235 F20101107_AAAKHV jayaraman_a_Page_160.pro
dbd687400e87243fc3d41239964a9648
5835d189709e4888efbc3b5831111b1169416967
51344 F20101107_AAAKIJ jayaraman_a_Page_048.pro
ce6d67d34ae86de3ccf2e85b74520cff
35005f09b3df2dba54a5a50f3ca41b13ea9ff433
36788 F20101107_AAAKHW jayaraman_a_Page_152.QC.jpg
fb0dbc420ebf622da7fb9a98288f148e
5aa360bfaf7192f922f74f501b4e2a0523c7ea9e
37031 F20101107_AAAKIK jayaraman_a_Page_102.QC.jpg
15bfe46094dc9533b1ecc55bba2d6aa2
18179ed560ff91dbff9c97e8f37236c62b2b67ba
9139 F20101107_AAAKHX jayaraman_a_Page_022thm.jpg
b0fab97e037ab51c992c15d280511023
fe32d3b7ec2972e6cf3c58c50814cd75d729508a
107489 F20101107_AAAKJA jayaraman_a_Page_020.jpg
cc33dac81dfac5a2bd1984c358d9a7c5
285808b486516ee52567b0f9a7308e31a61971d7
1535 F20101107_AAAKIL jayaraman_a_Page_076.pro
d1008e069943be685200bd6d71fe6d53
7d25974798428f7827735ddf1377270852476d3b
2692 F20101107_AAAKHY jayaraman_a_Page_164.txt
06ff9068d091d2ce58320eec58cf32e6
9dfd45d94b3e5eceb32e72c47ee56d8851d3b3fd
111912 F20101107_AAAKJB jayaraman_a_Page_024.jpg
df6acecf29d0e71d53af58628ff5ca4d
3c6417d27df185d31aaafd12e48d8968bbea83a5
275476 F20101107_AAAKIM UFE0022057_00001.xml FULL
3d0fc7b5e34c1c97e3176f33d24c5a96
4181d03451d7ff4157a58139bdeb2ede633f1132
1051902 F20101107_AAAKHZ jayaraman_a_Page_133.jp2
402fe35eeacb8869fe5e34306715cdfb
da01a4855e09f2c37c03f4d0f4f553ea4d56acfe
110398 F20101107_AAAKJC jayaraman_a_Page_025.jpg
306e5160ccf2fab250b194afbeac6acc
dd949e332b604b02074551379248c5b98853a884
111513 F20101107_AAAKJD jayaraman_a_Page_026.jpg
1aa105785aaa2e33af8ced5b8601d0af
0bf8f7662105c2a2957fb7439816ad59e31a73d4
109964 F20101107_AAAKJE jayaraman_a_Page_027.jpg
c4c1c04d21f7a17ab2d06ff0f313c015
8c29a0ca881a67780c66b0f6cb15b11c4a5e8974
3814 F20101107_AAAKIP jayaraman_a_Page_002.jpg
cf9719b397e36bc315c86a630f02add3
586631948365fc65974407b3ab8e6b8aa19b3ba1
112314 F20101107_AAAKJF jayaraman_a_Page_028.jpg
abb9afc56305ef791b5bc5e9063e97d5
9f04cf7783d0be592c086fee9e798eb55d56d630
40155 F20101107_AAAKIQ jayaraman_a_Page_005.jpg
810baa6f2379db601d97c7f3e0e7b564
ecd3ed61609a9325d669e6ff4b5bdbbbaaa0ffe9
118265 F20101107_AAAKJG jayaraman_a_Page_030.jpg
68bd23a2825242f1d8ad2faff213c982
8c91804d3ead47d3b06fe3566da7bc6ec479cd95
143892 F20101107_AAAKIR jayaraman_a_Page_007.jpg
dce964d89fa40f2ddf2c9ed45a6103a9
cb1b6cf7501929e3a3e31aa75271eb05d4f918c6
101299 F20101107_AAAKJH jayaraman_a_Page_032.jpg
226d4f6e64957fa0b3b598e7d9149124
d6711ca7fd6ab32edcf117b41bb2ef6ba8009f0d
147790 F20101107_AAAKIS jayaraman_a_Page_008.jpg
66344529782705edbdd3e6370509a87d
b9bc966895dce5663e3ea72e264b380755ebb6a1
113927 F20101107_AAAKJI jayaraman_a_Page_035.jpg
2eb4d2ed41dc0a76df90afa5cfa4562b
85b807757600e3de0e385c27fdf45f5f80b81bac
98293 F20101107_AAAKIT jayaraman_a_Page_013.jpg
c6567b47ba51f7c12184b4f42d36e370
d4515127229164e5288e90652e4fa679bc9dc7d2
113856 F20101107_AAAKJJ jayaraman_a_Page_036.jpg
5587e781cf43b5da588317526a47af51
d4e35f6ef7cf260c8b693d9c2c35dc34d6f393b7
62163 F20101107_AAAKIU jayaraman_a_Page_014.jpg
2e541d5c2b13206b467432e7ad87b101
5a4ccd0559a7feeabb5b24b27a8390210216b391
101927 F20101107_AAAKIV jayaraman_a_Page_015.jpg
82d90c9c134cb151e7c6f46b8d747c75
dcdbbb4788055bdd2ebcfc5a6299503a2da7beb1
114596 F20101107_AAAKJK jayaraman_a_Page_037.jpg
da45930155768cce5e2448dadf88641d
4c809b37aba4bebf947d1b4874c2ae3618d2787f
97761 F20101107_AAAKIW jayaraman_a_Page_016.jpg
6f9017b9e9bb1d44ab679de673d6ed4c
ea37a66fdbab5ada27d70d5546dc3df67ad722b6
100814 F20101107_AAAKKA jayaraman_a_Page_064.jpg
6eb799c8212baa7c20df9192f330a952
2ce0dcffa0f5c39cd97a66a2a776c20a3578eb32
116292 F20101107_AAAKJL jayaraman_a_Page_038.jpg
5c14c30fd606222e899c32602501b755
6e8cab9492519d8d80b15cce0b9fcb9e51ee511c
103977 F20101107_AAAKIX jayaraman_a_Page_017.jpg
99f450c1c729439a9dd35e80a4a3bae0
938fb27ddf5964f1ee9487298a4f294224ed7206
111354 F20101107_AAAKKB jayaraman_a_Page_065.jpg
2817d20fa94f8b641d765008692ec117
7f6626e247bc38e2a8e153a80a11e5c7bf6cba59
112802 F20101107_AAAKJM jayaraman_a_Page_041.jpg
f5293bf766749f7060773c506f844f93
468f56846e218db89fa0679ec427b689a5218100
111383 F20101107_AAAKIY jayaraman_a_Page_018.jpg
aac1d87fb2f5be1d0c52cb7e050f3ecb
acc94ea551bdc6430561c3d36b5aad8ada3a92b8
96423 F20101107_AAAKKC jayaraman_a_Page_067.jpg
541945036bf8b95eebbc63857373306e
6c855453ee929d2d2792064804dcde499c115098
113449 F20101107_AAAKJN jayaraman_a_Page_042.jpg
db9f18eb3bca650809f1222eedef3124
2b5ba88c4f71a981bad64e1190e2d6685d0d6ee1
108851 F20101107_AAAKIZ jayaraman_a_Page_019.jpg
4389919d06b65821f607cd5139dddd10
0969386e821bb8e050a677c586c208f29f1fce73
107747 F20101107_AAAKKD jayaraman_a_Page_068.jpg
5c2c5081756d852b09ea0794f5a71f64
7665e8cb10eae1766fc0b94d990ecf25f3462d51
110410 F20101107_AAAKJO jayaraman_a_Page_043.jpg
ce866958b67d1996d1b2d74d48a3a0c9
cf34ca41892a6df56749f340de0ff536e46bf079
102349 F20101107_AAAKKE jayaraman_a_Page_069.jpg
dfaf943c3b22fc97bdc2594c80f0071e
ff815e7a0159cddf5078ebcebe609d85a3ce9514
110260 F20101107_AAAKJP jayaraman_a_Page_045.jpg
4668bade6868c143c244b7241cb68c11
3a9ea7f480570d0474d89fc332760d902500d9e7
110782 F20101107_AAAKKF jayaraman_a_Page_071.jpg
ff43ddee92f7c09371e2f0252b1edfc2
fc69825036ae27cfa133b3b4f26e0bab86f1959b
103675 F20101107_AAAKJQ jayaraman_a_Page_048.jpg
14fb4eb4682c06c9cc77ca5cded4246c
da6dfa79e44210248739433033ac36892e549f65
105977 F20101107_AAAKKG jayaraman_a_Page_073.jpg
f709fc61def3b67545f59cd51650fb41
0fa2527de95ea10eb4daa2a1c51c0c2ae4fbd4ae
113622 F20101107_AAAKJR jayaraman_a_Page_051.jpg
082b8a380d53625ee1d856c3f1586fa8
63333e7d136705cffddf278bcd81dadf01b877a9
62219 F20101107_AAAKKH jayaraman_a_Page_075.jpg
956ae01398274768f35a5a3ef762877f
7d25c5b65fb165de0626cd5da97c2d806f67e102
111896 F20101107_AAAKJS jayaraman_a_Page_054.jpg
ed052a895e223b48ded11e03fca0db37
4c7576bc5f1d6ef141b71079e6892a7c0c3a5497
21220 F20101107_AAAKKI jayaraman_a_Page_076.jpg
7ec96f15a9b22195a0749c0220d287b8
379b59d423a5dc0914cb9c88faab1dde28e6f6a4
108076 F20101107_AAAKJT jayaraman_a_Page_055.jpg
781e153496bd67637aa76004ddcb34cc
7b556f36421349b2a54bf917346bd19189e2749c
93964 F20101107_AAAKKJ jayaraman_a_Page_079.jpg
af051969f5eb71a48b63670b6d2da2ed
6bc306aba895d6f7b4ae7261d773f29ba53770bd
83615 F20101107_AAAKJU jayaraman_a_Page_056.jpg
b3910f820ea8f58c4714af3944a3504c
9c7b7fc04d11bc0568725341cf3c23cdc195cc65
93880 F20101107_AAAKKK jayaraman_a_Page_082.jpg
f7214d06c60d04a33736875c99d30177
e1c1bf296f880bcc77f63a335090cb2fcd815925
47236 F20101107_AAAKJV jayaraman_a_Page_057.jpg
bf29713f672011f9e030c5601d37e815
47c6a2040ec671604126f16ee9bd728c58ca0497
66697 F20101107_AAAKJW jayaraman_a_Page_058.jpg
b33ad9b1ca7112d4fa2e1f86153ab2c5
54eb2fd3db050f8b25661230effce8d73359483c
106880 F20101107_AAAKKL jayaraman_a_Page_083.jpg
6147cffe7de74e8ae4a8dd7271c7088b
7430523a95ea61394690241cd8dd7d77ab3c1df1
83367 F20101107_AAAKJX jayaraman_a_Page_059.jpg
3bca2b1739f2be030cfea699b1fb92f2
4b61052ab48f3472a2cf39a6c73125f81f26967f
49455 F20101107_AAAKLA jayaraman_a_Page_108.jpg
0e08d502ed1d9d7524c794f3e799e5ac
e675f23fa6dabfabd7e511e676d5f30042bf8a07
102423 F20101107_AAAKKM jayaraman_a_Page_084.jpg
46a08c75cb4c5eda6426bee6abfe4155
bd5dd152338c9a76abc00f60a03a5c8b7ade1e76
102560 F20101107_AAAKJY jayaraman_a_Page_061.jpg
e21ce0a8d8682deadbf938a3b31c7ec9
2162d5c36169ba117aca0912470386ecfbb94dd2
32377 F20101107_AAAKLB jayaraman_a_Page_109.jpg
07298e7b6e32a6851bacf9ee111be5a7
e7e1d5f8f2d85d552e71766b83045c1bc65c6151
103491 F20101107_AAAKKN jayaraman_a_Page_087.jpg
bfc748a40d2f6e69bddfb334c29627fa
5b70099e5707dd8ccb3fe802a6fae7a88228a11c
94975 F20101107_AAAKJZ jayaraman_a_Page_062.jpg
f63fa23cc5a04a1e174c9687fdde4743
b1536735eacce6a0e039db4e99d8114d0c3415ad
32373 F20101107_AAAKLC jayaraman_a_Page_110.jpg
0c44f0b41a26a0b6e749cbe760240f22
aecd108c9102ed181ab989a91f0cac6a81289d66
61888 F20101107_AAAKKO jayaraman_a_Page_088.jpg
f10e52d7d58f177e356c2ae76f734b47
f18a176601f1362c8a0080f94faa2941952c7b4b
108123 F20101107_AAAKLD jayaraman_a_Page_114.jpg
c52c72f4e7625d24ebe46eb7746cf60f
d97b21e11fa584e79e59a3c78796761440f93f20
35036 F20101107_AAAKKP jayaraman_a_Page_089.jpg
f544535c88dcc8d334602896fae992f6
05447e0b1aa2b7e1b214cb2906cc4dd5ae795aac
112038 F20101107_AAAKLE jayaraman_a_Page_115.jpg
e579dd4dca647298eee7d93336dcf670
aaba6ab2afdf93719f3c37c0c5a99ac347db0208
39154 F20101107_AAAKKQ jayaraman_a_Page_090.jpg
2fe51679650dd4e0f4dce961e079c5f9
232f9bcb109d015307f48ac78314da4babdc34db
96164 F20101107_AAAKLF jayaraman_a_Page_117.jpg
db4ed50d0971b5e97f96d4815928a09a
a666de8ae8798c59fdf53c27ce08e2378a2fc38b
44645 F20101107_AAAKKR jayaraman_a_Page_091.jpg
4250d2732a44797ef6bc9397646de49b
34d6ee62a5c2121bcded2aa2151af9bbfda3e394
109996 F20101107_AAAKLG jayaraman_a_Page_119.jpg
054afc49e57879348fffad5619f30f84
a5a858d727236edf5265958515b4c6b261c6af15
29982 F20101107_AAAKKS jayaraman_a_Page_094.jpg
a05f9785ee671cba6f899e1e2c8ad632
58f161b9d5f88e49a2101d0fdaa2dc4c5202ff8b
107338 F20101107_AAAKLH jayaraman_a_Page_120.jpg
e59c8dba3dadee675a313e3939d994f8
175c7b264a9140e8f3ec3fd7164ecc33594b8bba
112466 F20101107_AAAKKT jayaraman_a_Page_095.jpg
582216d252b9e44b14030d1071533803
5e89c9ec80a139dbb3d34f13eb306c988f33a677
114490 F20101107_AAAKLI jayaraman_a_Page_121.jpg
44477f04fdccfd9c815c4683de6d929e
2673ce908f6da1cefa3b064e21c1f624146b3471
111396 F20101107_AAAKKU jayaraman_a_Page_097.jpg
2125f9642542d6fd5decde0f36ee11ce
0f60ca675828fe1767e2048f37d3711e92bab46a
52207 F20101107_AAAKLJ jayaraman_a_Page_123.jpg
34b50ef38100b3069bd6f61860ab02d8
4537aa9aaa1c3e20b39f19087df77966f12b177a
95152 F20101107_AAAKKV jayaraman_a_Page_099.jpg
ef304c23bfb68996e698e5f16905c0de
5092faa39f7cc11dc45a5bc99288f7e2056094c3
35380 F20101107_AAAKLK jayaraman_a_Page_124.jpg
d456eea74a7dffabddce8fbba18011a6
26f5a195885d98fad318e0128fd2a1a16faa9ccf
114027 F20101107_AAAKKW jayaraman_a_Page_102.jpg
314777374414da1b086d54aaa9158e2d
a6dc2efb906becb4930874336dd30c882cc9dc33
113129 F20101107_AAAKLL jayaraman_a_Page_125.jpg
eb7638fae60773c919ec1e63ddb125e6
c2c78e1832172c4a15a568d820239c70ae5a1f77
108616 F20101107_AAAKKX jayaraman_a_Page_103.jpg
e0cf767bf0553a9c19266b1a5515e4b4
966689cae0d321f294bde7646d335106b7f3da8e
109156 F20101107_AAAKMA jayaraman_a_Page_155.jpg
8b794c700702f437036892c11c1f0de1
47bc7426c1196bbd5066bef29a04d1c27822903c
61922 F20101107_AAAKKY jayaraman_a_Page_106.jpg
b7d5ca63879896675c1e16441e567f5a
df997d259a3747fc93934f64f641ccb82744076c
37240 F20101107_AAAKMB jayaraman_a_Page_158.jpg
9c5ca966725d6d2aa162fa429af5b78a
69f46b8b6befab53d4f85269dea03d12ac93a21b
103921 F20101107_AAAKLM jayaraman_a_Page_127.jpg
4c2ddb739e55ae617fb284b864e8a41f
47cf6d493f3837445b569259d227fcec43af3dcd
53886 F20101107_AAAKKZ jayaraman_a_Page_107.jpg
5ce8e7ae39088aaca84d05e80085340f
f6d9cd0dc3b97882459e2c67de3c3b39dd900507
39460 F20101107_AAAKMC jayaraman_a_Page_159.jpg
c6fc1a333fbcab7f3a515948ae607d9e
3929f66e3c23652707ca9888bbcaaeb78d89ac6c
99921 F20101107_AAAKLN jayaraman_a_Page_128.jpg
eda600a5e45a783c227b808aad2172f7
95f736712eeba6280a294ef585381e46ec58a72b
45050 F20101107_AAAKMD jayaraman_a_Page_160.jpg
0f4bc0c05f12db0e066bcc959e448c92
bb936f0d84fde7f9584d616b956d86dfbeabc349
111994 F20101107_AAAKLO jayaraman_a_Page_129.jpg
d1ead1f684d6ac0baf03fa52d85246f7
e953dc755825013d1c09d020070f0ca07163545b
31287 F20101107_AAAKME jayaraman_a_Page_161.jpg
02d04b99bacd36431bade18074949c2b
8476846032f6c7260d7b4e8c5b6e1f74af4b482b
114290 F20101107_AAAKLP jayaraman_a_Page_131.jpg
8e9894bae841fe32d657d5f2342a61e1
c74cfd9a59d9f3ed380ccf6f1aa5d8c036914ef8
38309 F20101107_AAAKMF jayaraman_a_Page_162.jpg
17341b706145c832a721004ac515ecfd
9a2b5fe7b98db072c57edc4e7db4931ab80cdbb0
112273 F20101107_AAAKLQ jayaraman_a_Page_132.jpg
a54d915a8caa5deeddceff1163f1f850
dee75881a4bc22b5f5a1ffb701e7d80675106752
134565 F20101107_AAAKMG jayaraman_a_Page_164.jpg
2a83aabebb83011417230ca4101e14b1
faeea23652a0007a9d9ad87814cbd63c14bcad28
113797 F20101107_AAAKLR jayaraman_a_Page_136.jpg
39a774183d040672e163f807c7d4a0dd
70844af7161c2452553a84c5778e1c44cca02b46
125894 F20101107_AAAKMH jayaraman_a_Page_166.jpg
4993ad47caec1ea3ce406609451baadc
aa05387415cc1ef89432c7f2d19cfd169c1d3dd0
112774 F20101107_AAAKLS jayaraman_a_Page_137.jpg
de359b3cd566f83f9508c5614b635738
6fa8b189f33ca23a903898556502a996eef3d897
128794 F20101107_AAAKMI jayaraman_a_Page_168.jpg
2cad9dd1a730f8da537afbe1cc51890e
fede4c7bbe8ab92b3045ee4a64167ce4623c6542
52633 F20101107_AAAKLT jayaraman_a_Page_140.jpg
f7bfd5335e9d42421ad8356a3fac569c
5fc8fb0780799b2b605e81ff427ff0931161d2ab
141816 F20101107_AAAKMJ jayaraman_a_Page_170.jpg
ab59f0ae1b6dbd862ff2a23135ba34fb
c8838cbf8bea1a64ea6b80d85af2a9917ec0e396
116937 F20101107_AAAKLU jayaraman_a_Page_143.jpg
496052e18b913875d94b09d1a3c4c8a9
dbaa1b7843288a359b60576591cc8b18bfaf170b
138649 F20101107_AAAKMK jayaraman_a_Page_174.jpg
c5dcf1e44f98bb638097dc0f884c7611
30f27832d62e2449a97bc58c596594f78f201100
103744 F20101107_AAAKLV jayaraman_a_Page_144.jpg
9b236ab147a7298274e1c327891506c6
c5f8f9a6c10b38ac821c7632f17e48298df615aa
138876 F20101107_AAAKML jayaraman_a_Page_176.jpg
dc251fa29df47ddf112a681d4db6aa78
00365f75935ead2bebe480e79d17310bfcdac423
108231 F20101107_AAAKLW jayaraman_a_Page_145.jpg
95f499ecfc9368b79f713aa13da08141
3dd3ea286f141e254b19bfe4e4a2e26b9de71fee
130421 F20101107_AAAKMM jayaraman_a_Page_177.jpg
22ef696b199d4352dc074e66e53a6d01
5209905cf22ed89fc4cd2af267270cdd7d9e5737
108302 F20101107_AAAKLX jayaraman_a_Page_149.jpg
f50b9c0e3be354c360dd143765e3dd2c
eb39b1c32cfd3aa17808db7ae0d4f5786fa1fbac
1051952 F20101107_AAAKNA jayaraman_a_Page_018.jp2
f9dd515e9f4d25ab0350be6d102897bd
45d257170e1784487a169408b93bd12a8466aac1
107894 F20101107_AAAKLY jayaraman_a_Page_150.jpg
4ac5821392f096ae79d9d1c0bff2464f
38203c9b94b2ef391992d848cd6e625e56cc32b0
1051985 F20101107_AAAKNB jayaraman_a_Page_022.jp2
3539c2cd16b13310f7176f892a279522
d1bd32152030663e4e6dac84fc85070ad1692c4d
129240 F20101107_AAAKMN jayaraman_a_Page_178.jpg
fb0826b571f082b1b923463c16d15181
9ae5b71b11be8b74a4ed51bded9b9a83e2cd4127
114456 F20101107_AAAKLZ jayaraman_a_Page_153.jpg
8ac57548a6e4b5f695dd233fcca6e7f2
3b3e9af5209ad1723b7ad951ed8db44bb6e0d350
F20101107_AAAKNC jayaraman_a_Page_024.jp2
736ff96f62e2df5163c3cfb0e8ca686f
ec125650b154cf6fa4d8d82e142ca87efeb0926c
29276 F20101107_AAAKMO jayaraman_a_Page_181.jpg
c925f83cca0e656ad112274e16c59a9d
6bd86542c95747b85be0e2e426a03673f61931d4
F20101107_AAAKND jayaraman_a_Page_025.jp2
fd455b694ddbce8b6e2eab63ab94692f
77ed0d9be5ba68f0e570117dc5ac8f36b84399fb
39084 F20101107_AAAKMP jayaraman_a_Page_182.jpg
00d4f1005d7e41a55154b5d6f965560e
7fe8a4c643c507edc85cc053dd20572ed90387f0
1051943 F20101107_AAAKNE jayaraman_a_Page_028.jp2
ac897bb49b1857669e17fd95273b495b
eff998b0bcefdce76bc3dcef144b7c01883e9376
255940 F20101107_AAAKMQ jayaraman_a_Page_001.jp2
d23351606ec0c0df88c31ff9be5f1507
019e3c7c91aa5c1705b73c99d4da4d7af096dd55
1051982 F20101107_AAAKNF jayaraman_a_Page_030.jp2
8b30d86efb7d6b741a264ecd576f75b3
68055deac433630883fd39c9035ae209940aa5e7
32296 F20101107_AAAKMR jayaraman_a_Page_003.jp2
571fac5f67b11002f8bbef4a6d2e8ad1
894d56323b3711ea29f2f5542665e09bda5b1438
F20101107_AAAKNG jayaraman_a_Page_031.jp2
d2bd2d9cdc18d6d40cec4b1941727bba
715a5f5cf64eee45933a2442a293ceb0296dfbf8
1051913 F20101107_AAAKMS jayaraman_a_Page_004.jp2
1f313866bab667b2af76ce1bf169b7b1
355acafd8dc19587ee8ddbf2bbc4e0190fda9797
1051966 F20101107_AAAKNH jayaraman_a_Page_034.jp2
c924f6551390613b6ed88cf8c2aecd93
128b105407c7bee9405438381b5a1cd1c0944c36
F20101107_AAAKMT jayaraman_a_Page_006.jp2
43349fdd3701b2db2a7f0f7f44bc8a6c
90afdd989e7624a3026d13595d41cff1198b3b41
F20101107_AAAKNI jayaraman_a_Page_035.jp2
23472ad33f0304445f00d0076fe32adf
3221dad8d84ebc0779e52d80d3dc8295256fa585
1051978 F20101107_AAAKMU jayaraman_a_Page_008.jp2
2beda12ba7ca006ce5a2724c1c9015e3
c4166f3860fdcef88c7a1b9e16edfa9e0e1f34fd
1051975 F20101107_AAAKNJ jayaraman_a_Page_036.jp2
300a88c403514653da5a436e3cf261c1
0dfce9c60a79a7aa0c05e12c11798dbfd80104ba
F20101107_AAAKMV jayaraman_a_Page_009.jp2
068e518d356a29cab0acfee208001207
486d3d699af5d0eec9a88edbb10b5bc0a85185c2
1051958 F20101107_AAAKNK jayaraman_a_Page_037.jp2
acbe8838c44b0e5252bdecdde04e84b0
0720b8cfe3d925c539aaee315b9537ec314933e5
590916 F20101107_AAAKMW jayaraman_a_Page_010.jp2
791191d3b817891982c85b68d452af0d
8fc2db78da4b54def58fdae7f496de29e0734701
8312 F20101107_AAAJKJ jayaraman_a_Page_039thm.jpg
d077d61b00abe6a578216755222dcf01
605d55f1b36123cd2556acbc5422e1050db7c4cb
F20101107_AAAKNL jayaraman_a_Page_038.jp2
ce97a2124c76830d84ce1586a3479ad0
1d046ccbfae95ff8de13aaef62ba6fb282d6148e
1051915 F20101107_AAAKMX jayaraman_a_Page_015.jp2
7ce98574c149e3da27cc440a16cdd37b
1c28cb7ab975c0472334c2e751c1f69635cb3072
880588 F20101107_AAAKOA jayaraman_a_Page_059.jp2
2b0cd111a5fc1a9f3ddf6b6c94fc0416
b3f1940ca49406b3752b19d69cb1a32e2898db3e
504774 F20101107_AAAJKK jayaraman_a_Page_108.jp2
ec5afa67d76ad4927af1ec45b528228f
4ae8bd89daa12d26ce24cd58ed3bf7584ba72ec4
1051937 F20101107_AAAKNM jayaraman_a_Page_040.jp2
266c28421a2853897e5f2cad2c7249d9
cf6b30d1fa88c5f6fc7e8f2319f99942e850e74e
F20101107_AAAKMY jayaraman_a_Page_016.jp2
c0d0a76b0283b72a60fede9ca2096f50
54ea0553255503130613f2b8c3c6ebc1b32e4506
1051960 F20101107_AAAKOB jayaraman_a_Page_061.jp2
fca91bf144fa0260281ea5b673129255
f86b735b4c48ef7cf4b77f8fc93499bf6955bcdc
2182 F20101107_AAAJKL jayaraman_a_Page_018.txt
6a68494eb4121eb4b42fd3da3c9550ca
d0d73481b49cdb39dc2b403cfd9c870a777fd000
1051933 F20101107_AAAKNN jayaraman_a_Page_041.jp2
ab7cd2b8d178a308fd50b2a678516790
3a7bd7c4609e37ebf69a666cf1b6cb0156610122
F20101107_AAAKMZ jayaraman_a_Page_017.jp2
34e1f3f68c522bc0bd67ae7dcac72184
753712c65e016ae647e6003acfb80628abea6aac
F20101107_AAAJLA jayaraman_a_Page_047.tif
2b6cf36621d786717e6f15800aab9d85
faa81809e32b5b5c347663085f95de57f8451685
1039157 F20101107_AAAKOC jayaraman_a_Page_062.jp2
85d231b60560eab58fed66a2f3298eb3
259a4c47509ac838449017c498c4551ffc2cee96
12843 F20101107_AAAJLB jayaraman_a_Page_141.pro
d68bb2cbbae9adfcdf9856bd72bc7670
21c7caafce507b09e5c67448971d6db642f2169b
1051979 F20101107_AAAKOD jayaraman_a_Page_065.jp2
3e6f8fc0d6842cb11fe350ffef360e48
5f38e3c387d88b10102ee195a7b7a8446dea307f
3378 F20101107_AAAJKM jayaraman_a_Page_110thm.jpg
0440753366b489007a4351d0348690ab
37f0cecacc1804335feed91228444b3ec69f52a0
1051932 F20101107_AAAKNO jayaraman_a_Page_042.jp2
06308b820fbc813ba3bc9dbd9943128c
0c1105d3ab8d55d6550b44fc90be8c9abcbbb516
45233 F20101107_AAAJLC jayaraman_a_Page_092.jpg
1c2dcc20db3c43aa5d44f688a585e531
055acfc2cf6093996b3391f5ab3318a7a43cc454
F20101107_AAAKOE jayaraman_a_Page_066.jp2
62e3f8d878ec2b26455adf122d24f501
13a56fc592dc5643b1c0a379c80cc597da31d0ea
5293 F20101107_AAAJKN jayaraman_a_Page_156.pro
2be3349269fd81eb598efc71ba7a48af
7b2219115fe1be32cfd9edf264f92755945759b6
1051944 F20101107_AAAKNP jayaraman_a_Page_043.jp2
bb2452c920d573e2a45f3a149bb5c19a
400774a24bdcd26ffe24abd69fb295944d049e86
F20101107_AAAJLD jayaraman_a_Page_128.jp2
4da117e0c43519caed78865ce68e8eac
16de5469b40a6d122b9fbfd88e1e042001b1b509
F20101107_AAAKOF jayaraman_a_Page_068.jp2
1085ef8493570713c3ac12ade984ef85
ad9066085d87d08c3009bdf217b5c53a4fb8f6e6
19256 F20101107_AAAJKO jayaraman_a_Page_108.QC.jpg
d780c590d57ea57d3d8cc8e24aed3dae
be9f73c65db956696ab94eb27f7aa4859ab9c619
1051951 F20101107_AAAKNQ jayaraman_a_Page_044.jp2
f28dff523a8359dd1f8c516742a81c9b
900e78d0b4a5a78a1eb0fb98c1c992cb6b157493
54409 F20101107_AAAJLE jayaraman_a_Page_100.pro
ac7e490347ed3392ebab99f165a42ae5
f4d9957a1247bc1583a8ce7ebe4e178d7bd32665
F20101107_AAAKOG jayaraman_a_Page_069.jp2
0399261790676746cf97e0b23cf4cf82
1126fa3c63ef463d784d2a1d4ab67c83ca059e3d
8605 F20101107_AAAJKP jayaraman_a_Page_103thm.jpg
83eb059b008bd4e4ad0a7784256ee05b
16e7e36904def778b5cdbd52ec1df623c194768a
F20101107_AAAKNR jayaraman_a_Page_045.jp2
8afaef3139de3ec16e5e67d266c10876
4f37cba508611cc16466e2f5a8089b86060b65fb
35975 F20101107_AAAJLF jayaraman_a_Page_104.QC.jpg
12be6d4219913b29f3a576d7cae7a41e
be47275882be4e3a3cfd0ca837af109dedb72e25
989792 F20101107_AAAKOH jayaraman_a_Page_070.jp2
ee5239bf6ef56dbbee0689202a5fd9f0
55e3b322a10cb69cb753d68168c92fd9ce27893b
587083 F20101107_AAAJKQ jayaraman_a_Page_092.jp2
8af84ed884435e02ce5fb1362792b658
536fc9482d1f86efa99bd3c539f8e08c0d314f32
1051967 F20101107_AAAKNS jayaraman_a_Page_046.jp2
1df7c4e87764ae39dde57b07cb55324a
36a26d68e066bf30592a252b62b400af9934f1f7
747 F20101107_AAAJLG jayaraman_a_Page_141.txt
fd70befd535798f075e36721b9b368d8
6d56cb58f0b7b9c9f32ce3f6fad9ea0306967fd6
F20101107_AAAKOI jayaraman_a_Page_071.jp2
06e80571a24045454d478ac7f3435a44
5722644383661651adcd7b603dfd9af259d2c430
F20101107_AAAJKR jayaraman_a_Page_134.jp2
fbe2d97ed799d65b1c323d8030cf1b25
db4a2ac7514dc19323fedb8df0918017654114df
1051984 F20101107_AAAKNT jayaraman_a_Page_049.jp2
80029bf48d98384ae931395ba03658da
b180908651ebead4138d9de37af918120df7eaa4
F20101107_AAAJLH jayaraman_a_Page_069.tif
336ebac153c9de3672c41fcae881f3f7
10b259fc0d50c83eb1a409de2804922fbb6fc44a
1051923 F20101107_AAAKOJ jayaraman_a_Page_072.jp2
c88ff688cc388186fd82d750aae3907f
d4cb46fb395d1f36500bb510874330d9ebe5e88b
35648 F20101107_AAAJKS jayaraman_a_Page_114.QC.jpg
dd7884cb130f783c55428bc57d5992c3
c04ca1c5b63956fbac1b26b875587fbe5323fe7b
1051930 F20101107_AAAKNU jayaraman_a_Page_050.jp2
c66b4f0c5ab53c1bd90dcabe1634cff6
718b606609e64d9620cd7f8358bd1e5bf71a6ee6
7198 F20101107_AAAJLI jayaraman_a_Page_006thm.jpg
17a2b62448ac3304e4cc494f277cbb40
71ac5568a61f8b20081a029fbb769e9b0463549c
244207 F20101107_AAAKOK jayaraman_a_Page_076.jp2
4581d0196a1f6fe9f5d8ce2f7c580b20
6422e45d4d3a38e96490eacdb82c4ffb529280c2
1051890 F20101107_AAAJKT jayaraman_a_Page_087.jp2
01b10be7815b920b3f9ce6356eb4c8ed
aed47b8550db6bfe6f4947f6fce93ff1eb91bcbd
1051953 F20101107_AAAKNV jayaraman_a_Page_051.jp2
ca7c6e1265356544f665396b772cf7d8
0775e16c1e39fd40b38c1805093c873fe9113bbb
8510 F20101107_AAAJLJ jayaraman_a_Page_130thm.jpg
757641a0a4f12159c7cae968caa4279d
078087f1c4262a62325ddf05cf15ccec11269b8d
F20101107_AAAKOL jayaraman_a_Page_078.jp2
b836729dc194cd7069338252c18524f8
be799b406c639e6ef0685fe121128c9443a94bab
F20101107_AAAJKU jayaraman_a_Page_058.tif
d28303d8bdd97aca4c95069be185ed37
4b98b3d48641e8909bdbebd73f1ae17458bd8b8b
1051959 F20101107_AAAKNW jayaraman_a_Page_052.jp2
5754fde4ae0064210887775cdabd4878
e225ce5baa78e77a48f84577a86f75fe20d96037
F20101107_AAAKPA jayaraman_a_Page_101.jp2
0c68800aa7e4de6c2c13dc9d5855959d
6ca325b5c6a88f280fbd3d9117445b7ce4ce4edc
F20101107_AAAJLK jayaraman_a_Page_046.tif
0c5024f5b175d3301eff47b938432fd7
9bee820f28f00b74fded921fef413fe931ad9a3e
1024259 F20101107_AAAKOM jayaraman_a_Page_079.jp2
c40e5c844d1589db10a93ed06ea39805
94434306c249074cff7aa84994aee261ce514a3c
7937 F20101107_AAAJKV jayaraman_a_Page_118thm.jpg
99d7760d45a4d3d97ef0460c93b9d8ae
9351cb316e6f74f7e5be59221605a69a8a7bbffa
1051925 F20101107_AAAKNX jayaraman_a_Page_054.jp2
cca99925f7591085578a874f92ee733e
cf6692f8d76979c6e51ba143b990bc9a3ee9e506
1051977 F20101107_AAAKPB jayaraman_a_Page_104.jp2
c0137bee5e5b0a179c53300d2aee7631
4a6a028c295fba26614055718392362f631bb594
108486 F20101107_AAAJLL jayaraman_a_Page_122.jpg
88e11f4727c37ee08582b4ede362d74d
e40e2fa839d6643cbe969c3e6d798bc3b406a9bc
995961 F20101107_AAAKON jayaraman_a_Page_081.jp2
7f245bd91a936d5732b1c2ce96d37f55
2a84f5125489b6f256a09424b7c0cce21f7a7cc3
36403 F20101107_AAAJKW jayaraman_a_Page_033.QC.jpg
b5e03527af9bf97f14418530997d296e
973dad58c890b16a329d740efb2931010c9cf164
F20101107_AAAKNY jayaraman_a_Page_055.jp2
b53178c1206985a1bbf6891fd93538d5
8707afb8e01ffd49f93bf7aa414ff0195e5faa3c
497383 F20101107_AAAKPC jayaraman_a_Page_105.jp2
50d8347b99a21bf0ed2b786e8825456f
e1dc84252aa1a2acc47a72292934be4a82ca1005
24677 F20101107_AAAJMA jayaraman_a_Page_002.jp2
bdcd7ea4a5d22511219e483ce884f4c9
f425b8eb8f549fab9ba61d3d4af34dfd706fc3d7
F20101107_AAAJLM jayaraman_a_Page_053.jp2
9ae2a97b471ecd3d3add94dd417e63ee
eb6afc8524cc2ed3d318e0775ddf60d93d581214
1016106 F20101107_AAAKOO jayaraman_a_Page_082.jp2
acc29859f33eac716cb72871a1433bea
e97738494226c738d0797aed76b245e06d6bcfb7
54475 F20101107_AAAJKX jayaraman_a_Page_125.pro
067db12ee2a6eb62f4052ac8d6d97c91
8480aa8f50da00abc37448b93113339b18b1bf74
469227 F20101107_AAAKNZ jayaraman_a_Page_057.jp2
7d4f5c2e70a6055253107b93de93d5f5
cbc13137e31d552b2da588add66eb26f415770c9
694317 F20101107_AAAKPD jayaraman_a_Page_107.jp2
ef93d3eed7e3d4d2737d433af1387c88
f1ccd19ef8c2ae7d0fab6747fa8f3efda79b0fe6
8787 F20101107_AAAJMB jayaraman_a_Page_125thm.jpg
e2810c5c4e4940051b7ef9afedfb1f38
4b7e852119931e8d186cbec61df5b5190abd5d7d
7898 F20101107_AAAJKY jayaraman_a_Page_128thm.jpg
a46dff92ec2cd73339189c44e61540d8
f2b31a3d2947c736478e7997b4ba9687ca87ad95
31137 F20101107_AAAKPE jayaraman_a_Page_110.jp2
6284603b76ced7d976d88dadae7b693a
3d550692746f1381b28f3f108a1b35e2a13a4cd3
F20101107_AAAKOP jayaraman_a_Page_083.jp2
f354a500ec2a0c1b078125d2c18b275f
b9b6919611c35dc0862f1bdb40935a719d4591a3
8998 F20101107_AAAJMC jayaraman_a_Page_115thm.jpg
0bc4fb60496cd48cf9a3185ce26e173f
073b615cf94e2be11302c55c21590e23d306984e
107599 F20101107_AAAJLN jayaraman_a_Page_049.jpg
2ce8c2434c049caa788165e8aadcc89f
0ee7f451be3f4a197e784f0e4fc3e5df0af140e8
59246 F20101107_AAAJKZ jayaraman_a_Page_030.pro
ec125fad7f80df02ab04a2a578d700c2
ecab8f3cd368b633bb1688095431920b97330fff
F20101107_AAAKPF jayaraman_a_Page_112.jp2
0ea9990f9f0d8c8f1ebe5904f8c4e2fe
5dc3023ca7d82b686a8d2b58db3d7034978495b1
F20101107_AAAKOQ jayaraman_a_Page_084.jp2
220d355272e25f6ca719185d752ba112
7eb6afa5d7deb39f6f110321768989aa02d2e6cd
F20101107_AAAJMD jayaraman_a_Page_060.tif
7eccc2b4dcea4a5e3ba1b0fb2e57a985
99a584db9425280a6681405de04a6d68f3c6d891
115069 F20101107_AAAJLO jayaraman_a_Page_011.jpg
2cccb8c2d82c9cc593156dcad53c2d57
d1ff2e599bc4767473d309944ceceb5e3be8e136
1051908 F20101107_AAAKPG jayaraman_a_Page_113.jp2
339b74340d805d16aa7689e553e91987
3ada2cb0ba3e29cfc1194239132a1dfa71c61947
F20101107_AAAKOR jayaraman_a_Page_085.jp2
dbfbe49cdd84b3be8428974cca3e2d81
c1c1044853243a9a812e88f78dbf1c9bad3ad548
10968 F20101107_AAAJME jayaraman_a_Page_109.QC.jpg
e772954fd3fb5c919885b37d75364473
10255039b5f08966d34acf8c7dcd509e7a76cfb7
1051955 F20101107_AAAJLP jayaraman_a_Page_021.jp2
738516071639eb5645d393e9f22838dd
c529acc0f16a743c00a13caffb19f0b9300a30bb
1051969 F20101107_AAAKPH jayaraman_a_Page_114.jp2
50bd20d7a0058d6ce2dfe328127579f5
d50fe1c476917af30fc33693885d7f55724812b3
1051980 F20101107_AAAKOS jayaraman_a_Page_086.jp2
936b606081ce4f867015ff97ba542c4b
3291b827d8d7ddbbaef4544f3a1a3c293e848562
54261 F20101107_AAAJMF jayaraman_a_Page_122.pro
f2ff2b4f922a4185d6cca582a349e5d4
8dcca18100d6d772993000218ae9400f5540fe9b
35742 F20101107_AAAJLQ jayaraman_a_Page_043.QC.jpg
9ce49e188d08e30b6b45254cf9c36a67
469fe0ea6b15272b7835d8c5baa1b4336da094b1
F20101107_AAAKPI jayaraman_a_Page_116.jp2
1af503a90b8415d3040c2816a157e8a6
f6a43ff312891715bc8c96d3e33275fd22fdf10b
590860 F20101107_AAAKOT jayaraman_a_Page_088.jp2
1c6f023794dbe502284db814aa1e6ce0
fee14cf33c34d15213cfe7816fd0973d70fe73e1
32991 F20101107_AAAJMG jayaraman_a_Page_163.QC.jpg
8177aa84ab741581238d830596004831
befeaa13e83a9f655979d651428c75876de1e924
36496 F20101107_AAAJLR jayaraman_a_Page_111.QC.jpg
cb29056e3aa23581d1d1832b3674ff11
6afaf07d446087f1ff432ce6633f85f8d351e842
1051965 F20101107_AAAKPJ jayaraman_a_Page_118.jp2
a5b0ea8ba81b753deb43fd7c75626fdb
67a135f4ae242d93dce760472783bd83a0d32a2f
445019 F20101107_AAAKOU jayaraman_a_Page_089.jp2
d82f3a5b877fb6d1e676fe5bbfb9ba8b
7047a04f519883728f340dfb199a3d084c245043
F20101107_AAAJMH jayaraman_a_Page_126.jp2
655776f095d267ae1c5206e4772f504b
d0ec81a12c01c95516e147b632e20e424a90f863
2213 F20101107_AAAJLS jayaraman_a_Page_102.txt
03aca386fd5c78dccc5252cf0b57a83f
048b802726d05affed34639e89b043963a47fdb7
1051971 F20101107_AAAKPK jayaraman_a_Page_120.jp2
067420bc77f5da17c73469b9143f2845
1e698eef09191af8cece68df2f97403367d17a31
457768 F20101107_AAAKOV jayaraman_a_Page_090.jp2
74ffa9b82d315a79689c712af0dbafc5
0ebf371a1efe844b2c2ff4698f49ec6809b8980c
3818 F20101107_AAAJMI jayaraman_a_Page_089thm.jpg
9ffc024c81825f17c0d37666337d69a9
ff144e887ab3c74081a8fc01bf97ca7fb9aec6fb
F20101107_AAAJLT jayaraman_a_Page_051.tif
2a80c7ff9d1aa2875584ed3526fe691d
b157afbf13a367f02961d08293dbe92c7d892a48
1051936 F20101107_AAAKPL jayaraman_a_Page_121.jp2
6d5abaa07194dc87e7eaf0c2b803e0fc
8010e1ee6d2964ae38b56bc28451150bec30d759
1051912 F20101107_AAAKOW jayaraman_a_Page_097.jp2
514a33561a1413277aa4a10be0922f9a
1359e41908eed5b2cfe33a7a58a2b07a88b800e9
F20101107_AAAJMJ jayaraman_a_Page_097.tif
38de900f946bd7fe1c3463e1ea26ffa9
f3bee58edc7bc535b00d93954149354818941c2d
1974 F20101107_AAAJLU jayaraman_a_Page_061.txt
08054da41a8a33ab1e7925ea1703c0ba
cf3d72d2778090f55b1819f04e4e7a4af4599a48
F20101107_AAAKQA jayaraman_a_Page_153.jp2
ea5ec5eb6655ae6a4e6028317dbc19f1
7fabf3d089d9f487dac251947d55275a8f20ef6a
1051968 F20101107_AAAKPM jayaraman_a_Page_122.jp2
d5bb827d0238648f1005f5d61774d2f2
dbafa10b34c78504d087deb30aac63b8d31d9f32
1045551 F20101107_AAAKOX jayaraman_a_Page_098.jp2
924d3cc6d87e718746d43114bee142c8
a43aa664675e0965e40b32e7f0729433a823bb1a
F20101107_AAAJMK jayaraman_a_Page_146.tif
25e301cb8a1be76fd7f5c612f51aa02e
f30c3bade7dff21562bd80beccb3335098e9d335
F20101107_AAAJLV jayaraman_a_Page_122.tif
d35bd532c6aa721febdbbcd443efa0c6
3e7b453e7d4bd19eaf5cf0066d193bb757877912
F20101107_AAAKQB jayaraman_a_Page_155.jp2
387776e1924e3bef6af6dd4e33eb0b4a
5205dbfa4c1de7c31f45505c0cb476a48b7f16c6
539857 F20101107_AAAKPN jayaraman_a_Page_123.jp2
f6695fc6065fae06ede75f3f5f183716
c9a9bde6e95545bff3aff50e7071f39fb5ba9df6
F20101107_AAAKOY jayaraman_a_Page_099.jp2
6de1834ba67487709796db7ec143241c
28f254d40267f387de6c4c2a663538d1c91ccead
8871 F20101107_AAAJML jayaraman_a_Page_017thm.jpg
7a61aa064265fdb7583e612a66e1009c
95a4d143c7cf57ecbda727d5f7d3fd39f3c3e87c
1886 F20101107_AAAJLW jayaraman_a_Page_117.txt
f5df4131b21305f547a8696981302b0e
5410db83d1c9d75ffe79c4ec75953f1d9d9a78da
275303 F20101107_AAAKQC jayaraman_a_Page_157.jp2
ae789973eba6c0d9dedbc1549806da7a
e472aee389fcb23dac4731912c6e7edad913f279
681128 F20101107_AAAKPO jayaraman_a_Page_124.jp2
5e9e7f24b20cfaa6f7865b009d8b611a
bdb980fdb6103523c14063deb201a4f1207cdd6f
F20101107_AAAKOZ jayaraman_a_Page_100.jp2
aadec4b218e40a27356ebc36e043401e
d8d35d15b3d9e765561cb710c23c7082fa3fab35
110326 F20101107_AAAJMM jayaraman_a_Page_134.jpg
77deff815e61ab94e2f4624750202aa5
0427028c4d0f77ab021b03dd55c7b4baacfb2762
1989 F20101107_AAAJLX jayaraman_a_Page_073.txt
f45251d89f139661bd765e00bfdda8ae
c32ca1193b811631707413b2c80a43e954d5b225
64330 F20101107_AAAJNA jayaraman_a_Page_168.pro
c1f802348ffe8fb374eea09baa0e6c94
4f61c1f0935fb38ca89dc43645510970413bc2ee
282517 F20101107_AAAKQD jayaraman_a_Page_158.jp2
ec00a2ba9b11af3b868b823c8aa871e8
6ff1b993c123b4ee56d444fac4f648897b2a3887
F20101107_AAAKPP jayaraman_a_Page_127.jp2
6010704fcfb3003e29b6ccc7156614fb
ca38ce469c2d072f59679035997c88860d710075
35268 F20101107_AAAJLY jayaraman_a_Page_083.QC.jpg
82f08a7cbbc70b884a903087192672d8
0daf75b89525cd019a155753aaf4c23426083df9
9269 F20101107_AAAJNB jayaraman_a_Page_035thm.jpg
b1c6c284c9c5ad9ac23c2075c792ca98
a635cc1cf40e7f86b3971b80968c7a5cc2b5b0d3
8587 F20101107_AAAJMN jayaraman_a_Page_058.pro
98322b7648b10ba0c9eb2f3cf138ad9d
8e4761a88beaaa08da8573930305b793d187a0cc
312861 F20101107_AAAKQE jayaraman_a_Page_162.jp2
a4a4993cf1776b1b6deb313cf2d5db77
ca43f2d96239900e9e54e20de4a172178a877676
F20101107_AAAJLZ jayaraman_a_Page_137.tif
c2fadda0d1f63a16deaee512c66f6de4
ea2a6845b8f44a0118eb0dece5894c479c816ef3
34707 F20101107_AAAJNC jayaraman_a_Page_017.QC.jpg
8c3b57e27f6cca8baf6660bc1f757783
2d3eff1486a9e60752b96089d450e14a78be53dd
F20101107_AAAKQF jayaraman_a_Page_165.jp2
ab04c776d453c6d5bda865c516e2a8a2
be7344fd6e75ffcc5bf8ef33f16d9db7959d56c3
F20101107_AAAKPQ jayaraman_a_Page_129.jp2
43adc75deb2dcaabac523a28e65f1315
3cf64ac3ec9f3b696f959b728f8fae9faafa78f0
44273 F20101107_AAAJND jayaraman_a_Page_081.pro
f2bb04b2c5de4b9f883d24b87ab1cc51
b6cd19cf18e7ffbc1e918865e3041523d1b94305
385085 F20101107_AAAJMO jayaraman_a_Page_160.jp2
1eba2a6b4c66ec9912bd78d8252f8b15
1a2d741325386c53ffc2e289dde242fc315cfdf2
F20101107_AAAKQG jayaraman_a_Page_167.jp2
ea1b68dbca7cd30f5f30619967150a2c
e2d6812a62de5219c037cf714820c3a6b4b4c60c
1051907 F20101107_AAAKPR jayaraman_a_Page_131.jp2
ac37e10fa6f8b0286c9b7fb34b4dff42
830bbbd93d5ea88f810ebec3287c7414763bbff1
55959 F20101107_AAAJNE jayaraman_a_Page_154.pro
10eb0b84df95834fc37343e9d8bd917f
cf7e0117b72b73b8b2d19d680749b3821ff22575
38657 F20101107_AAAJMP jayaraman_a_Page_022.QC.jpg
04cc8c5a682ef9063e94a21b85a8d5b1
3fe881c4c5f15a5a780f63ecd2601f3cdc72c9f9
1051924 F20101107_AAAKQH jayaraman_a_Page_168.jp2
7fbaa544513133278eca44bffd7e5377
ba1eed5f2647160113c413ce1fbfefb98b76a85f
F20101107_AAAKPS jayaraman_a_Page_135.jp2
5cf57497eaf0474ea8e0264d8178252c
b12fb1b1be0bb59c4361ac8e8df9354cf3f37b53
8833 F20101107_AAAJNF jayaraman_a_Page_161.pro
de6541590ace4079760a15b258a53a94
892521c94a2b31b346c0253cf3fae03a27b35d70
1784 F20101107_AAAJMQ jayaraman_a_Page_070.txt
b8f74206531549aeb4621429d7d5a01a
6662446e47e69ba01214854cb9ce81a1975debb1
1051949 F20101107_AAAKQI jayaraman_a_Page_169.jp2
557c44be5c14096d26b0304f491f894a
9f5ead9b9958dd5367a35d4a268cbb11dcefd201
1051938 F20101107_AAAKPT jayaraman_a_Page_137.jp2
753eb3706957656ad5c7fc46132e8af6
1cadedc25f7536ae0af7ebbbf19d805e0c2ddfce
8793 F20101107_AAAJNG jayaraman_a_Page_018thm.jpg
37b0da3b186581b5a62900eae2766730
762d052a69295da46b15ae2c7cefb0f43c961194
8739 F20101107_AAAJMR jayaraman_a_Page_040thm.jpg
f0092c2cb2cdc1c1773e70d1dca632e3
5f1226d54482fc831c2c5cc579308ccdf701eb2d
F20101107_AAAKQJ jayaraman_a_Page_171.jp2
197f5a8f7de5f001b97f6cf131ef098a
c54f34840134b453399c91ff41b8d355eee7bce7
99414 F20101107_AAAKPU jayaraman_a_Page_138.jp2
4ea234f94a8570ae0cb1d1f65e1c37aa
a049ac9251e060bb4a7caa0ed014fa8758470243
4314 F20101107_AAAJNH jayaraman_a_Page_012thm.jpg
d339b5f47df42d9cc1c37de365138c98
359bb4d57c60537863eff8d038dfb1375fe1a6be
101560 F20101107_AAAJMS jayaraman_a_Page_118.jpg
e490e0e0a71dfdb5105e0b75ef931094
3418437bad616b233d4f8d174f1e5bed63f9ff6b
F20101107_AAAKQK jayaraman_a_Page_172.jp2
56fe8e38159856ddb723850924dc9887
e5fa763e961ef6a504a08469fae2ed059f56a61d
412784 F20101107_AAAKPV jayaraman_a_Page_142.jp2
bbcc1712e99665a72ec26c04278275c6
aa1cfac3e429ce55615d4b7033049c9ae6f580fa
53577 F20101107_AAAJNI jayaraman_a_Page_111.pro
c6cd1f9dcebb59053f43f5e834bf2ae0
9ed960de29f99a7c39b1f04988ad7bb0acc9e0e9
130628 F20101107_AAAJMT jayaraman_a_Page_173.jpg
2bbea635090eec70d67ead487372d2db
1a9a25c049f5332cb47077b5f61f74a0e24b8d3b
1051903 F20101107_AAAKQL jayaraman_a_Page_174.jp2
1544cf47736478168e372b9327077237
d153ea1d4fad005d8eafb4b134d83aaa3397356f
F20101107_AAAKPW jayaraman_a_Page_143.jp2
49c887e225bc3de767afd16faafd5007
4ce2d74e54b2af4887a095363b49cbcaf11b98d1
108113 F20101107_AAAJNJ jayaraman_a_Page_021.jpg
609e53d988e7cd13b21058c5a553dfe8
e9e243defa92ccd9c3eebd71f587fd8ee2e4eb0e
3442 F20101107_AAAJMU jayaraman_a_Page_005thm.jpg
dbb7584aa52759fd5ad3b1488e0bfc01
f1924ba100606c959115d6c79d42b53873c77af3
F20101107_AAAKRA jayaraman_a_Page_014.tif
5f6f8cb352c1f92fe783968dae356ecd
c656bad6702d96abe8e684b8fdf2ede896ae8f1a
F20101107_AAAKQM jayaraman_a_Page_175.jp2
a0298f24c03dd832ca852a9001652a86
7c2e77b4160871e3ecf0003068d94bad6522f87c
1051922 F20101107_AAAKPX jayaraman_a_Page_144.jp2
24d195855ad933e005ce25b05b475a3f
ccb59501fa1107cf187740bedeb7ad351aadee11
33964 F20101107_AAAJNK jayaraman_a_Page_084.QC.jpg
32ba8eb5c63d73a90712ad54f0795d24
419754fff336cbb45895e16faf03ad9bf3739040
2744 F20101107_AAAJMV jayaraman_a_Page_167.txt
7799cbc13255d98c6be671c712f6d87c
9591bcbbcbc06edc20cab0f9d324b23f0c59f511
F20101107_AAAKRB jayaraman_a_Page_017.tif
d9d0276d26d969b2913942d854f8f24e
e49d42128bbd5ca4a860e232321119e8a621e095
F20101107_AAAKQN jayaraman_a_Page_176.jp2
fea79e915efff8d55f77ab931ae46eeb
a71d51fc51a019dc97910a31d8b602608dc2ea1d
1012203 F20101107_AAAKPY jayaraman_a_Page_146.jp2
f5c58b580b9e2f042f5d6bc450f7d663
172fd2575992accd42e542530f46741e076342b2
2158 F20101107_AAAJNL jayaraman_a_Page_065.txt
bf2330d32120c3077ac28639c6967069
7d03bcbcb135c2ba0bb7a38a5df1beb935f83867
13074 F20101107_AAAJMW jayaraman_a_Page_092.QC.jpg
7888de93c130a506f14d1a541ecd9906
02a3a21b66a3ef69016a6913e6d93550df2a3a54
F20101107_AAAKRC jayaraman_a_Page_018.tif
6a9f0a1b8d9917af5a309b0d57fc27e3
8063751a7f446496c25ffd664114981836950d85
1051793 F20101107_AAAKQO jayaraman_a_Page_178.jp2
64595b237f7bc13904388964f7264697
93307007481bc184894bf92748f05c7a9df35aae
1051942 F20101107_AAAKPZ jayaraman_a_Page_151.jp2
2fb4c5df6fc66f6f80e7391cbc91afbe
6b7a1450a35c0920342b27479ed92e031bb950ed
F20101107_AAAJOA jayaraman_a_Page_150.tif
09d86076c97a5363a32c0df042815e8b
3e97759d93a82caef92b3a3a92e5eb4be5572010
2889 F20101107_AAAJNM jayaraman_a_Page_170.txt
f89e8c4b35df7b5888a95ca811372715
9de686602ba94092d0ab7b3758487da3b0ce3494
36704 F20101107_AAAJMX jayaraman_a_Page_154.QC.jpg
f80de90e2b42d6ce579dfd4b2c74dc25
f79e8bce17dd0d667a3469a1698cc814a859c73c
F20101107_AAAKRD jayaraman_a_Page_021.tif
690f464fd986f82beb1358a973a078a8
8a8722b38483066e61cf837b979f5326f96dfb96
F20101107_AAAKQP jayaraman_a_Page_179.jp2
29e939b88d974f7c5ae1856d36694da8
a29217d85617a17b41bf711f055bb8a305cac4ba
35758 F20101107_AAAJOB jayaraman_a_Page_149.QC.jpg
8dc4afbffadc7916789ffaa45164a9e6
0b2dbba6bc207f96f98cd25792b92baf4e8542d0
113453 F20101107_AAAJNN jayaraman_a_Page_072.jpg
3194d6cbd423b64acd0442d747c09cac
8c0652ed43a2fd3f025c2be67218d6ab47fe133d
7058 F20101107_AAAJMY jayaraman_a_Page_157.pro
9a0533e007ef4d63c419a1abd977aaed
9a4e206454fa99c003bb0c46d2cc3222737b4e0d
F20101107_AAAKRE jayaraman_a_Page_022.tif
2feda6e13d42aa5158f835d3fb19cc59
0b3955041aec57640211eb3b7b49baf332bdb71f
F20101107_AAAKQQ jayaraman_a_Page_180.jp2
fe5afa859a58a9185f9486cb33fd5cf7
41f660e2f9b84dfd37886299a77038963a12d408
1322 F20101107_AAAJOC jayaraman_a_Page_156thm.jpg
e99795c651eb567112d8dc38a2b6b2dd
fa8831e69b143c78d3644746d7f9a447c6e5a1bb
38296 F20101107_AAAJNO jayaraman_a_Page_176.QC.jpg
c9fbd507509f2dcad51dd1280e683443
ac543f2020f3bde50558ad49dbb39922c621af93
F20101107_AAAJMZ jayaraman_a_Page_109.pro
b60bd5978db9e5dec9254041daad8e8d
99012ff5c06935ee3755a2495c4aa62ee9b2b51d
F20101107_AAAKRF jayaraman_a_Page_023.tif
3c391c8f7594aa97e6e98475019f30d8
63844678980cc22492e0134e34c158d7791e2835
113655 F20101107_AAAJOD jayaraman_a_Page_135.jpg
fc7a5c9335aba27457f716ac335a5c24
1de7a14a9f65c5fbb8244925f5f8578e551c17ae
F20101107_AAAKRG jayaraman_a_Page_025.tif
5ca8be8ab5f881c53fb4c7261ca58e44
43c13c2d3ef580a841f49c86789bca84cb64510d
297492 F20101107_AAAKQR jayaraman_a_Page_181.jp2
109acec49eacdf33d8bd8b42bec411cf
ca69c326817234668b465fce66f38bbee3b749ec
1105 F20101107_AAAJOE jayaraman_a_Page_139.txt
1283301f4b466e188845695653ce16fc
5176700588c06575a639dbfb9f6366b9229e50d6
15150 F20101107_AAAJNP jayaraman_a_Page_158.QC.jpg
6b7bb0f6fc3dc3d51abf5e7bc2ec7cd3
940047aed645d65e455ac28ff6357179e6f9c3e8
F20101107_AAAKRH jayaraman_a_Page_026.tif
659bf21e095429e32bf00f534fbad9a0
146350dd8195daae519fbe0238a304c23b3cfb70
388197 F20101107_AAAKQS jayaraman_a_Page_182.jp2
f81549f098066b1c662500644a0e758b
4dc791343d52e1d0912487e5e8274f6c89729f19
8767 F20101107_AAAJOF jayaraman_a_Page_119thm.jpg
fa3bd2ef6de23806dc567bf1a8244484
b20c5f33410c0d5310451ada7d32b5007448e628
2174 F20101107_AAAJNQ jayaraman_a_Page_134.txt
95b07277a2f8244c1597a3aaf3249c80
f4d6732499d217528c0e2e8f8dce22fcac9b03dd
F20101107_AAAKRI jayaraman_a_Page_028.tif
c3b23721527a2ec56f1376b25a305509
bee63aded2676b01a2dfda9d5488fa46adb28868
F20101107_AAAKQT jayaraman_a_Page_001.tif
8feca0c5a6c5b65a61b7cdcf847555b0
47f59a2a818bb8a129237a8a50e80654e3662fc0
F20101107_AAAJOG jayaraman_a_Page_024.tif
8ded4a9214ddde1b9bac0151e1607371
43020c176f26faf70d8d252586fa3476ac1cb919
F20101107_AAAJNR jayaraman_a_Page_166.tif
5468bff6492907c072409aa23e30b602
d61dfb1d37fa7409b59e8c803dd2e413f7b56057
F20101107_AAAKRJ jayaraman_a_Page_030.tif
aba7bceb7191e8f88a3251035beeb5b0
cb14e8f7d1cfd326a60c14c790afdc9125d311fe
F20101107_AAAKQU jayaraman_a_Page_003.tif
421a4501e6f4b5438ccf9d1d9e16b18b
5cfa382d38bec4a0675e898bd9ccb3506f73c667
114614 F20101107_AAAJOH jayaraman_a_Page_154.jpg
57295786cbaf731d0636d30587f22ff7
46a18fa3f40f71b1e2d2dc7483df932f3e6cb2fa
464 F20101107_AAAJNS jayaraman_a_Page_001.txt
2eaaef7ae90305218030d05ed5b45b49
f7a82a6dd02efb170c7da6ad3c4186d01f650f03
F20101107_AAAKRK jayaraman_a_Page_033.tif
8940df7742c373c89faf2feb8bc88ab5
9d160526aec6e83da30f1637d193629bd93587e3
F20101107_AAAKQV jayaraman_a_Page_007.tif
729313d023fe016ff8c352176b31347a
e650b1969155f5b80952780d392b4e1d9b5010b1
2011 F20101107_AAAJOI jayaraman_a_Page_118.txt
102779215793b37ee03b559fc87527dc
9e01581d0bbca0c6d0c0dc5895ebe68ce3990b13
F20101107_AAAJNT jayaraman_a_Page_012.jp2
d61fcb723325528afd5a3e2f33bb3455
07addf17c3d286023708ba4f859ad6f12b90bb4b
F20101107_AAAKRL jayaraman_a_Page_034.tif
4ad0f69f34660729b95e999bc4455421
6213cf2ffa9da86aa47f5d7a4906e03ec2d680a2
F20101107_AAAKQW jayaraman_a_Page_010.tif
cbac2b5da11db99351a3fce7a26404cd
c8572d73748392d735f78818eeb4ba4bffde0bdb
13058 F20101107_AAAJOJ jayaraman_a_Page_091.QC.jpg
e9ca68bffffd28e62bbcdec76ccb6e4f
c6a83e3230dd819baafb81c07a46e0414fc2269a
3133 F20101107_AAAJNU jayaraman_a_Page_182thm.jpg
fbe8902b3eb383f4add8955726230e2c
fd798c343e5094d06abab7de786b7337a38e3c3b
F20101107_AAAKSA jayaraman_a_Page_059.tif
3a3708e553d973146b3dd2bf600c5bac
66be2f2fab3ebb1a4e07c14b9f6b33537b3552ae
F20101107_AAAKRM jayaraman_a_Page_037.tif
f852b1ce8646f7f9df21d99eee67da7d
4a188c6774982e05872700c3bc6b885afe0d16f3
F20101107_AAAKQX jayaraman_a_Page_011.tif
6f39710bb0ac321684fc3163a52b5b49
9b58169c25b2ca971c39dcfdc102ce8471e6cfc9
740082 F20101107_AAAJOK jayaraman_a_Page_058.jp2
1e1dd0463d1c395c49a6ebf099105bdf
6038bcbca064a49912530c982ce5f336af21390a
2249 F20101107_AAAJNV jayaraman_a_Page_095.txt
88b5f755cbfafe2af622e32378453b09
1e4efb88394f1409af66e31245b6589ed6edfe2f
F20101107_AAAKSB jayaraman_a_Page_065.tif
5570eb7b4db07efd75ad6fefa185d62f
9a0b3b2b303ce8e4eb95c940afae8baec7dd0c39
F20101107_AAAKRN jayaraman_a_Page_038.tif
6d8d6f11f7a252fe4adf6c9e8f7fb6a1
9fa709d974b13257c0954ee3df54d393af03d42b
F20101107_AAAKQY jayaraman_a_Page_012.tif
9127b1b317fe4103dd7cb649fd80a646
aec796c8226c64e211db3160a5a5cbf8d11b42e7
1051973 F20101107_AAAJOL jayaraman_a_Page_150.jp2
a4e164fad31b8080d6f8aed16a108009
c07236e20431a6d8bf53e86e4b528a635127b658
F20101107_AAAJNW jayaraman_a_Page_120.tif
aafa1e183a36ba8fb0949380298b7072
ad658732a1a3ccd4961665feddbe1bd378cfbf17
F20101107_AAAKSC jayaraman_a_Page_066.tif
5e72b0e9f61ec48fafd860555dc593ff
4afed2be2d6a61c14ecaf353ae0d0bb6f6bfe65a
F20101107_AAAKRO jayaraman_a_Page_040.tif
8f1db7b7ba38522fb6998fbbd198de2d
20bdee411031d71e98d3b345f346e50a327ee91f
F20101107_AAAKQZ jayaraman_a_Page_013.tif
aee37c19e6346c960130792637d47d9a
d3441dd09a515de5eac931691bd7c504b02b7e7b
F20101107_AAAJOM jayaraman_a_Page_119.tif
cb8084a1de47c3cb59c6b157285b2891
495ba1e52a87020069133d8c9be02bd8e26d64d6
1051963 F20101107_AAAJNX jayaraman_a_Page_125.jp2
8e13969a05ea076c1a1d75969fc6b16c
6822ff43beea53e79c1b77c60521732bc8a5cb9c
105252 F20101107_AAAJPA jayaraman_a_Page_116.jpg
26df079537615196c9b5d7bf8d9d760c
0911205768159d4270019f3e76cb3550fd418ca3
F20101107_AAAKSD jayaraman_a_Page_070.tif
f38bd155b5d7a00681b76031813654e2
7c2d4a5ef9cb57acc56cc5c3ad87cbc0ff4df3fe
F20101107_AAAKRP jayaraman_a_Page_041.tif
c7395ead9bf9f6c1a9a4b9d4bb390879
2e9aeff80daeb8eb2762a1f8e2f985e665137563
35494 F20101107_AAAJON jayaraman_a_Page_172.QC.jpg
4cd1968119a712d233fcebc28f4027bd
e8bc18b4da0b972e09ac620709f843a824140e48
8863 F20101107_AAAJNY jayaraman_a_Page_151thm.jpg
9ef59a2acc9574f5ba2ee82bd3a4c7f7
4753f42b54e60f1e329f3c62874236fbc703d494
671 F20101107_AAAJPB jayaraman_a_Page_091.txt
29f638045dc8ca76591d1966ed61b517
0f8dddd359c7f7444e8b678c6753feb044ce7467
F20101107_AAAKSE jayaraman_a_Page_071.tif
d0652d802164361d3f703a4fd864e7b1
4f5e11b03aaf7294a8e6f936bd0108367d3d39d9
F20101107_AAAKRQ jayaraman_a_Page_042.tif
c9c1516c00dc44872d7a3bd5ecafef7e
e73b475e539164d89d205922c76d9731075d1f3e
1586 F20101107_AAAJOO jayaraman_a_Page_124.pro
a6fec7cb30ffda4ca95665448b8e6d20
b48a2f057b026e16c59d9972a8ccdddf100927e1
130829 F20101107_AAAJNZ jayaraman_a_Page_180.jpg
b79f937386e1d04372f16536b0bafb47
ef1adf3c0fd9d37e6fe94909c65165901f4cb1e2
F20101107_AAAJPC jayaraman_a_Page_045.tif
031306fdff7ee7e823a4d6420c266513
216c89ce67e1b560c133dc122eae2c5ea538681a
F20101107_AAAKSF jayaraman_a_Page_074.tif
2ca5a775a7e48272fa0c35ff57b04a20
c4addb00ecb1d6469fffb79de613a28899569cff
F20101107_AAAKRR jayaraman_a_Page_043.tif
d5953c546dfd6f81fa78b59955f701b4
fbae029bdcbd2e3a52bce7a73db60a40efc963d8
9525 F20101107_AAAJOP jayaraman_a_Page_176thm.jpg
131ae6e5ba5969e912e638c10fab7d9e
a219411a4e8983080a433fbb1c6a8b27a778ba82
F20101107_AAAJPD jayaraman_a_Page_020.jp2
ac19e2d651c3868ad4c2bb3ebccecf5d
3a8d1d87ea5206073092c210f19b10783e411a3f
F20101107_AAAKSG jayaraman_a_Page_076.tif
3b66992c1985f250dee7e6d5ce331c63
963257b7c4c718657bf06432b5e831a54159262f
7288 F20101107_AAAJPE jayaraman_a_Page_056thm.jpg
8ac8ed581f0368688bae613118ea2bb2
fbcb34dcf5703101943c6176aea40c7601174027
F20101107_AAAKSH jayaraman_a_Page_077.tif
6d69ba12ff861c8afe9cb9bf5c15258e
d7418c02073ef7e1d360a4b179ae1cdad9eace1b
F20101107_AAAKRS jayaraman_a_Page_044.tif
3b028ed290de00562b26ecef3f91e2db
01c84d1c145aaa39dadcd5daafb95a26b96f85f6
35560 F20101107_AAAJOQ jayaraman_a_Page_155.QC.jpg
cd6a609d7b2afb414975e41b9e8f784b
8cfa0567b2a1c4defc5cbfd0bdf5f1f9675e5ef8
30337 F20101107_AAAJPF jayaraman_a_Page_079.QC.jpg
4a945cc77b5fa754e0c9ccd5c56a54d1
471ca5c9e544e0a1550344dd0d7d17e88d78fc04
F20101107_AAAKSI jayaraman_a_Page_082.tif
c64d21407e7de8c37455f8af810d0396
8d07ff810ce7da92800844c5d76a4a2617c630e8
F20101107_AAAKRT jayaraman_a_Page_048.tif
8e41b2c573082f40b10572b30a7ba2b1
11b3b0cdc986e9fd704682bee08a061bf493a25d
45563 F20101107_AAAJOR jayaraman_a_Page_142.jpg
2d3df0e38deab0163bc5e1aa2f30ac88
ac24778cf8860a3f9cbdade2c498f8d79c4f4e15
2079 F20101107_AAAJPG jayaraman_a_Page_147.txt
3996693fca002c5463d426eeb3f3ea5e
2a5a14299cfa8fc6e0e1f02c5060a4946d51787f
F20101107_AAAKSJ jayaraman_a_Page_084.tif
86688f7f66402ef550e1a7020d762f33
c43921696bb31b6b9a3b49087f4f2854b2ff8454
F20101107_AAAKRU jayaraman_a_Page_050.tif
0dce735c6cc405214242ff6a2c1e6bed
a4e54d3e6b83a617a72970210a47a9f8f976c9a3
105795 F20101107_AAAJOS jayaraman_a_Page_151.jpg
a9194cb47431dec2ca6db4fe6c9a0a1b
6ea9ab0836ac5c58ac09d2f036472be5a5c8be1c
36688 F20101107_AAAJPH jayaraman_a_Page_086.QC.jpg
cc6e243ab33b690eeb00fe9b794b7295
0626d3e77f4db452ed79140a4fb28a818be5ff70
F20101107_AAAKSK jayaraman_a_Page_085.tif
9426479926f0d258db7fcfea2eda838e
c000dbdb667500d0033604c7620a740f01740351
F20101107_AAAKRV jayaraman_a_Page_052.tif
6b6385aa898506b7da3c54aacc7452ff
0c35f628227fd2391726dc1006acbded16736589
2214 F20101107_AAAJOT jayaraman_a_Page_042.txt
448e3e7a5f40cf26dd68ebeb85b82271
242b7adf48ca8a07796f1fcbe8da5281bd5c8aea
33164 F20101107_AAAJPI jayaraman_a_Page_048.QC.jpg
581c826d06eeb3cd7acf8512baf1554b
d6375046d55ed8a24d098ec04de728e9007a5df3
F20101107_AAAKSL jayaraman_a_Page_086.tif
9303d567e382b15e6907a55bc0ffcc08
36fd17cfc2bce06c6cf710f74889d0eb596a0cd3
F20101107_AAAKRW jayaraman_a_Page_053.tif
45fb18355d9dbaaf5a01ec003757dc59
f95257bfe5adc333e4db2477135464b351f4eadd
F20101107_AAAJOU jayaraman_a_Page_068.tif
a64479ae5342e279dabf017da9f4b9cd
833674666fa551f32abb60e72da3be7723eb4315
F20101107_AAAJPJ jayaraman_a_Page_174.tif
8353c52ff5ea2ac40c414a3a16ed6b22
9c57adb44e77f825cae783a760c87974263ccf16
F20101107_AAAKTA jayaraman_a_Page_121.tif
292536df30114882c5e4f9b2060da954
03e2187a9847553e9466e0e6e44c94cac315686e
F20101107_AAAKSM jayaraman_a_Page_087.tif
6b00dfaecbf565b4976a0b5a02b9ac73
593ac6a423fbf578c1697132dd5c0355478fb7a6
F20101107_AAAKRX jayaraman_a_Page_054.tif
1eb9b19bb26817dee3b765e05e565cd3
c7ca1e70227e62c33e4ddcaa01b5ce3ec1fb3425
255074 F20101107_AAAJOV jayaraman_a_Page_161.jp2
74934453f47240137b5f2eb1a0ee78a5
e5ba162416c69dee4b87d8eb8c353c4bd920ca90
F20101107_AAAJPK jayaraman_a_Page_117.tif
374925ebab66e4a207827addfcda50f4
1afe6535097a8c5c3d24dc6217a59b47dc27d3c3
F20101107_AAAKTB jayaraman_a_Page_123.tif
3352d57b667f829b18baf0f3f735ad20
8e591c718f0134d2f2a1d82642680ad1b79a6d93
F20101107_AAAKSN jayaraman_a_Page_089.tif
d06f872fd68e52dfe4c82c746f4bab35
16c48e1ef240985e85e91e30a50e6207d5e11f9f
F20101107_AAAKRY jayaraman_a_Page_055.tif
ce70a1e07e104a08a9b34c0606aba116
db030769ff460d41377e1358e85422521ae1105f
F20101107_AAAJOW jayaraman_a_Page_031.tif
1da027341b6dc4d6e64ed8e1b5daceac
e9625e505a19b101ad7859dd740728ebc4f158e8
1822 F20101107_AAAJPL jayaraman_a_Page_079.txt
0ad7057a693f78f5d352aa1514659cf4
0b32d488e24e350f96808820aa0d9b50eedf52a6
F20101107_AAAKTC jayaraman_a_Page_124.tif
bac66df444f82eff00a054c9f8279986
902f9c8e6019c5b92b846d66d2860f5f11bfdeb1
F20101107_AAAKSO jayaraman_a_Page_090.tif
fb36a84431d81b82ca60f628501268c3
9271c134b858f9173303ffa7d0952d98768a9be9
F20101107_AAAKRZ jayaraman_a_Page_057.tif
ff47c436a29ed6946b659a72ae34bc12
f1970344dcb9005ca4d6592073f2019813eafbe9
9276 F20101107_AAAJOX jayaraman_a_Page_180thm.jpg
ada65b5951919a0be18e6ce3ba7d5d3e
a0a602575be2c5ee88adf3427fdba3916c56e97d
34352 F20101107_AAAJQA jayaraman_a_Page_004.QC.jpg
ce03f2cf4f8fd12eb37a76300dee867c
ed9cc47f5d5c19c6493f8aa5a178b21d5bf729e5
1985 F20101107_AAAJPM jayaraman_a_Page_127.txt
5539bced59e70141d7412f903106ebb6
c73b500e6e3b09804f6e99b2bf27c847368627c6
F20101107_AAAKTD jayaraman_a_Page_126.tif
9f50df44b4d3824faf6c4e7da6a85d26
09532a5a8d18284cc79d246706ff3b4a5f71af44
F20101107_AAAKSP jayaraman_a_Page_091.tif
8524db7cff3944c031a39638a13bccc1
042b663d51f6237ea827f408ac5a0eec676af56c
2070 F20101107_AAAJOY jayaraman_a_Page_116.txt
385a1e39e3cf6ed8a6333ef29f10f903
c47eb44fbab0ac6757b088db28bda5a0c422fe78
33302 F20101107_AAAJQB jayaraman_a_Page_061.QC.jpg
06945b9d683933c4de82259182b8fadb
c694040db7d8cf51f907cb0399fdcda8e9e06d3d
2008 F20101107_AAAJPN jayaraman_a_Page_084.txt
f638dc568b8655ef05dda9ae88859bb4
ed0ef2e9a5d34987915ea2f0b6ba5794cc232e0a
F20101107_AAAKTE jayaraman_a_Page_127.tif
f8300506dffdc25ebe72520f9724ea24
a674315ee48bd7c9f2e1121ea572cb548731def4
F20101107_AAAKSQ jayaraman_a_Page_094.tif
a2690f526413f353bd031c9b02ddbffc
5edcead035ea6ef58d889124eabfdfefe88c6b1b
F20101107_AAAJOZ jayaraman_a_Page_180.tif
80f480f07e26ccaa12d0634c67b26067
0525c2dfdc1f83bb460f9b58cae0aae592838a5c
32627 F20101107_AAAJQC jayaraman_a_Page_157.jpg
bb83aa226fddcfc37691f9de3e89f270
40bdf22a96e4f62265e99a2f911aa499dee48410
F20101107_AAAJPO jayaraman_a_Page_015.tif
6a79b2c8a4dace63ccf45b5a64aa0c20
d67ca52b833c373dd8af4c46a7c0b34787c847c6
F20101107_AAAKTF jayaraman_a_Page_128.tif
436051c9f85c96593ca87d3ad069583a
69f5ef3a891298346c745373349eda12a94144f4
F20101107_AAAKSR jayaraman_a_Page_096.tif
59920ce7f3b52561eec37155983ce0c2
cbb21856234138f703d412d6e20efa0d1ad72abf
55977 F20101107_AAAJQD jayaraman_a_Page_133.pro
3a4f10fa9a4903d22cbcb788dbb04e03
341da0b8554b2f9b30e0dd18791e4ca51ffd87c1
112062 F20101107_AAAJPP jayaraman_a_Page_152.jpg
c9cb37cffac3f3de17b9b31b0400f434
0395d6bf0e082f06664c184e609cfd962b5b17cc
F20101107_AAAKTG jayaraman_a_Page_129.tif
7890a5eb1aa2f8000e59b70a1c8ce0fe
be7a2ab07d3fc6051ba4bd1fc8b28c437d2b05be
F20101107_AAAKSS jayaraman_a_Page_099.tif
4b407f16edb054127016948a32f56a12
259108ec37891f533781d7d20a4a175b96fd666c
F20101107_AAAJQE jayaraman_a_Page_039.tif
f70514034393529db82daeaa138c5a72
005a6e48701bf1820339bf306f27c589e3096f56
8819 F20101107_AAAJPQ jayaraman_a_Page_122thm.jpg
bc2bd429a579e6ec82c7c4ac7fd0e152
4c39a466edd81cb53969abccdd45649e19dd3e53
F20101107_AAAKTH jayaraman_a_Page_130.tif
25dd3b01e062e0656eda3bd88b962154
3cc9e91680afa602cb296e9ed49a77699fad74af
136640 F20101107_AAAJQF jayaraman_a_Page_171.jpg
7e62c263a399ed386ec811a117a0029e
1c1c280e172d7eab4902bf0ccde0a1c68afdfa34
F20101107_AAAKTI jayaraman_a_Page_132.tif
bc5697ae5fcb78cd9ff0f28fd7c5229a
4defb843134aa9fe915ecf89cb06c5dd272abc77
F20101107_AAAKST jayaraman_a_Page_100.tif
29c6a4e7da64bba370fe05d818749530
fcbe35f97c51173e8504c775e71d6126785bd906
1938 F20101107_AAAJQG jayaraman_a_Page_128.txt
e49ccdb55c382f6b16bc7d20c143dc88
2fdda808215ca0ac0295641127a5561db531a389
1429 F20101107_AAAJPR jayaraman_a_Page_106.txt
67c83bfba3f4280dc2de43881e68a64f
f351cad33ec0b92a75bd3c8a0c8ab615720ef3db
F20101107_AAAKTJ jayaraman_a_Page_133.tif
98f5e72065d4859073fa46591f95df77
9faff43fb0a44a6e2182b0e3b15c2e012483c52f
F20101107_AAAKSU jayaraman_a_Page_103.tif
29ac82ce44baf105bf3e98aafbb38bf2
9523abaefc4376361a527fe1ddf73ece7e244916
12099 F20101107_AAAJQH jayaraman_a_Page_089.QC.jpg
5a2679d205f654b0c5b82172b6ed82bf
32a15f403575ba15178dbe2a95ab8f23b0b83d13
2144 F20101107_AAAJPS jayaraman_a_Page_027.txt
b6cbf1e082cd93d2a0d2885ed189e037
c38d2323d62864446e1222449790ae7594120d9e
F20101107_AAAKTK jayaraman_a_Page_138.tif
8c128b1ccbe82887557fa104ae87bd9c
5885fb1edbfebcd2e6d28ec82651f1604778b1a2
F20101107_AAAKSV jayaraman_a_Page_104.tif
5634e67f840c87fa1104413a98f706f6
0dcf3d24d2789a845984657d708dacf449e22b5d
F20101107_AAAJQI jayaraman_a_Page_008.tif
6c53cd5bc423163b231f36c5d7d85645
1f3a6632e73f7e12c3837ebc584e6a1b794b6dc2
55845 F20101107_AAAJPT jayaraman_a_Page_035.pro
20cd7c3c92fa4d4c04babc075a5f1af3
43edc4ee154d1fbbbb51639fafd9c591f198c8d3
F20101107_AAAKTL jayaraman_a_Page_139.tif
eeba2d9f44d7a9227fe8012e3aa8ce2c
4688a8681bdb130cfb50b7fe7b71cb52db19145b
F20101107_AAAKSW jayaraman_a_Page_106.tif
fd6ff33a371826ac1efa8de415994c04
08de7d993e1bd44c861e881cbbc8ba3bf1010515
585802 F20101107_AAAJQJ jayaraman_a_Page_091.jp2
a3079e0d79196b9025897778a3c8cc45
c545ca1bf44088ee6ec08d85c98768c797ff6182
39312 F20101107_AAAJPU jayaraman_a_Page_170.QC.jpg
1cdca57e3ae3b02021669d5dc2156dc6
aabd554f87c845d56917336c64c94ba36d89e8ed
F20101107_AAAKUA jayaraman_a_Page_158.tif
858f5d9c501449e76c6eb8935dd3cab5
d6f1c870437f764f9cc8e875a81657338dbc6a7c
F20101107_AAAKTM jayaraman_a_Page_142.tif
5e3ca6e07f53a1ecda29c5e7b22d039b
916c987d9e25a90a76a05bae30cadf28a01b30e3
F20101107_AAAKSX jayaraman_a_Page_107.tif
33a8b9fbe01ff83412f2fa7f055919bb
d1ca902ebed773c767bc6f25225367f424b5e399
49538 F20101107_AAAJQK jayaraman_a_Page_061.pro
2547e54a2580396e14cd7c3f364c07dc
4c0485a7ec799e09680ebac8b78f542616b7a2cb
8374 F20101107_AAAJPV jayaraman_a_Page_048thm.jpg
df7e1787e0e75e9cd6a180fb048714f7
4651b239ec16b9f7ade01e7c98a447aba5de04c6
F20101107_AAAKUB jayaraman_a_Page_159.tif
46efa5e002601fce59c876b2ccdb7054
93f86b2a34bfc6cff693d7100c62480fa217142c
F20101107_AAAKTN jayaraman_a_Page_143.tif
b4c3849254ad95282c5140e21e584c24
2ef78133a725349c60dad43f697d39ddac472b67
F20101107_AAAKSY jayaraman_a_Page_111.tif
f76663c50a60630c3b8e9e8ddf5f81fe
dc8ca86d94529d94f177363bb5ea7412737b65bf
37206 F20101107_AAAJQL jayaraman_a_Page_031.QC.jpg
39b744155553ca88407861535e065f72
06a185d2fd69d44e6e68d7bc8f366b3c0e70a755
7642 F20101107_AAAJPW jayaraman_a_Page_080thm.jpg
71cc768a0a4eef5301bb569f22b7a8d7
c184bd26d99716f421cf71788f93d396bcf5d699
F20101107_AAAKUC jayaraman_a_Page_160.tif
16aeed8b7239d2d5ec32846fa46faf42
af23f12a0736d84072f6e03d25fac22a24ab827c
F20101107_AAAKTO jayaraman_a_Page_144.tif
1fcac8acfd0a89bbf4cc6af452487324
4276220457a06fd767f2b9ee7fddef5621204dbd
F20101107_AAAKSZ jayaraman_a_Page_115.tif
7653064f6367d38837ba7b3c7941c921
828894da2ebc6f4a670d084cd7b8e26f606bee75
8953 F20101107_AAAJRA jayaraman_a_Page_097thm.jpg
963ad884221cd0d07ef6bf1e1f2cc2d6
aa2d8fc99bf5ea6f08dc6be0828df5889a85d005
36247 F20101107_AAAJQM jayaraman_a_Page_147.QC.jpg
8f63e71ff8b2e15fa5c3a7103591e9a4
9f44829ae29a809f6214cb72ab9703914631c9e7
F20101107_AAAJPX jayaraman_a_Page_163.jp2
0b0c18b1ab2b3744a15e6dd6f022bf2c
8bee2d26b557f9b8965e890c008950bd48e85dc9
F20101107_AAAKUD jayaraman_a_Page_161.tif
5864a51ccc781fe03474b87c059918c1
603c61c85fb526243d1134468b8dce434dbe0fd4
F20101107_AAAKTP jayaraman_a_Page_145.tif
a3a7d4475f1f98ff5d2de207ff359267
31459ce468bb53723536bc9f939078184f05a1a3
F20101107_AAAJRB jayaraman_a_Page_049.tif
26b5a3827c969c3161ba467a392f2bd8
c6097f98698e3da1c8680589cb878af790a20991
F20101107_AAAJQN jayaraman_a_Page_103.jp2
1f378427a7d1fdfa63493a13a30c83b0
475457b43982643278f858deb3458ebdec800176
2538 F20101107_AAAJPY jayaraman_a_Page_178.txt
8139ee86046bab00ea1ff9d8401d3cea
19c253ac34d27ab3ec908fd26c6ee7b2731f1e4d
F20101107_AAAKUE jayaraman_a_Page_162.tif
824311f5303b902a5ee9b85a2d2f890d
1752fa7f6eedb3dfd1796298a85d3cbed35074fc
F20101107_AAAKTQ jayaraman_a_Page_147.tif
59897aa4c36040144b91d1819bcb7b16
31377abf4c950beff35885c473cab1234ba54290
F20101107_AAAJRC jayaraman_a_Page_165.QC.jpg
d33e737fe523ebd003fa2c44d4b1c5ef
6ade06ef112315f7ef6948489d7343bcf18cdc9e
4855 F20101107_AAAJQO jayaraman_a_Page_105thm.jpg
576d082b9b02583b603ec150a8928e12
894fffdcedb4522c9b5bfd9ad53db561a96d25d4
125494 F20101107_AAAJPZ jayaraman_a_Page_156.jp2
3006823d06b2d4f0b7a32800ba83d09e
32042db1499927d97e9f2db0542bd3243787fb8b
F20101107_AAAKUF jayaraman_a_Page_163.tif
8db38f91e797f49039847d640b5ac130
0a005441a07160e4157d379e99e075a9c8d307f3
F20101107_AAAKTR jayaraman_a_Page_148.tif
3a573fedffb04df4dcd2a0032e1858f9
f4a8da78a32e80ee87dee93e0621808fe4dbb004
109195 F20101107_AAAJRD jayaraman_a_Page_040.jpg
acbbfd68f561b08012f4d55a8a544825
047eca6620ab576e85ed856bc41410d1a2a998e1
36728 F20101107_AAAJQP jayaraman_a_Page_177.QC.jpg
00e13325314240b1c26c8a7cc00b2f4a
6c8fd3b36aa5c53da8e641ee22e8136361dacb06
986 F20101107_AAALAA jayaraman_a_Page_140.txt
fc3fd197ff770ee548e4c7d29d3d3a6f
c219d2b44c0ef31f708f6d2340a4b8afe6f55ef2
F20101107_AAAKUG jayaraman_a_Page_164.tif
8acc7666c1b062a8e21b2eb4c09ec15e
fd3cfe5a2b28aa59f76823283c9963fe113cebb8
F20101107_AAAKTS jayaraman_a_Page_149.tif
4f20545086788ff17ec1530da221db2e
4e575fe8caa0a39bbc80c6460620ea2f47269641
6649 F20101107_AAAJRE jayaraman_a_Page_106thm.jpg
b7d153c9366ce973be86a1eb1f56d70f
7d8b210a1d79cce9b1bccf9ace95871a27c8a18d
F20101107_AAAJQQ jayaraman_a_Page_083.tif
0d9f87e514d216e23a4bceeb517c9115
11a881451ed7593ea0849d9669d0abcb3ebb2386
653 F20101107_AAALAB jayaraman_a_Page_142.txt
89ab2eac58c84f404a9fed77eb0dd8ce
f87d8d6a65915754f532f61df3af6253af8221af
F20101107_AAAKUH jayaraman_a_Page_165.tif
cffcc80dcb5ec353c54f4ff780ee76a2
f86e5f9beff700e379f4112cebb7c564f2e2a97c
F20101107_AAAKTT jayaraman_a_Page_151.tif
32dbec845f05168cd9717d57ef8d2b5e
918738aced947b5179c9378ddf20594a478eaca3
114609 F20101107_AAAJRF jayaraman_a_Page_052.jpg
9e7947718715ae5e1e3a95465fe6d0ab
7f03fe357ebed51fcf88ab2dff409d8a6dfdd091
F20101107_AAAJQR jayaraman_a_Page_132.jp2
3d6bdf3afc8d0903ecada2633ef188b4
a5440fa0ff937955f1c7f62317cee83165dd8cf3
2082 F20101107_AAALAC jayaraman_a_Page_145.txt
74811b9bb346fa25a4b5135040365908
31492097b8b531ed8a160ba4b97ddcd95cae91de
F20101107_AAAKUI jayaraman_a_Page_168.tif
f6e5939ac888ee8f69d145d785e3805a
d1ba97a3c359bd461cb6eb7d89b4b5b9c39438a8
1051964 F20101107_AAAJRG jayaraman_a_Page_152.jp2
e224acc39f105dbb16f01fa84755195c
1cee2a027d351dd58883abb82f35f0c07a2ac3df
1809 F20101107_AAALAD jayaraman_a_Page_146.txt
2b4996e756db92518f4cfb61d687be4b
4216e3c7d77c1bf2bfc31b5d49e3ba8b8c19fa74
F20101107_AAAKUJ jayaraman_a_Page_171.tif
75eb72ce00c98a573ae612931680971a
0c7acb589b2e3884b5f110d34b552b64185dc1b4
F20101107_AAAKTU jayaraman_a_Page_152.tif
63516c498896bd84940bc218df42d9a3
b0e148ccb2ca7044a9992e5193ab6db7a716c86b
488079 F20101107_AAAJRH jayaraman_a_Page_140.jp2
b0b8bd993cb30e46fc882d5f0350b0e6
6cbbaae7ecbd76f144846ef1940bfa85a369ada7
54661 F20101107_AAAJQS jayaraman_a_Page_095.pro
ccd8ce069ef7fc1bcda233524ff12c30
852734dc0e4a525cb58da5c77f38c9ffb1d7fb94
1959 F20101107_AAALAE jayaraman_a_Page_148.txt
63655f5da2e7efe00ec733bc31dc3f97
ab718e6600bb118a450735fc8e91a65da6eb19a2
F20101107_AAAKUK jayaraman_a_Page_175.tif
6c137e6e9c582e6eea8eb0f3e3fe2705
82e7677f4530349114438785036627bb005b076a
F20101107_AAAKTV jayaraman_a_Page_153.tif
a0ee53152ff117f1daa507154606abde
515eac3db50cfd9001c4a9d6660e61cc03c17750
35834 F20101107_AAAJRI jayaraman_a_Page_166.QC.jpg
daf204582517ab925433fbfd820358c6
5f25200f17e35b4acfb51d7c89a69d934c2d3aea
8975 F20101107_AAAJQT jayaraman_a_Page_136thm.jpg
70a278b28d097fc75b20bb853fea24b0
6ec4277d8a6ce072e8ec55c5a0529b8b1248baae
2067 F20101107_AAALAF jayaraman_a_Page_150.txt
af86c93b9ab98f130fe8f662df2aca13
7987bb9b4b05b7be9325c88bdd8ba9e0ff848f47
F20101107_AAAKUL jayaraman_a_Page_178.tif
227baa36d446659b38528382b771a9e0
bb3237c5fe16a590e991e82ccdf57c43439c3f9e
F20101107_AAAKTW jayaraman_a_Page_154.tif
8bb313be0c80910e405f5869a740dc3a
04f55f5c34747a6b72ba6e3224bee9e7fd40d8de
103011 F20101107_AAAJRJ jayaraman_a_Page_148.jpg
6b0260a2a49d5cb17c5a6f6e4e7d1067
3f5bb702c5193de7dd0a7900c9164885d617e67d
9103 F20101107_AAAJQU jayaraman_a_Page_132thm.jpg
dace47af53c5c9e00209f43ff1c6cef6
34c374699f024f071cb04bd0c70784fea646a6d0
2116 F20101107_AAALAG jayaraman_a_Page_151.txt
6a43203db4c1fc3ada6f02ef49130891
8bea4f3aa722b1ca0f32211ca2d754ee8dfd1ab0
53263 F20101107_AAAKVA jayaraman_a_Page_020.pro
2bcca7042ecd27400569be1d0f5b3dd1
adbe66a0bd22170703551b49a5887bf2ddbf2a53
F20101107_AAAKUM jayaraman_a_Page_179.tif
8f9679719bc5bd9009fc8dc561d230e0
1690d747b3c1461c7f4dc80cfc8e4b9a1cdbc3cf
F20101107_AAAKTX jayaraman_a_Page_155.tif
2bb44a51e93879f11e11517a049cd9c9
bbe903e518094f141b8783acf41a4dd12a6a733e
8201 F20101107_AAAJQV jayaraman_a_Page_078thm.jpg
b64bfec90414a163372f696fbae845ca
683bfba7255d39dc4db7a84e30c87c2559474b9d
F20101107_AAAJRK jayaraman_a_Page_136.txt
612cb0b4179f637eafd2866f25364e0a
be76e6af4d0373f34a29e3776b8b97819db0b930
2222 F20101107_AAALAH jayaraman_a_Page_152.txt
66315f61f9beab375cfe06d73dcd44cc
8be786ddd1aedb3d11d7169666b85b8f7544c456
57168 F20101107_AAAKVB jayaraman_a_Page_022.pro
5de3f30dc4be5f16e66ae80ac8063bff
cb8fe19580d607396942e3943b82f55137b22ad0
8327 F20101107_AAAKUN jayaraman_a_Page_001.pro
b928fcd2e02e96d8b92eb4120ca16a9a
3ff0c0862ac847df35ffde52c0b0a904d2a4d5ba
F20101107_AAAKTY jayaraman_a_Page_156.tif
aed6116b6a4d6bb1668b097b8e0105af
ec358390bd5530892f615e23fa715cf739681999
8351 F20101107_AAAJQW jayaraman_a_Page_114thm.jpg
2be00cbaef447b79a306a15c5a898d5e
60fd53251ccca492aaab7ffe8a1d76d0fb26f23f
2118 F20101107_AAAJRL jayaraman_a_Page_029.txt
7bc0645d6e549b2c9d371ca949d1fa98
a3025b8e9817b4fd79c84eb2c6c3d32656e1bbb1
2190 F20101107_AAALAI jayaraman_a_Page_154.txt
4e8419852dd51f1685b64c8b4bba7116
b6f78cb734c430554539816992f55e7c349e04f0
56898 F20101107_AAAKVC jayaraman_a_Page_023.pro
b732a8d65c5191e14501039c1d6e6cb5
d7a7c28a01c965e68a8da426a59aad28d09134df
833 F20101107_AAAKUO jayaraman_a_Page_002.pro
deb6d640a4bd6be46d5ea8bb14a65ead
0b5eaac56842b69398d86aea6f5728686313c516
F20101107_AAAKTZ jayaraman_a_Page_157.tif
641afa7174e529c8855cf2eaa3a65cbd
3b455c0b70aae1782d9508181b60fbbd54218ee4
37236 F20101107_AAAJQX jayaraman_a_Page_023.QC.jpg
dbcb4d845b7dd147751a584ef6c9070f
a8c15ff597741d1814cf3fd37cd222ef93f219e4
54693 F20101107_AAAJSA jayaraman_a_Page_034.pro
68c9bc7229bd4bda73d446aee92db404
4cf5b5117e188d3bec80c25782a5f192321e33cd
F20101107_AAAJRM jayaraman_a_Page_154.jp2
9d182253be5de493267ce29343862b51
0f078a3dea5a9a1f8047147558a3f16b03157b83
212 F20101107_AAALAJ jayaraman_a_Page_156.txt
25d9632c0c32a7aeabb4503bb5985493
f0b155e5824184d9d596f7280ff73f992915cc6d
57474 F20101107_AAAKVD jayaraman_a_Page_024.pro
04b0134e93d7b1e8574d8d8d9acf5be5
dcd6e291b90d8a5375a8fcecd534efe6c281d4ee
1223 F20101107_AAAKUP jayaraman_a_Page_003.pro
dd175192bb95af74a2e38516c741a9b0
bc0aef050c85e771b0434267652e9b834ed3c007
22004 F20101107_AAAJQY jayaraman_a_Page_058.QC.jpg
465454836060342aa71402408e63c2d3
92ad208c31cec9103e50c356516a27b132461322
F20101107_AAAJSB jayaraman_a_Page_075.tif
3496da3beac060cd9e3e11f7e96392bd
15b9e615af9b58211f53281452dfdb64c28d1e02
59212 F20101107_AAAJRN jayaraman_a_Page_172.pro
89f1d14949bbed073b70e94b8e202d02
12947eeb34533ca7ae843ef23bc1ad1376d3e1c6
310 F20101107_AAALAK jayaraman_a_Page_157.txt
534f34b65bf2d3afdda1f78a703a19b9
33fc41e7dc546bef37b1aef8650aba390c9af4db
54355 F20101107_AAAKVE jayaraman_a_Page_025.pro
0abcfc989150907352a9972ce0e7f5a1
b580ebe1d0b3b83ec76f3ba52203e7f9153592e5
51607 F20101107_AAAKUQ jayaraman_a_Page_004.pro
fba88e98a7a2810a70aa04a24b4b73ad
a28871d6530b5615008719374ddcf179ef01d78f
7384 F20101107_AAAJQZ jayaraman_a_Page_082thm.jpg
395fb227f0c1053ead74494f764d4ec7
78a8edd361cab48bf07bfc30aa7ec33f025c86fe
47000 F20101107_AAAJSC jayaraman_a_Page_013.pro
fdda806b7a1f331523f3baa87413e813
668d9de8bef25ea775cc2044e01fba4ece62232d
10783 F20101107_AAAJRO jayaraman_a_Page_074.pro
c055236e4f5954463645379da2b7b396
d36b189afd4602aed7f02c2390675ea2bea8cee9
1326 F20101107_AAALBA jayaraman_a_Page_003.QC.jpg
be2624f1f513bf87c79907328646b612
0332169402c18ea71fdc950a9a20513d03b286b4
1013 F20101107_AAALAL jayaraman_a_Page_158.txt
a0fe2ed88d13049198dc693ef32ac9f6
01f57b01c9dd77689019da2018dfa1a1d36eb85b
F20101107_AAAKVF jayaraman_a_Page_026.pro
4c9ed7ee1f28de00f004b4d996bf4d73
fb0773beb25e57cb772cd790b7c2603e087ba618
83234 F20101107_AAAKUR jayaraman_a_Page_006.pro
23cae5e45a1056a9065719ffd54e7ed7
d49f330a4bc83f4a8859f794777ab17dff5a5336
5227 F20101107_AAAJSD jayaraman_a_Page_156.QC.jpg
d6b1235b0f4b99492450bfb4db61ced5
3b94b7228634510bdf4488d86f6595e847398ffe
55686 F20101107_AAAJRP jayaraman_a_Page_050.pro
fcd1b4ab5424506457de95348a6c5d1c
1bb5049f24741b8073189c142f2ba1fccb92af35
374 F20101107_AAALAM jayaraman_a_Page_159.txt
09821bf592560f9bb56d6828b0988435
f4f997ac0a0e2479ca6da49ae72ef896951223de
50428 F20101107_AAAKVG jayaraman_a_Page_032.pro
ab6a23b93beec8c53232016c50420857
1de5f929f89ab9b442cbefeec4918dc033decaeb
86461 F20101107_AAAKUS jayaraman_a_Page_007.pro
62ee37dea39ebe2f14889f48686af1f2
f94bda2dfdcb9a0eb72a1d721b0a56fa5bdc0ae6
F20101107_AAAJSE jayaraman_a_Page_077.jp2
878c3eeb30c5ff6ea4ce5ab377c759a8
d880bdd0ce2c239442fda7e438aec9422cdf7d07
928258 F20101107_AAAJRQ jayaraman_a_Page_060.jp2
c3a370dfaee5a2bc6c462b83c854cba5
e6e26060a22ff60afed4a55467bd8b211560238c
596 F20101107_AAALBB jayaraman_a_Page_003thm.jpg
9a536fbc3099c76264e29bc7d3c85294
3365e01bd0fcd75748fb2b23de57ee851e0a7bf4
2246 F20101107_AAALAN jayaraman_a_Page_163.txt
8b356280fadb5049568cac6f179c5bf1
f1ae563d71f06abe3ec4484f562f6ca0a410eea6
54603 F20101107_AAAKVH jayaraman_a_Page_036.pro
941cc06311691f5b78595919b3333181
8fff5dd77ea8682ed6e2c760487ffbbefc90f183
95114 F20101107_AAAKUT jayaraman_a_Page_008.pro
3895d8ddcd0e58b91c9c513321e456ba
d81564504ae32a98d0a9de056a004ab22cb87798
51093 F20101107_AAAJSF jayaraman_a_Page_066.pro
4a1da1394ca71be7062515278db20aa0
cf6d16c688444e73a9a73385375be535661e8789
F20101107_AAAJRR jayaraman_a_Page_081.tif
c98f11a0dcc832f463684d7d47a578d6
d55ef447e9267c3a678066ef0ad109e804383fe2
8838 F20101107_AAALBC jayaraman_a_Page_004thm.jpg
1e35ac3e0c7a1d1f06ee50fff01cbca9
b24178f71f3cc6f3ba3505c93264ab87b6e8a8e5
2789 F20101107_AAALAO jayaraman_a_Page_165.txt
16e4cb32cf715a1a0575f60f427deea5
5c3a1c745ce6fa321efbb1b72f6a1b691efe2f88
54034 F20101107_AAAKVI jayaraman_a_Page_040.pro
b3bc87b7bd0edac4813fa6b349d373bb
ffef71394c9b50a8c000b5715888f2162f378226
79637 F20101107_AAAKUU jayaraman_a_Page_009.pro
594a20e15cc1f364701e6e15a9b98d0e
2543d968bb34f7c6685fe74b6f5439a5ba9626eb
149287 F20101107_AAAJSG jayaraman_a_Page_175.jpg
88f651da752ff01aa5853ed97471212a
9b1f22a4b5cbf8da4635c1d096e6f6fe89fdc4ad
11749 F20101107_AAAJRS jayaraman_a_Page_162.pro
dd95b2fe27e713c64ac3d5af762dfa7c
09462ae6cd40f6624e60c039039fe3f0ee7d72b8
13886 F20101107_AAALBD jayaraman_a_Page_005.QC.jpg
79d758422934481c2e6f40d84731fd3f
ca0820fa7024aad226794ac05c915c65f7c7ff96
2497 F20101107_AAALAP jayaraman_a_Page_166.txt
8aa0ad15968be65dc8e11cc943c11835
17f935327f6a480d7a3dba244cfeea46be6b14ea
55190 F20101107_AAAKVJ jayaraman_a_Page_041.pro
8d0c9be34bc981cf7212120b9b9152e5
0c30de8e156ba32c48fd9b892d794731d6da0dd9
48971 F20101107_AAAJSH jayaraman_a_Page_078.pro
51f60b15ac2c1ebbf97df9ff683ba98a
2ca3e22713be3fe217d4dd9385a9a5f4e1689cee
30052 F20101107_AAALBE jayaraman_a_Page_006.QC.jpg
e7a88b0279f6643573bc220749ac40a6
9312c020ee344938c640a45f1c6d7a1bd556e639
2634 F20101107_AAALAQ jayaraman_a_Page_168.txt
def69484db9b6c56cb75d9b793c5211a
22d36142040befe8293b67d8b5b8913b6d2c51fb
56525 F20101107_AAAKVK jayaraman_a_Page_044.pro
3d25eef3d67bd80c63310727e9e9b410
d644423c525080c2a26aa0eced9de3d21b51c011
33325 F20101107_AAAKUV jayaraman_a_Page_012.pro
bedea70a871ce37cb88087e387df659a
4e8c818effde296649e809afe2a2c4fd0ad8223d
2123 F20101107_AAAJSI jayaraman_a_Page_040.txt
14b4048190e80cb2a7e3e15ba6c3dd73
236d0d4c3e06e48cc505616efed52a526104115b
95375 F20101107_AAAJRT jayaraman_a_Page_098.jpg
e16de6fbc0b7b4a677abfba779a01cd6
b84d96897a7f96c97cad9125bf9a40e3b9a9c402
32003 F20101107_AAALBF jayaraman_a_Page_008.QC.jpg
240f34c5658f05a1455f435425710223
d16f2522a07a43b71c8180e431f722e17e1047b0
2661 F20101107_AAALAR jayaraman_a_Page_169.txt
bca1ec6561bcca415a028efc8510dbab
d333a06883c2f4246210771ad4fa2e472a422be9
55220 F20101107_AAAKVL jayaraman_a_Page_045.pro
a8f5f1ba4b241192cd07be3a9d508e18
2da5f7416a96a4c99b40bbbacae915e06aa97578
50311 F20101107_AAAKUW jayaraman_a_Page_015.pro
625d5b0423f976863ec7c6af5155a724
8a472a6e9554be2caa1ac3ff0f05b3966fdb3f24
1051962 F20101107_AAAJSJ jayaraman_a_Page_013.jp2
0ea9ea0fd24d3c209cb1a0c0c434f4b3
26aa3b5bb89a21cac9ca954bd31823d5e040d097
5404 F20101107_AAAJRU jayaraman_a_Page_158thm.jpg
aabfa48c2c06f86ae63296d751eeb0fe
f03f5aa754640b6069b3496c90f98decabc39d33
8263 F20101107_AAALBG jayaraman_a_Page_009thm.jpg
a02f544eef4a01fe09e577a2a66f7c5f
656df8d6577f78d3fd3ed7b95553ec073b354b59
2720 F20101107_AAALAS jayaraman_a_Page_171.txt
9d5ff9f64de76f1180cc27e0f2f8c691
4d7a14d5c60e936be20693f239cdec671e3a940d
44414 F20101107_AAAKWA jayaraman_a_Page_070.pro
4972004c9c4fc973b9e51134d5a98cb0
6e4f3efcb65799a1105947f90c124055a864ff7c
56293 F20101107_AAAKVM jayaraman_a_Page_047.pro
e96e0ba05d658c344a7220f15bdb5b34
1a5f32f51a6d73e8751e90dc6598d1f3e24d4d63
51866 F20101107_AAAKUX jayaraman_a_Page_017.pro
114edbb066d8672e0f09dc41fcf78159
492c7d5e18baf2d7f40a2ab76c3e95aa479ce1ce
F20101107_AAAJSK jayaraman_a_Page_035.tif
e5763e0b6aa93311ef2a370c076c239b
0ce181afa0d55b6b854d82e09eef30699fce74aa
6986 F20101107_AAAJRV jayaraman_a_Page_059thm.jpg
320e61e46a7739144f3f5f374913659a
38a917d28750c5a6178a9b99fa073cfa8abd495a
2676 F20101107_AAALBH jayaraman_a_Page_010thm.jpg
879565078d3a42d73912014df9209f91
5037916dee21044c3374441a6b0be6b35851cdf9
2617 F20101107_AAALAT jayaraman_a_Page_173.txt
d77d56b25d4edd248c14ef802ad6879c
91826fb1496c1a1ba44fa45d81a36b206b2a59da
50498 F20101107_AAAKWB jayaraman_a_Page_073.pro
6de5d4ffe2f203437c857de80fc57de6
74f5c2573e3698adf468422d72f978c666a9016e
58179 F20101107_AAAKVN jayaraman_a_Page_051.pro
5966b2d796236c921af0433626b023ed
c64163aca1aab5efc7b2333db28e3a6b31ef1ead
55475 F20101107_AAAKUY jayaraman_a_Page_018.pro
16366774f6493f76ea546f0cc1845c74
efdd89470565a4d4b52ed474d1c7139406916ea5
104157 F20101107_AAAJSL jayaraman_a_Page_126.jpg
ea2a89b2925f491803fcbbaed332e08e
f001cbfc29b589bd126f9ac310052700047c413a
33991 F20101107_AAAJRW jayaraman_a_Page_010.jpg
4d9dcca95846633413dd627fad8143d9
0d17a454254f3b2568dd98f5976980abd2c955b2
32789 F20101107_AAALBI jayaraman_a_Page_011.QC.jpg
d043c006ef82b78165b4d6dec9ec0fb8
36749993d8e769362bfb6a8c9ad916eb059ddc9e
2837 F20101107_AAALAU jayaraman_a_Page_174.txt
7dc7f39dd20e03c01dda99f5cc7b1726
40015d3c054582dfc00dd00e235edc3e74bf0aac
54934 F20101107_AAAKWC jayaraman_a_Page_077.pro
92c04194ab64e3fcb92d94e9715a58c4
b68955707134be1b0798fef88043838c3869869d
57740 F20101107_AAAKVO jayaraman_a_Page_052.pro
dfcf1651c77cb5ef99a7365410e30801
d7d5d96e2f7f92e32a7c6379f0fe392a8f2769b7
54607 F20101107_AAAKUZ jayaraman_a_Page_019.pro
ac5e9bb172e1ad743ffad919782af936
c75ff913ba412dbeb3c07f7827eae9462dcfe9ad
F20101107_AAAJTA jayaraman_a_Page_032.jp2
5fa6e739b586fbe4c38da2a6204d02b9
21b36de9d4df323ac8cf5e4dceea8fce3353432e
2320 F20101107_AAAJSM jayaraman_a_Page_143.txt
9616be7738c816caec88e1a1dd808575
0e4dab653c24e7f27fcf05fef624d3509735274a
4708 F20101107_AAAJRX jayaraman_a_Page_162thm.jpg
bc8303a495b9eb6d0b959ad7410a3f44
ddf6ed177e24e8ca95e1daad6fa56f99185f4839
8132 F20101107_AAALBJ jayaraman_a_Page_011thm.jpg
b77598861827bde91c2181abb8381a87
7f0ddcfde84986170b633c8e043d632d2f7ca9bf
2822 F20101107_AAALAV jayaraman_a_Page_176.txt
d2fd83804e7e4383048ae8f814b6288e
29a4e7fa611adaa650fd4d29a130ba89534f496a
45946 F20101107_AAAKWD jayaraman_a_Page_079.pro
789a0c53acfdd56151669b2924fe2ff4
507301c2bca0a8a681cacac67cb706001d2041c5
54580 F20101107_AAAKVP jayaraman_a_Page_054.pro
9b8bb0d13e6f736fe0d50ba311365480
3321e9c250aa41cbdd7517c92a66cc4afaa0e0a1
1573 F20101107_AAAJTB jayaraman_a_Page_063.txt
3152bc47911d66b5017b35f5655b4732
8af5c9a9527b153130168fade28564cd7bc0f51b
F20101107_AAAJSN jayaraman_a_Page_027.tif
b05d175a4a9225c59bcbbb9281e5adfd
89407b272e0547cf8ac4a5a342e81b3d5cc8651c
2177 F20101107_AAAJRY jayaraman_a_Page_104.txt
abe6358f0b821d4783156f470199385c
e7a3c08d6bef7de2faee5c46e21289d99f042389
17445 F20101107_AAALBK jayaraman_a_Page_012.QC.jpg
c59a7660484648d21eb37d0fc5b94688
e816b70c4afcb354da36aea267c85d4f3059eb72
704 F20101107_AAALAW jayaraman_a_Page_182.txt
3d294b3a9c22707688e4efe097d3ce85
a6e9455be4f2feb5aaf93f84c2c76888246a22a2
47584 F20101107_AAAKWE jayaraman_a_Page_080.pro
b05e06266f1371eec6bc64270f4e82af
2b465290c031abe2eb210377e65c21c1096d6b29
53359 F20101107_AAAKVQ jayaraman_a_Page_055.pro
7e6a1bfb61afc62f863112a81fa45fea
a3b316ad706eff2072e6ca8a947db6f2d5accc22
9262 F20101107_AAAJTC jayaraman_a_Page_133thm.jpg
665ec3a6d419e6e9044cba1c2b6702b4
39e9dc62c5bd007c3c0f0c90d07d2c508e5f276d
17987 F20101107_AAAJSO jayaraman_a_Page_140.QC.jpg
f53db5b5730daaf3151581728cbd654b
becf4badfaf4da6e7d061cc6e40e86f373fc7be0
8611 F20101107_AAAJRZ jayaraman_a_Page_148thm.jpg
b5b2cfc253b1b65515973ce1026240f5
5405bd8a34b05346031fe050231d5f9172b72437
33072 F20101107_AAALCA jayaraman_a_Page_032.QC.jpg
68dae3be9f92f0f67027e6dd5528d13a
ad45a60b2a389841ecfac9d09cf8a7126308b4a1
30187 F20101107_AAALBL jayaraman_a_Page_013.QC.jpg
e93040a16f925dbeb442c6ecb19fba1a
6abbfbb0d7528499fa794dd09ae96df3aba3560d
1292018 F20101107_AAALAX jayaraman_a.pdf
e07f3cc19834184b84f5ee26daf2627d
ed6f1733147235cb6a462ec9c69860fc4b6274cc
45237 F20101107_AAAKWF jayaraman_a_Page_082.pro
4be8fc694d81153105d23b3cdbd6cf68
deab606aa6d6e24cf3a30bde7a4b877736b5419c
35167 F20101107_AAAKVR jayaraman_a_Page_056.pro
754aaeacc3cf2bbd6b4c99837539725a
2e168768473ded837a8195f72a12f2b693ea6862
34809 F20101107_AAAJTD jayaraman_a_Page_040.QC.jpg
0a905846c40fb083f93f27b2101590e7
887c32684570028aa214b6d51f77c38f0761deb6
58219 F20101107_AAAJSP jayaraman_a_Page_031.pro
231b2731eb7ad0167b92e98549894781
ba297c23c6cbb638036b2b56056f2a0d71dcd027
8708 F20101107_AAALCB jayaraman_a_Page_033thm.jpg
bdd639fb7553d1d0f66fd3d88d966dec
8c290603e17e3fbb6cd0e13f1d8b60f12879209e
4874 F20101107_AAALBM jayaraman_a_Page_014thm.jpg
6c2088356457b47f3d4d300e872dbc7f
27904a8946aa2fe7ed9b1b386eb141216ba991ff
8128 F20101107_AAALAY jayaraman_a_Page_001.QC.jpg
8083a62f7b5c3f30cad9d7bde4f09675
6266c3748fd7be06393ba5f2947d223721f2d5ce
53356 F20101107_AAAKWG jayaraman_a_Page_083.pro
fe0a6703a39e4c9bbdef6e0d14944712
ca3a57ca9f5eb9a1bcf85e2cbb79b8952a7a7290
19844 F20101107_AAAKVS jayaraman_a_Page_057.pro
832fb807da288a6b890f6cb56578fdfb
84ec6c83050bf7269f3bf76e01798d1477ec61ad
8138 F20101107_AAAJTE jayaraman_a_Page_016thm.jpg
005c4a9265a1f75e15d1bdd64220c374
9eeaf9b946b99c22b7eb6e2358d0967b6abfed1b
F20101107_AAAJSQ jayaraman_a_Page_136.tif
6c25cbc687b4b7bed577ffc9612d4ede
c240a3355bd73dc0b3475284e2a171b58fa07474
8381 F20101107_AAALBN jayaraman_a_Page_015thm.jpg
16e093502da4323dff8ec5a41538eb09
8ba17e3efc7ce23dc352383a4998284f9ec2c057
1156 F20101107_AAALAZ jayaraman_a_Page_002.QC.jpg
9fb7904b1c17c514c767525f130e13fc
d0aab478056822632e82afb9ae01f3b4a5c9827f
57447 F20101107_AAAKWH jayaraman_a_Page_085.pro
0e5bd43053ae3301b801431af41a8ba7
693a421b4ef77ba0305eecf3b0a61b94c42e295b
38753 F20101107_AAAKVT jayaraman_a_Page_059.pro
f00efe1c1fb27b8ca795cb70d01457e0
719ce74882ea25761c2aeaf945d89a78ed48c0ea
35572 F20101107_AAAJTF jayaraman_a_Page_145.QC.jpg
17883dc48ea53025a31b505f6a2f24e0
6979ddede81de5fd1ac30e9c9690f1996bcd86a0
8651 F20101107_AAAJSR jayaraman_a_Page_149thm.jpg
05bbb5257c4a0d40b42d4e1436fc1bc4
611fc4d68d99e54f15577e40a2b5cde0495d1b9b
8733 F20101107_AAALCC jayaraman_a_Page_034thm.jpg
e6f78f70221dcf84a3e4cebc84ece044
089940d43052d6a6b2e23e10adc44cf0882a800d
37638 F20101107_AAALBO jayaraman_a_Page_018.QC.jpg
ba61b1f4caba31dc7bdcfc6657621f6a
9aed46c37ca2c668bffca15c31abb086e0ab4d44
51596 F20101107_AAAKWI jayaraman_a_Page_087.pro
935a668cbec8764333ec910052261318
18cb440334ba48e66e9d98e8fe86ffa208db5d39
41200 F20101107_AAAKVU jayaraman_a_Page_060.pro
ec287d3f7e6fd45c92aefe00c7d517be
64c36fd9e40def5675c52bbea45cfcac720858c2
113003 F20101107_AAAJTG jayaraman_a_Page_047.jpg
a69cee8c48f9447bd753f8e3d68dff3a
3d5f79ccc0508ded261038f2e229cff399b8d9ab
F20101107_AAAJSS jayaraman_a_Page_136.jp2
5a4f5345e8eb14363e6eee2d06aae129
84f0ff23b2e9cacf1bde12066712bf50b29ca0ab
36907 F20101107_AAALCD jayaraman_a_Page_035.QC.jpg
378e38c6ddfd32700b3a1200fd431b06
a0e78ce6110c79a652b8ca882429e19c7986a6e8
35488 F20101107_AAALBP jayaraman_a_Page_019.QC.jpg
644e78b80caaae312828a909ee10e014
452fb13407c6ec834c3dc0eef9e3ea3413683811
25845 F20101107_AAAKWJ jayaraman_a_Page_088.pro
78813794de1a985fb23ca984574500e1
98b210dc94711f40878d3d1b8b1a5f386bea8f63
46964 F20101107_AAAKVV jayaraman_a_Page_062.pro
f03d1854f83a3fabce65e7f8430a9a42
91467fe3df98b4c2bac1e5e6403f066f87fc823e
36690 F20101107_AAAJTH jayaraman_a_Page_029.QC.jpg
52a5229f6330a31aed3e87052806f629
f25fa0e31d1c391b3bbfe56b42ad8375eb501282
3464 F20101107_AAAJST jayaraman_a_Page_009.txt
7faa4790b1f0e520c1d87862dac92956
36d9675f9acdc8dd3b7d746c86ff911e19b7edbc
37459 F20101107_AAALCE jayaraman_a_Page_036.QC.jpg
73b0b0eb8883bfefc56db11fa29cde3a
78ce9867e0ddf9db75b3f60771bf6fbfa133934f
8612 F20101107_AAALBQ jayaraman_a_Page_019thm.jpg
abd9ab432b0d1287c562f1758dd22d60
5227231553376b9bd955696f523d9e6748c722a7
9021 F20101107_AAAKWK jayaraman_a_Page_089.pro
296fa1d35902a511f66824f600939aa8
c1a0909e66df3a7cb811b4dc9b5d7d2e69e9904e
F20101107_AAAJTI jayaraman_a_Page_116.tif
0d77ca0d5fe8287e005709e08d773b8a
a03689c1720841317392621335a701184a734cf4
F20101107_AAALCF jayaraman_a_Page_036thm.jpg
f012b92b425aabfb505f69416c73bd61
f56b3d3ae21294989c58dc97d791eee9106ec608
36012 F20101107_AAALBR jayaraman_a_Page_020.QC.jpg
63ebafc0b3b6638873b31c2f333094c6
e6703826a77a110fd98bdc63b763ba13a048879a
13759 F20101107_AAAKWL jayaraman_a_Page_090.pro
e49ad36fd9f54ca619961b7698566871
fd123157ee8876b06c38086362c7d796ff2fb7a0
38773 F20101107_AAAKVW jayaraman_a_Page_063.pro
d75c70365e971d7dee77c23333ba8752
a24b1b47d4e2efb0770ac7a9a7e1d26748c7c727
F20101107_AAAJTJ jayaraman_a_Page_019.jp2
e1bf941daf2cb2c0ec3cb5d7b0e8f8fa
361c913a1a8acc68aa4ee83ea23b0710d8566119
386316 F20101107_AAAJSU jayaraman_a_Page_141.jp2
bf861eeddd805c7781f9033608f507b5
3d5f65c3eee2f8eaa3ec9c6dbf30f2e08a9b29da
9121 F20101107_AAALCG jayaraman_a_Page_037thm.jpg
0e5d150c8020e379ff9d21f0e5bf1d42
bb61cc6a10efadac97d34af0e42fbe6769812ac0
8941 F20101107_AAALBS jayaraman_a_Page_020thm.jpg
72b36cdc57865a9d68467349c4a0cfb6
c637407f97c922586e9d4e046d5469fbd800971b
55520 F20101107_AAAKXA jayaraman_a_Page_134.pro
924e88cf2a43e0b11815192a185b885a
3b8089aa3d0944905f85adb64a696e089de9b8f9
14533 F20101107_AAAKWM jayaraman_a_Page_093.pro
9d608754c565a4e13dde8443170ff0dd
00c42c1e17179cd222a6f5996548fa20d8828bf0
49551 F20101107_AAAKVX jayaraman_a_Page_064.pro
436f27ef0e78f61287136e32a1127ca4
bca965653d36c757c771330cccf46799bcdfdff4
8347 F20101107_AAAJTK jayaraman_a_Page_068thm.jpg
cf2069724cf51e48b788d2f662f9f1a0
6bc7580c94f85c338e06755ff04cfc0ebac11dea
40901 F20101107_AAAJSV jayaraman_a_Page_093.jpg
43004425a83b0bd34644252f4a8bad60
cc071e3cd2089083855675d8ecc95d3612587d48
39052 F20101107_AAALCH jayaraman_a_Page_038.QC.jpg
fb9578e56189e1b174040b3971e9f19c
84c077e6b3287ceb0e4738f9d3edfdb700732dc8
8901 F20101107_AAALBT jayaraman_a_Page_023thm.jpg
ea089cae3f210160d9bf16bae2ba1d51
9d109288360a0f478c3dd1bb41c62e1a659bf1aa
56500 F20101107_AAAKXB jayaraman_a_Page_136.pro
b2bf29f13e86655db278d621c55a377c
56afd09f8a536dac4181389f1c89cbddfccb2e0d
11223 F20101107_AAAKWN jayaraman_a_Page_094.pro
f70226f7786b49970c4c62cda221c041
fce30067b3fa673e6cd3125f8f7c8a541bd8e3a9
55222 F20101107_AAAKVY jayaraman_a_Page_065.pro
af0efe815bec897f61d2b07841ebb291
0b01c26f95579af6ea1d61d82cdd654435dda2de
F20101107_AAAJTL jayaraman_a_Page_141.tif
f4a48c106253ebe1fd5adbd0523148de
b834bcbe5940bb6bd94a2604f45b83b00d781eeb
1995 F20101107_AAAJSW jayaraman_a_Page_032.txt
db27236d6cea83428e7ee94b37036bea
0b08faaf40bb50f98cd2076238bf28e807a70291
9031 F20101107_AAALCI jayaraman_a_Page_038thm.jpg
362156799a36dd24b794478e6e39b40c
18853ca88db9820483adab1e3613e1f8b9773e54
35974 F20101107_AAALBU jayaraman_a_Page_025.QC.jpg
fd218f998190ca7f476f8f09c53d68e9
f13d3735b6b277211c9d6480f33c429471fe6f1f
56193 F20101107_AAAKXC jayaraman_a_Page_137.pro
5437e4fe55403189021e57dd768b5afa
2922b621eb918d00a5f26d2a2328755ef533ee79
55245 F20101107_AAAKWO jayaraman_a_Page_097.pro
09ac7b3c5e387fc3391ebf7ebd172e85
7b859a59f9cc747692cd55b12397b645d93f40c9
53360 F20101107_AAAKVZ jayaraman_a_Page_068.pro
5bf149364c3ac50b568d8dc351358e01
a967a3dba42a8387fc5efbcccd7f9ea6afd0645b
F20101107_AAAJTM jayaraman_a_Page_098.tif
01a01bbcbf23b991df94517f833e3a9e
9aaf4e2e05421c3d1aabbfcd26538de428e4a732
2132 F20101107_AAAJSX jayaraman_a_Page_025.txt
42d0c7e0b7e27cd0c7e4688d8827a388
8292f84acb8a1c20db335d858be556368ce752dc
8951 F20101107_AAAJUA jayaraman_a_Page_102thm.jpg
38e7b551efcd55d528267dce705630e2
aff7e3a54ba8f25359a10d7a1a35c4105fae046e
33901 F20101107_AAALCJ jayaraman_a_Page_039.QC.jpg
06f5458d89a182d93f5e1ff64c448751
e554d09256f3315b7f666c03b5616a759d7373cc
8761 F20101107_AAALBV jayaraman_a_Page_025thm.jpg
9250252952d66566c0ab20dbdf0b4877
cbf92ea2be673b888669143b84b995d68452690e
18938 F20101107_AAAKXD jayaraman_a_Page_139.pro
420dc1409ecd2481d9272e756b4ce727
55053542366654ac76aa09b230a1026e3a23d7b2
46977 F20101107_AAAKWP jayaraman_a_Page_099.pro
574d0d75b1c58a5b1bb96fa5a4b2dcaa
b628c8a47952474a3175e407f633d33c104a1677
66547 F20101107_AAAJTN jayaraman_a_Page_171.pro
2ffb1cf38ea5236d646e9cae70337dec
c2b764cc3dc7e97690eab4f2ba0da047b35afba9
4833 F20101107_AAAJSY jayaraman_a_Page_123thm.jpg
947014313ad2f72fc42e2c83c90b3f9f
99cc5368ca20e14d7d41aff94b07460b0f9b2760
112627 F20101107_AAAJUB jayaraman_a_Page_077.jpg
a0c92279743f4c43d2747369807aa8e3
dac1b5b548d2925d48a2ad5837ac57d912b1db44
37551 F20101107_AAALCK jayaraman_a_Page_042.QC.jpg
80023431b4f41ed554c863dd02159dab
3f6b6d7aff1886348d58d10fc7f5432210748b2d
37315 F20101107_AAALBW jayaraman_a_Page_028.QC.jpg
245693596faa948a6f66d92e2adebfa3
dd0356b1f290bcfd45b9e66a11360805320814d6
51820 F20101107_AAAKXE jayaraman_a_Page_144.pro
fa42e29a405133d39bad23318585f77a
2b6656b926b357b57fd09dff7df47c50653fafb9
51473 F20101107_AAAKWQ jayaraman_a_Page_101.pro
7d2479e87cc8a6df1161cbf8e9a15b20
3c0b0fc81332620f616c68acb1e3f6dc54b962d3
114531 F20101107_AAAJTO jayaraman_a_Page_133.jpg
d8fe9ef4db63194f64b2b8fc06d33717
f9f4f2d802eac2e30efdb94b84d9b733593aa896
4744 F20101107_AAAJSZ jayaraman_a_Page_003.jpg
b56c8b4225095a2674cb66069e83adc9
5fdb71b3995f43cc84da6d3ec79bf3a083b26638
1141 F20101107_AAAJUC jayaraman_a_Page_138thm.jpg
4ec32fe7c4a5f865914bdda6d493efa5
cf989fd9016a215754ad9624f3bf9c7232d2ba80
4265 F20101107_AAALDA jayaraman_a_Page_057thm.jpg
120e489c94b8254595449319af731835
f7ee415729151d773d0da62276823cabbd3e6626
8712 F20101107_AAALCL jayaraman_a_Page_042thm.jpg
a8a980d27f6ece9c76bd38b739aad570
b55302d19d5651a9cc0b37dac4e45764dcbbd3d0
8788 F20101107_AAALBX jayaraman_a_Page_028thm.jpg
402db848892c47cf80d9f487d9fc02c5
bbb1ec12f78b51bce716246a5de6c2977f090875
52921 F20101107_AAAKXF jayaraman_a_Page_147.pro
1a2cb1ad4d4715945e4181c9f53c7445
cb36100db8c3e6d3554f30cc2281162272cf06d5
56441 F20101107_AAAKWR jayaraman_a_Page_102.pro
09b1cdccb082ff0fec302be6c01c224b
41efb065bc14ca7017a32609ba218fe25e4cd128
7660 F20101107_AAAJTP jayaraman_a_Page_008thm.jpg
0c1a97c7e4bdf4b3e8fbf0576d03b42c
8bd157c5a48f5986ec38bbd0f36173640cc4b813
21082 F20101107_AAAJUD jayaraman_a_Page_075.QC.jpg
2376f61745a8bffee3dd5baba7753f76
6aa03a18b8259e8205d089797d5342dc3c8f084d
6539 F20101107_AAALDB jayaraman_a_Page_058thm.jpg
bbf627782fae327c6779c55bdc2ec965
e11a1e1e244c23945ad76056ca8924460be79700
F20101107_AAALCM jayaraman_a_Page_043thm.jpg
888e70b372aefed4a70cb460843a23cc
e3ac51dfe1430e737d894fbf5f2ef46403395b92
8955 F20101107_AAALBY jayaraman_a_Page_029thm.jpg
00bdd8ac79d3746ac315d7e750eaf0e5
fc4c0897a4edfd364c35c4c813502015c8253525
49693 F20101107_AAAKXG jayaraman_a_Page_148.pro
326103b6ece0c2a86a8ca9e22b9bb9dc
bbbc17de9ebe716bebcfed9d663848f2b6448e77
25860 F20101107_AAAKWS jayaraman_a_Page_106.pro
10949fa8d857494fb82290e1d046e8f8
484d286e9f39d8b03f3dcf6a2707959230a9ebdd
9191 F20101107_AAAJTQ jayaraman_a_Page_143thm.jpg
86ec6123d80b23647ce563e9a73a69ef
b1c73fd534c4a1fa11e18c468b26c0ed0701a8b6
7950 F20101107_AAAJUE jayaraman_a_Page_081thm.jpg
caf4cf352d16d59a4bc2a9f98359eb23
dbe8509fa39ffe1a3c9c511813552dab383c6d4e
28272 F20101107_AAALDC jayaraman_a_Page_060.QC.jpg
49b82ebe3ff11719f3b4e41a7ebb6132
227a5b64895fbda7fd18b16968fe2545a6e86251
9122 F20101107_AAALCN jayaraman_a_Page_044thm.jpg
3e637c7b41ea99eb21ed5a352fd9dfb1
ca6753892739bf440dd7ff56636e6c975676e615
39136 F20101107_AAALBZ jayaraman_a_Page_030.QC.jpg
89a2861bcb5a45209979f3182d49c76b
9252cece194999a58a6caa49029525485c881d50
53181 F20101107_AAAKXH jayaraman_a_Page_149.pro
5de6f4b8906d44bc65dbf1397cdf5ea6
9238166846f68a20ed313199927cdbda58651500
11126 F20101107_AAAKWT jayaraman_a_Page_107.pro
46330d42853e0394dd8222adc98c598b
fa2c7a63382f1cf31cdd1a998b39ce9f1dae2392
F20101107_AAAJTR jayaraman_a_Page_148.jp2
3e2aa65ba0145306f5741f6bbbc0d04a
cfcc8eebe2f0265cc53fdd66fb881b8613e7e723
F20101107_AAAKAA jayaraman_a_Page_124.txt
78d55db1c1ba5dc8350a81b457ad0ec3
9e202a60a600ae15be13c42f5715b0f342ed67d1
F20101107_AAAJUF jayaraman_a_Page_101.tif
0b8725fd60a90d53fbfe4d1f114d78b5
5bd548458f6869c7230aba0d324da1ba2a0f0839
36428 F20101107_AAALCO jayaraman_a_Page_045.QC.jpg
5dd6a710297bd37187e81deb5df3a6cd
8c36096be07182e92c225184c99fbad9a609bcee
52523 F20101107_AAAKXI jayaraman_a_Page_150.pro
71df5f7b97ca6f726dc77849ddf1291a
621e02882b5f9b174ed7b833ec5391e7b0eb9845
51453 F20101107_AAAKWU jayaraman_a_Page_113.pro
dd3f5ddec4b714a3696b27c8460efefe
16ebaa5048d0315c7153a479d7a86129e281c63c
F20101107_AAAJTS jayaraman_a_Page_118.tif
ba51c3bc4949246eaf8c4aacbe783611
b11a0e95ef5d09dbf97ed3274314ca3f1b75ddd6
F20101107_AAAJUG jayaraman_a_Page_095.tif
fc2b02019885f112d93cf66ca666bdcc
0be74ab55b8a79e8da62136ef8434b127baff39e
7005 F20101107_AAALDD jayaraman_a_Page_060thm.jpg
7814f027941c72c92fbfa256e10bb938
cd61d8fed03d45d6f8ec49d5e98618b30f8f41bc
8667 F20101107_AAALCP jayaraman_a_Page_045thm.jpg
4f1d170631078156bd44349e81cbdc87
137a3db77120617ec5248c06a17411427f5df3fc
56759 F20101107_AAAKXJ jayaraman_a_Page_152.pro
ad0b5d789265cd97e0d1f9e9e613429f
998f1d6e0db66aaf07b8aee55a8d89c1184cafd7
55900 F20101107_AAAKWV jayaraman_a_Page_115.pro
94e7b69cad5494c2a31b74446933ad9c
7e15eb537f6d0eaac1d8139e1ad076d93522e964
F20101107_AAAJTT jayaraman_a_Page_009.tif
62f6a03a87de9dcc3b5b8526556be24f
b7430ec6866e1934fe530706375ec67abcc2f0f8
56789 F20101107_AAAKAB jayaraman_a_Page_112.pro
314d1a870599eb0390dcfc23abb4384e
941cb05bee50a358bc90c3a628a4f2f419a37418
50370 F20101107_AAAJUH jayaraman_a_Page_069.pro
7826e34892f708397c916bc2f061bd93
0f3a59723f17ef1f8152e6c9a4cb85b2e94ac7dd
8243 F20101107_AAALDE jayaraman_a_Page_061thm.jpg
97c85740e7c88981caa59845a28799c3
91b2db8a519b89700e16feb9065b91750a3499e1
38573 F20101107_AAALCQ jayaraman_a_Page_046.QC.jpg
5a5b1dfb142644cbeecbca5aee206693
6558ad759610d3b4f32c1a35530152766cd4b506
54901 F20101107_AAAKXK jayaraman_a_Page_155.pro
5a4b954b5bb826f6cf9cd9ba74cdf387
f0350c578a7d59582cf4deac0d78951e39ab2348
54113 F20101107_AAAKWW jayaraman_a_Page_120.pro
90a0373c52b99cfa11050348268f5c9e
c6169e705af7fdbe30e2436873d19ca8eb44b053
36994 F20101107_AAAJTU jayaraman_a_Page_034.QC.jpg
88bf13453392c86ee05697e344ca4b49
ddc8360a2ae4f3863d20da44ea05adbd447ada20
36717 F20101107_AAAKAC jayaraman_a_Page_136.QC.jpg
ed47e778308e1d2a14d6549153463829
ae459021b197ac7125b5e1e16f545728912d5efb
56788 F20101107_AAAJUI jayaraman_a_Page_153.pro
65dadf1e388884fd8ad22acba1a3805b
601d8b9f934ab596bd3b10a376f142842c0101bb
7828 F20101107_AAALDF jayaraman_a_Page_062thm.jpg
301b4ceb0e65ed0684321dbd26ca7b44
49e2a4fdeca0ffafe173eedab61db98de611d8d7
9096 F20101107_AAALCR jayaraman_a_Page_046thm.jpg
9cdf9e6f7b065f1ae9cb34de26f3fe16
c3e7f0639e39554d70a1a6445aa2beb4bbf8c409
6468 F20101107_AAAKXL jayaraman_a_Page_159.pro
af4620a54b35f5cf92aaf77aef602338
980ae5ddef9d15a934f09308138564a9522585e7
8982 F20101107_AAAKAD jayaraman_a_Page_047thm.jpg
66958d743b26640945e3eb71335e23ed
c28a1a8452d534e67728a21984f2f4b764659bfa
56058 F20101107_AAAJUJ jayaraman_a_Page_131.pro
c06808ecafb08776c768e97c13d6b5de
2949da412945c6e0575d55192a32ca0a130c7c85
26900 F20101107_AAALDG jayaraman_a_Page_063.QC.jpg
202d8b3f5f60fc5e54bd09bcc7e2661b
46ec8b6f7095b1fef0b0891c8c802379ab649a23
37857 F20101107_AAALCS jayaraman_a_Page_047.QC.jpg
8978850e3e29181a042e1d7ddaeb8a4a
6f5ea23ca4b9757ef736ce96ca3db20e0c0efefb
F20101107_AAAKYA jayaraman_a_Page_015.txt
daff9c9be12557be668aaf48eb754ba6
4b80d6b561d2053ef12da53703802328fce04313
65785 F20101107_AAAKXM jayaraman_a_Page_164.pro
6d321fcc2730244d919aeeb6051dd88a
1ead0d51b64e9d781501190727d01de1d1e63d73
51329 F20101107_AAAKWX jayaraman_a_Page_126.pro
fc96c6b8ec1edf75685d3f716e787e16
a48902219bdf022457eb20458c658c7e95c6457f
2232 F20101107_AAAJTV jayaraman_a_Page_023.txt
804456f61b8f39351f896c307ce36c00
22782fbf7ee7d132de8933426eadd0e7e662e80c
F20101107_AAAKAE jayaraman_a_Page_011.jp2
321d5df499fc75a33ccada7f878288e6
60d963333f63071b3c6ed2a3057b8bd086ba1616
57067 F20101107_AAAJUK jayaraman_a_Page_121.pro
fcfc1cf825d6a0eeba9f27c2b9b1ae47
86c7a2c51839a78068c4ec56f3a72301483eb25f
8155 F20101107_AAALDH jayaraman_a_Page_064thm.jpg
966734f345f83d474ee6993bcbc9a379
09074a2ec783a6d480c7baad9542a938743d8077
8713 F20101107_AAALCT jayaraman_a_Page_049thm.jpg
8f4b056fa5d3422eb9a4d7b392d7b503
9c47ebf4d117a58de9e4625bfa0058e100271f49
1888 F20101107_AAAKYB jayaraman_a_Page_016.txt
d0ca4b3adea0a59a29df0ed16b025741
a86bf2ca0005923c0b7ad28b28edfa4520a5a948
60336 F20101107_AAAKXN jayaraman_a_Page_166.pro
f9f572c8f72b559b1e19a606312661a7
d04ebf4ee3520114eadef5ad6c77711f825eeab1
50353 F20101107_AAAKWY jayaraman_a_Page_127.pro
f1136a1e5f1f3ed01f97957c40c10e52
54195f2daf74911b581516470766cd69abbc9322
52278 F20101107_AAAJTW jayaraman_a_Page_116.pro
196e1df0170318e3d14707ee956f7186
b7ca7774153881f2c26bef6b006806860adbc259
18618 F20101107_AAAKAF jayaraman_a_Page_005.pro
662e99b606cc4e191751b2de78daada2
89685c1ac8fa837a6ab465298d96d21ff096a502
F20101107_AAAJUL jayaraman_a_Page_144thm.jpg
2873a571bb1f242f54458b87a78509f8
fab82156351f757c0bc6c6423843cb88a42e718d
36835 F20101107_AAALDI jayaraman_a_Page_065.QC.jpg
b183c899cd28c205c3b0dcf4594a74ed
6c2d70eef42fac92ba011620c72bcfdddcccc3e8
37259 F20101107_AAALCU jayaraman_a_Page_051.QC.jpg
fbb2358b082bf72a5954f18ddeb87a1d
c1a41af14931c4f1b2c875d76cf3c2301dd15e73
2141 F20101107_AAAKYC jayaraman_a_Page_019.txt
fcc50f72aa900eb2ad36f620e21bcad2
bfb100c641bfca047d24b71da49d97c9db252744
64085 F20101107_AAAKXO jayaraman_a_Page_173.pro
84ddc19d6589763a2512b0ec6c8cc4d8
1714c4ac49c243284c9062e33f4959475c229c44
48936 F20101107_AAAKWZ jayaraman_a_Page_128.pro
ee53934e373af761676902ae5bd94040
c30fbc93fb59e088623a03af6cca4d1dc38efb52
30954 F20101107_AAAJTX jayaraman_a_Page_007.QC.jpg
1628e23d0f25ff1d62f0c9c94dc36343
25b2336de5bd02d6826c702a400d24576044ba7b
1051954 F20101107_AAAKAG jayaraman_a_Page_177.jp2
7be291e7e849298a7ba73640d432494b
7bcd3f206882c54ba89a98f44562b8c2e8c896fc
8950 F20101107_AAAJVA jayaraman_a_Page_104thm.jpg
5129bc74a0eec93806817c7b56c2b1c3
c5ee8eafcc560ab655f7d8469bb0a242d5ee64c4
2967 F20101107_AAAJUM jayaraman_a_Page_175.txt
9e387f5d14c59891ad891af1be5cb86e
bb60e055722842b0d17624623b506ac4be28ae77
7851 F20101107_AAALDJ jayaraman_a_Page_067thm.jpg
f7aef994cfddc1bbce1025b89563e8db
e8567d02073aaa51c2741394f4f41f1e6714caaf
8973 F20101107_AAALCV jayaraman_a_Page_051thm.jpg
a923e48235d6fabfadc177bcb18eef27
3c96dbec32da99f18b05e8085093196a630eb321
2122 F20101107_AAAKYD jayaraman_a_Page_021.txt
9500d5c72641645341e10f6f422e1d71
b006608aafd564f564526939068019deb1e2579c
68813 F20101107_AAAKXP jayaraman_a_Page_174.pro
decc4383ded7172d26ac953c5b14b46d
31135dc51d46eaeaf3e8efad033c8b88a579479f
F20101107_AAAJTY jayaraman_a_Page_113.tif
407394b8016c6353c4fdac700a1fcee8
80afe47f8876a4a1029ef6b1103fa9b697a67b4a
114357 F20101107_AAAKAH jayaraman_a_Page_044.jpg
e66627fa38970c68b097a92b3bd42534
e7f203d6c7f7c7a9660738e9de027fdda7f863b9
8704 F20101107_AAAJVB jayaraman_a_Page_120thm.jpg
deb3ef55bcbfa639a36435ff5e96810f
b235a224ddf5c91865cfe847ab45f1bee8b0e848
F20101107_AAAJUN jayaraman_a_Page_078.tif
3e775cd4881253e29fabdea19a3eb93f
632f85b1b44393789e136c96d27bc432d5a0a12c
32957 F20101107_AAALDK jayaraman_a_Page_069.QC.jpg
545a4b9e3f5d80fa3a6696328b940846
4307c55549b5920cce56bd69de0d518ad13166a0
37290 F20101107_AAALCW jayaraman_a_Page_052.QC.jpg
6a991d03e8d08b7ff655920bb04f3acc
a7bcae693569069869c814d089e924d94a50998a
F20101107_AAAKYE jayaraman_a_Page_024.txt
410e4aee9c4a4156496aca2d9d6e8ef9
7fd91be5973afddc7bdc51e5a11d7c852b5c6cf3
66377 F20101107_AAAKXQ jayaraman_a_Page_177.pro
8a88f630b002421d10fd93655b1b1305
1394665fe4bb79125b017f5936e1d000cec258c2
1053954 F20101107_AAAJTZ jayaraman_a_Page_110.tif
c52a78d4027de56d15d0dd9bbb7bb97c
a70a60099eca3e19cc3f5099bec3f18cce5dda26
53045 F20101107_AAAKAI jayaraman_a_Page_114.pro
93fe27969bee2881825b35c87c9b9001
2e5dace7a54205983229b99350a829fd91443838
9110 F20101107_AAAJVC jayaraman_a_Page_053thm.jpg
a2b02d19a06e28cc3ec41923f96795ae
9566a284a4e4233929e666f35845f0a08ecfa81d
F20101107_AAAJUO jayaraman_a_Page_177.tif
9767f5ac5e7056c62ba6a2246b3d9310
434e10dd51fb2e30060fbce26e51f6594919db0a
5494 F20101107_AAALEA jayaraman_a_Page_088thm.jpg
ec70edbffa62c7e81fc52bbae9e5b532
a86d8f0e29da286aaf9ecfbf241d6c8cae920e96
30196 F20101107_AAALDL jayaraman_a_Page_070.QC.jpg
6d2b580e605f1cfa875d163e62d462f6
66f41b168c2ce1240d340880df542b10853ac2cd
36877 F20101107_AAALCX jayaraman_a_Page_054.QC.jpg
1a06de5c91899685fe05b3507e6f0471
bb610bbea879c3b590c8a7f3b7ba21c2e10ee933
2280 F20101107_AAAKYF jayaraman_a_Page_031.txt
010f393bcd2a5f73f755d9da3b88916c
e7ab9e449ed515efc63df24a179084dd8ac1eb3c
61836 F20101107_AAAKXR jayaraman_a_Page_178.pro
ebff249e63b8086bf97d5b2a7660da97
48ec6e7101696b576413d716550db0ba4c08696d
36073 F20101107_AAAKAJ jayaraman_a_Page_021.QC.jpg
2624855b9fe289bd0d06e503f608077f
546aaede6c93b60c46d2c52766e99ec46a8f68de
55543 F20101107_AAAJVD jayaraman_a_Page_072.pro
1b62883440f83e8c63f11ee662d5fc8d
3e2fbf0499fcdb741208b3ed6dae2a6c6f5f0c66
F20101107_AAAJUP jayaraman_a_Page_102.jp2
e9cf9182a1fb8b86d9407b8067f2d543
0112da84a5e4dac5da5991a2020ee7f11fb81384
4178 F20101107_AAALEB jayaraman_a_Page_091thm.jpg
ae83a8d33fcd1675417333cd2d1600fe
029b487f12d73420e24c670018a0058fda3c29d8
7553 F20101107_AAALDM jayaraman_a_Page_070thm.jpg
a1310b2b7504420b261992744c27bbd6
32e965720c993911edd866035e23a83d89652061
8624 F20101107_AAALCY jayaraman_a_Page_055thm.jpg
bc7dbdd51f1160e4c9fd5eaef287111e
0077b3a6419569bfa4205fa790e63e0567705005
2160 F20101107_AAAKYG jayaraman_a_Page_033.txt
b21fb15ba8000a010e1860c500fccb6b
130630079b66fa940837666e01060e9c0932aa50
62974 F20101107_AAAKXS jayaraman_a_Page_179.pro
10f785accff54b03493131e44ffc3adf
7e0e7964b00b3e4641ae361f75cf34259e55f7e4
2130 F20101107_AAAKAK jayaraman_a_Page_122.txt
264a665724464c84f217324d6df291d0
b9c032f3477b1c182afaed9729987f2559fe4700
66864 F20101107_AAAJVE jayaraman_a_Page_167.pro
1b109e27c6c8de735830f05103e2fdb5
8858f77190f986da3e5dcb33862f5eb76d3dd706
F20101107_AAAJUQ jayaraman_a_Page_036.tif
fe32f2223a0deb5f909c527472447eca
9068e11b9122ef70c3bd9785db3b0ae6e3441af3
4112 F20101107_AAALEC jayaraman_a_Page_092thm.jpg
837cfe75e6e3f5c7b8059f14fde22b86
0877706fe834a0cff9e5ac5792c5ed793da03d20
36663 F20101107_AAALDN jayaraman_a_Page_071.QC.jpg
a65bbe4d2d0039b985f03f65dee1bf29
a6f98b0eaa5300059e45f71b36e99023e47bc1db
26732 F20101107_AAALCZ jayaraman_a_Page_056.QC.jpg
bfa3d98ef9fb9e06e298d252c7f75984
82fc8517eb86d35d95978ec930fbc29f1f781aaf
2143 F20101107_AAAKYH jayaraman_a_Page_036.txt
cc86c51a73970e1854fa010593e161d7
44b7212b6519779e899bc782c98f01c8e0acc219
12751 F20101107_AAAKXT jayaraman_a_Page_181.pro
5c334b8bd242435ade7a5324b5c9ae49
1104cfcaaf21201a0a443c1dac6a959c41f15cfc
8002 F20101107_AAAKAL jayaraman_a_Page_158.pro
065f107ef220c270cbc695a4db1b9ba6
e3b240839b5294ba9919439e3881930cc1140b7c
8303 F20101107_AAAJVF jayaraman_a_Page_113thm.jpg
4ad6636b052feac9a84ccc51f3ac78ea
4773db89c365e54c1513de1de3eedc56986d0eed
111773 F20101107_AAAJUR jayaraman_a_Page_033.jpg
a3468f09b978fbdfd46fbfaddf0bcc7b
c51ba759a05333236eec0d9e68377c8c327562e6
1049456 F20101107_AAAKBA jayaraman_a_Page_117.jp2
41610f5a12dd9d17d7113ddfd0800a23
f35647389f7f021f9387e3d01ae695ae3eb2e649
12563 F20101107_AAALED jayaraman_a_Page_093.QC.jpg
a83f0dedd0e4b3d2077dfd14394b6035
600ce29b71e8d1b02089c4541f7822f41c879b12
37944 F20101107_AAALDO jayaraman_a_Page_072.QC.jpg
3a699623b7d46fb8d97c68a0e3823771
1f763ceb0958c7ab1a1e2c143156e2adb3a69240
F20101107_AAAKYI jayaraman_a_Page_044.txt
a3653debc232e25ae765b0eab65e296c
292bcd30bb4df5484ad4998453c2291ee80d120b
16772 F20101107_AAAKXU jayaraman_a_Page_182.pro
fa61015cfa7f5402df497353df5dac1e
e38fdf10e7ea3c7b61ebad06caa5e79a89aeb84a
F20101107_AAAKAM jayaraman_a_Page_114.tif
77b5ff0ecc5138f47360c7756705ddd4
bfebecd9b711b9bcc86e01e7dbdaa5cffb6f5b62
2168 F20101107_AAAJVG jayaraman_a_Page_041.txt
8522c27a783d08ad29dae8aa17f8a85d
63e66a8f4b1c345d33efeee37632408b974026bd
F20101107_AAAJUS jayaraman_a_Page_125.tif
ccedae2c5e352a5f26d2a831ae9827b0
e5b94bd3a24bd40827c5efa6e0b2463691e9aad4
7276 F20101107_AAAKBB jayaraman_a_Page_007thm.jpg
804b12db71633c28b59673f5655c92b5
f549f66814ac911dd44092ef70265042cac5fa17
9017 F20101107_AAALDP jayaraman_a_Page_072thm.jpg
188435e885b3cdeaa294c5b436dd578e
ba022ea2fc5a477ac7733f2d2708b069772b6295
2027 F20101107_AAAKYJ jayaraman_a_Page_048.txt
af20729c548a301a5df6e45b1b6e7cdc
54e70ec714e55495ac08bc74ac02fe962f364709
90 F20101107_AAAKXV jayaraman_a_Page_002.txt
eee0b3a631c0fbed6c0e07badd34dcd4
f891f2e792636d010b2294bc364ed6152d8255f2
F20101107_AAAKAN jayaraman_a_Page_140.tif
5d3b0503464edf89fb85174d432890fa
120df5eb1984d554375e14c44036d8d1c17c90a1
105 F20101107_AAAJVH jayaraman_a_Page_003.txt
ca0a40c768e43da4c00feb483ec12545
850acfbfec9a6ad762e31b6333fe0e0aae31600b
53996 F20101107_AAAJUT jayaraman_a_Page_029.pro
b779fb70ff79c6df677be467fea8e759
e250da4a832fcec188891d6553ed4c41ff97926a
3771 F20101107_AAALEE jayaraman_a_Page_093thm.jpg
737dfbee3ba1341b2b1df46d74934f60
25a880f9fffd3d818a5ea807e0a80ecc502132b5
8521 F20101107_AAALDQ jayaraman_a_Page_073thm.jpg
1892fab6d434c9b43e69e0d160c0d39c
93f868d79d4045b1d2bad93db517ab58faa32604
F20101107_AAAKYK jayaraman_a_Page_049.txt
7ff455c659107f8a3cc18681ccc127f6
cce56180e60d98afef2af8faffad6e4bb8b515ae
739 F20101107_AAAKXW jayaraman_a_Page_005.txt
a4762876c9fea90402a44122613c617e
18024c7e0e9d6d8a4c38898091484e8b9540f528
F20101107_AAAKAO jayaraman_a_Page_170.tif
4fd96c8c2ace57e4d4fef66391acd9c3
bd448a105f9e61f7f99b6d42c188e8cd2ccfdaea
54375 F20101107_AAAJVI jayaraman_a_Page_028.pro
234aae4ea3b749f5220dab2dece05b7e
c747fd5b6bf765544bb1d615d1f31ebd7fc9c957
136137 F20101107_AAAJUU jayaraman_a_Page_165.jpg
c92f96b3f96d1f1e26d1b1ffb89dba3d
a81330f9aacf5ebc50a1d1da874aeddeab6660ed
15490 F20101107_AAAKBC jayaraman_a_Page_010.pro
55c1223349698a3116d2074c9d7584e3
582ec92f60b348b88e84f231894f08bcf478ef4e
9984 F20101107_AAALEF jayaraman_a_Page_094.QC.jpg
bc398c1f7b7a91884cdd62800f61c595
175576d8fe84d65710fb15f8257d9b7cba6a263d
27522 F20101107_AAALDR jayaraman_a_Page_074.QC.jpg
ff9661179059dee4dacb411ab39215b5
0975e9eeed0bd46d78d9ab2e8e7552c57219cc6e
2188 F20101107_AAAKYL jayaraman_a_Page_050.txt
60a7474305fa4e5aa89fac01bc790f06
445987a97f26caf97df6dbe076bb330f47b3b4c6
1409 F20101107_AAAKXX jayaraman_a_Page_012.txt
c080cc017c01d8cab8c39ea730e28209
35fc92696d18656372ee055d4d829a53e7e0349e
F20101107_AAAKAP jayaraman_a_Page_027.jp2
cad3f5f59eb76335915c914657789051
63a46380c0883651b2f2ba6ce56260d9f7cc8519
390323 F20101107_AAAJVJ jayaraman_a_Page_093.jp2
41e86293b4c206307a7338d68df36f32
e55b5404c3147e17bdee7fdfca024e7f4b92011f
F20101107_AAAJUV jayaraman_a_Page_006.tif
ce32cff6c00a270f4ba63f97aa877afb
84edf6231386f14a49cfa958b9c477de19b30c0a
2434 F20101107_AAAKBD jayaraman_a_Page_172.txt
2e02d1cc8a9f314c244b69b6be0ab6cf
2be52f9a2f772649f4ae116059510569bc5c9049
2971 F20101107_AAALEG jayaraman_a_Page_094thm.jpg
24264501aa4d9fcf4425c9c32c309e6f
ee7a29af8f4ab325cb02b46a18429e5a2215f05e
6381 F20101107_AAALDS jayaraman_a_Page_075thm.jpg
c1fcff208b17cbab9606099555bded97
4b56da09d3de57ac9cda472fcc62551f645a8240
2254 F20101107_AAAKZA jayaraman_a_Page_085.txt
f7f549eaa7455769ae829300d9de11b8
05ca72b328983691af89d12b54c7242b6116eb3f
2279 F20101107_AAAKYM jayaraman_a_Page_051.txt
4f873d39cb1111e4bcdf5df6f9d62b5c
8fedf86be55cebcb362da20910848fab8d37065d
55462 F20101107_AAAKAQ jayaraman_a_Page_104.pro
db709a7e7b74a3e6fb8e3eaf4ac9ff2d
6c88eee17d02e136fa26ce361eee8e8a89173921
F20101107_AAAJVK jayaraman_a_Page_064.tif
4cb72bd8e925f167c9478211688ec5fa
d77b1d5919b26a3642f97fbdb5abf02db9f036b7
F20101107_AAAKBE jayaraman_a_Page_004.tif
2213dc864824db4703cbaf7312059376
cb00b50d2efe4a4e12eb63f9ba8d39d5def43979
37292 F20101107_AAALEH jayaraman_a_Page_095.QC.jpg
8f3b1f9e2a9f2094742082b6cdc55365
52179909b63cd66d56ab8de3e6c627df7161897a
37070 F20101107_AAALDT jayaraman_a_Page_077.QC.jpg
446b0dc4f27e4466aab0457cb8e0cd79
56dda452c3d8807cb7abb604c93ae1fd219a30a2
2032 F20101107_AAAKZB jayaraman_a_Page_087.txt
e7c567427f2f8f34b1104179cc94188e
5aa1c0e68c810b93d8d71c5010dbe43fae5f8e06
2193 F20101107_AAAKYN jayaraman_a_Page_053.txt
2d7971f624d7f18d27b1eb4d85ddccc9
4670e01cea2d53e33d360108d14f0bf7a674060f
2049 F20101107_AAAKXY jayaraman_a_Page_013.txt
dbf74f4f1f56aa86ea7232d0b8c4b78b
2ccc1a58655bdf0e0b55f8b0d46d0b9a73b50531
54271 F20101107_AAAKAR jayaraman_a_Page_129.pro
b4ae5ae3ffbda67ab7d34a3294936e4e
467fa9e3b3bc76346636bc27b58969e2b3226996
1901 F20101107_AAAJVL jayaraman_a_Page_080.txt
87e9b557867b745ac61aa06ab3898578
9bfc156bd5741b12486913260de26e46063c2997
52912 F20101107_AAAJUW jayaraman_a_Page_151.pro
9c9ae10cd9f4f3f9b11e8b56ba26ae1e
114e520f751485d9a9756d873562138c0caca7b9
2240 F20101107_AAAKBF jayaraman_a_Page_022.txt
019825a01c085d5c854a8c54b115f4b4
4b5481c780da8f490e7c5dae0a2802ba891e2026
8791 F20101107_AAALEI jayaraman_a_Page_095thm.jpg
1b725e94c7a8647b88fa62e323cad490
8a9619858a170eaa56d3d4e1e4345fec61a9defe
8937 F20101107_AAALDU jayaraman_a_Page_077thm.jpg
dd48a89efe59ddcaf73bc942c29e769f
e3620c9278fc9346009a62dae12c93ca23e00474
1157 F20101107_AAAKZC jayaraman_a_Page_088.txt
35abaaa78c33970990ff82e1894b968c
1852766a025a63c2d9517c57e86869c315371284
F20101107_AAAKYO jayaraman_a_Page_054.txt
3742d78d1fde3fceb62415e462631613
d2221027b20ffec45d28426ab787874196716a11
1181 F20101107_AAAKXZ jayaraman_a_Page_014.txt
aa9c890145c141b2b50255105ce664ad
0c46d9d7cfaf2f13ff88605d0b2bf268c7b9a7af
8307 F20101107_AAAJWA jayaraman_a_Page_032thm.jpg
7282fc66d181c5c2ac4735faf6c1af97
acac9c935d6e49f1205015533a948bd988f1e091
674245 F20101107_AAAKAS jayaraman_a_Page_014.jp2
d99ce5e1cd44bb4c8f0cb32eaf36c066
ee96236d1a7bffc53a6d5e5383ca0587a184463a
F20101107_AAAJVM jayaraman_a_Page_092.tif
c3f3051c8fd1f902240dea2da9c53352
b4d321341a528588f68bb07003884245e6d57bbe
113690 F20101107_AAAJUX jayaraman_a_Page_053.jpg
d1cb5bd64420c96f83b3c1cf3c4cb5e0
e77c82939693c0e14f334518834c3f399926411e
34975 F20101107_AAAKBG jayaraman_a_Page_068.QC.jpg
d39b1667fbac1b542720df524df09b1e
b542ec1b96a4e5571ab5916d7c0551003918321f
36194 F20101107_AAALEJ jayaraman_a_Page_096.QC.jpg
65cdf5508d37db0db8a2c2a3eaa248ae
4404963b8acd5a9043ba9a05a37aee95204db616
8600 F20101107_AAALDV jayaraman_a_Page_083thm.jpg
1c6ae7631a7e672b1fde131b02d3d6d2
9681e569818dfd50ecd1a4246bcf9767ca611fbe
472 F20101107_AAAKZD jayaraman_a_Page_089.txt
edb40d0703a73c6398d34c600e7fce61
19ff21e9a4bbd4c7f79096dbf6bb21184fa5def2
1495 F20101107_AAAKYP jayaraman_a_Page_056.txt
269b2fe51b6febbbfdafc23108c8c4f4
dcce65655dbf6c52a013db780d944f11e1ba749d
9385 F20101107_AAAJWB jayaraman_a_Page_170thm.jpg
7e039744f0ea2ae313ebc3ccf66463e7
f9dd2d2cc2a65710568b8cf2601a61e01bc18a44
54862 F20101107_AAAKAT jayaraman_a_Page_119.pro
c6334d053e3edacb181a33217a47e9b9
49dedf243b0be0c4364312345b9c42a52b588507
F20101107_AAAJVN jayaraman_a_Page_149.jp2
6bef83af19b64242a037a60598ffc0dc
a57f2e9e2790cb8fe026ba3bd3c3346669068c9f
17863 F20101107_AAAJUY jayaraman_a_Page_140.pro
a0471426c2ea95b7300bb03996a6b2dc
d296f4d86153f009f1fb75c3bb3c606ab68a3cb3
F20101107_AAAKBH jayaraman_a_Page_102.tif
3cf593c082e00f4dcff5e1e028a66752
3c4084147f592c1fb883cf504c2de2f8837f0d54
8365 F20101107_AAALEK jayaraman_a_Page_096thm.jpg
9aed33b02a188dbe26551808332352b9
4c4f0e9b40828284328f52704b0a60a8bc25c683
8337 F20101107_AAALDW jayaraman_a_Page_084thm.jpg
9b0ed7175186372254f504b7a015293b
e5659a592f5674304557dcae9706c018ce155a74
607 F20101107_AAAKZE jayaraman_a_Page_090.txt
5d4bd64c8a91416ec6a1e10ca783a57c
83fd7a3634b63a321ab2831b03a59ef8af930598
488 F20101107_AAAKYQ jayaraman_a_Page_058.txt
0d60d7ee9ed162b17284c4b180c82e86
98ed639b3eb4615eaa903ed888680902480d1d15
1973 F20101107_AAAJWC jayaraman_a_Page_078.txt
57b3c919f4615df2dcde73a1d5f20e70
26f3796f1b75d06d124b61e314c02d83e37c29fc
109808 F20101107_AAAKAU jayaraman_a_Page_034.jpg
f48f6ecec9997bcca9c604a3a8af29dd
d3dfd6b7916ec603677b15d1bcf3ef23199a0f7c
34726 F20101107_AAAJVO jayaraman_a_Page_055.QC.jpg
f3d902d27e750b8945fc8c6abc810527
ebcbd3f7b320280de6978e73f572991fee9489e5
F20101107_AAAJUZ jayaraman_a_Page_167.tif
2234de8b55259e976904d6d6c3733dfe
f48fc91b807a5768055128bbd47282bc9827a938
148513 F20101107_AAAKBI jayaraman_a_Page_009.jpg
3b978974ea3142cb3f6eed8bfcff567a
775c7db02d042fafa05798f9661308507eaa1373
8934 F20101107_AAALFA jayaraman_a_Page_112thm.jpg
c4d349a1a549d73bc6ee6bc90c9f308f
4b0cc74f829724cd651afc728eb956a9b681da27
35902 F20101107_AAALEL jayaraman_a_Page_097.QC.jpg
af2d30b0c85520bbb415d8251d9c70ac
edae8c2bf6e6dae6b58dc838d5d5beef235da20f
9024 F20101107_AAALDX jayaraman_a_Page_086thm.jpg
caf00f46c429fe05f2b5def5c78e8f38
e4d6faa5c45e2c8235eb1e6a56427c70b30a76be
753 F20101107_AAAKZF jayaraman_a_Page_092.txt
94555abbc8b52aeb9d740540915f9cef
34a14d413f6d7a825ea564a5bdaebdf729bdad35
1700 F20101107_AAAKYR jayaraman_a_Page_059.txt
0daebe19398314a1ca4eb12b59b4c71d
5fb6800385fcfd1c9d2b20c904651fbb3db82ebd
52894 F20101107_AAAJWD jayaraman_a_Page_021.pro
6013a6037093130cc2493888afb97e26
8450aec0f28c9be30393421d4ecbc38b225730bb
F20101107_AAAKAV jayaraman_a_Page_063.tif
4c42e21e67ffe0d89b1461bfd5a7c4c8
1f5bbaa4f92af2ac94dea9cf7bacd87543ce9f82
3986 F20101107_AAAJVP jayaraman_a_Page_124thm.jpg
3a1ea96c12c3700c2050772e1f147973
180f2fdc8453b63e945a0834fc6a7f27da3d40dd
F20101107_AAAKBJ jayaraman_a_Page_047.jp2
508868e640ed4e6999542b6d2376b672
7b03b35429f13bc296d18f5b9369e73e4c69e1e6
31719 F20101107_AAALFB jayaraman_a_Page_117.QC.jpg
5fc3d3e8a4b905dba955351db8166935
16a5913f00011827b5dd2e4cabd34ffb22979cc6
7489 F20101107_AAALEM jayaraman_a_Page_099thm.jpg
92bcd1c69e204fcf3cd945dff3fc1511
813ede4c642cba4bafc2fd3c44c82ed6b8763546
8142 F20101107_AAALDY jayaraman_a_Page_087thm.jpg
4d8bfd0213da35374e67dd5dd9cee229
67b13c29743368ace0dac12bd98cd5adf377f2ce
554 F20101107_AAAKZG jayaraman_a_Page_094.txt
b061fea3461042bf8f5210c1b42f7844
d07bfe6d6d497b0291aaa3d32066aaa7679d471e
1695 F20101107_AAAKYS jayaraman_a_Page_060.txt
fcc6d0844a90753b917a31345a1324d0
405d1be4be1e6ba4755063a088d2cb1df56a5293
37098 F20101107_AAAJWE jayaraman_a_Page_044.QC.jpg
af5bfe73037f0e4ba63a76c93550e140
1945a6d815a0aa686da042005b2087753e307037
9104 F20101107_AAAKAW jayaraman_a_Page_137thm.jpg
70ac680a4835e8f8cb46cee2c553b18f
ba35098ff5b5b0ff7f91ff73c6e375c12a2e66bd
54720 F20101107_AAAJVQ jayaraman_a_Page_071.pro
b7320e828d35077a89bac0e75ac9f3cc
d7f3e80b611d9b776ec079c2456986de53902070
2259 F20101107_AAAKBK jayaraman_a_Page_038.txt
e7e99423ccccc17b1e20682e85ff5a99
0f392be2e42c0db99964ce89aac4e6f4e3662b1b
33281 F20101107_AAALFC jayaraman_a_Page_118.QC.jpg
7c5fb0278dba301c5c5e52183c6e95ed
ef5615e3498a76aeabf96e06d5a8e7b1d973306f
35524 F20101107_AAALEN jayaraman_a_Page_100.QC.jpg
cb643ea6de4111bd940a2fc143abb885
de8f2cb161bb0c65e8f87811ba7522d8ac4bdad7
19305 F20101107_AAALDZ jayaraman_a_Page_088.QC.jpg
8a4f3378a9aed1ee35bcea5ade18840c
59d3e63100b3e4759b945eead959c9fb4a819157
1854 F20101107_AAAKZH jayaraman_a_Page_098.txt
4c9948acdf978f09492a8061ba48f938
8c373b5d7a8abcba6b67cb0ec4ba6bc3ec0b5a29
1906 F20101107_AAAKYT jayaraman_a_Page_062.txt
4083346cc21b43ed5379dec58a4fc19d
3e10ab983cea52d835bd2e1475b4ace6e7c70501
909 F20101107_AAAJWF jayaraman_a_Page_057.txt
447c6bae5d91f2532fdfd3c061b07015
ea15d31794aaaf0203bcf4a853eb61e80b734f66
33248 F20101107_AAAKAX jayaraman_a_Page_123.pro
0bfeceb9a269b2d1bd87f2054411ceb5
f04e153660d31bca6fb057f596df9b59d38ed2af
299304 F20101107_AAAJVR jayaraman_a_Page_159.jp2
5cf7303b2284183426cb14ac429035c6
cea1a9ce70d1407e47bd5229f6f6bedcd609f849
9200 F20101107_AAAKCA jayaraman_a_Page_024thm.jpg
9d59ec17dc7b7b1a98c292a5a5e55010
1d0f7ac71085357e86d65cbf5232d1e4bd7e6cb2
8925 F20101107_AAAKBL jayaraman_a_Page_027thm.jpg
3e5a38a9334a6f208ad66e51d7702c28
1a8773004d6312a2b2c2fd87621aa38eb206a89d
35251 F20101107_AAALFD jayaraman_a_Page_120.QC.jpg
f7465474953fed26bbc6fd2477025cd2
8425196638d66bdb3dd0872d4e5e5bbf40c2ab2c
8914 F20101107_AAALEO jayaraman_a_Page_100thm.jpg
c6530d23c0c587e1d5c9f30bad2657e2
e64b7666122ead94461565831dc46f3da7e514dc
2135 F20101107_AAAKZI jayaraman_a_Page_100.txt
62ced59ef3352ece4abbe7bbc61f1dc3
e0d7d9a90059810d733d17efd1b38b84a20ba336
2048 F20101107_AAAKYU jayaraman_a_Page_064.txt
26a9fa236a24aaf0c77587ce6939614d
e09aa4ed4905fceb0bf524b1e464bdbc198b03d9
113276 F20101107_AAAJWG jayaraman_a_Page_086.jpg
c49a1e7c6559951e239ae1f078f5fb35
edc3e92bff656e57b04de27fbbbc927d21c649ca
90440 F20101107_AAAKAY jayaraman_a_Page_070.jpg
2cdc4f98c5c1a773f709328993c77677
dabbdb36215a36cc06371acd8f93a28c773d1568
31735 F20101107_AAAJVS jayaraman_a_Page_062.QC.jpg
5c9a3678f5f4f2e2734e4da11c8dd88a
bb57395dca934e8c424e6597083a5cb0a1c1c14b
34252 F20101107_AAAKCB jayaraman_a_Page_073.QC.jpg
1a23fd258632e441851f3fa787f9ae31
bc25b49585f31070eea900d0e575f54eb415bb5d
824664 F20101107_AAAKBM jayaraman_a_Page_075.jp2
7db5dc9abe6b6be2b405d13259a8a5eb
b7f438c4974277006541eed3d749a3679b418184
37149 F20101107_AAALFE jayaraman_a_Page_121.QC.jpg
61f6c4830f10b12d189cfc4a582e1b31
81117df6505fb68537161cb4596592a3a26ee0a5
32921 F20101107_AAALEP jayaraman_a_Page_101.QC.jpg
1651985a72779a59905ccf151af10534
3e3304ee0aae7b3dba3a281c8172f928e58e1aaf
2145 F20101107_AAAKZJ jayaraman_a_Page_103.txt
ee7ff1f36befc4a78a19ade37db6701d
a64b2921eff926aecb20611cbebd08699be5f054
F20101107_AAAKYV jayaraman_a_Page_067.txt
900df33d48a2da04cd671b359ce12fab
2333e71797a1c1163184ee9e33cf79ca54963e88
64670 F20101107_AAAKAZ jayaraman_a_Page_169.pro
4572701685531d5fc6cb92708fbfec40
17635b6a1f6499802d1ea48b2f5958e837d3f6c0
8881 F20101107_AAAJVT jayaraman_a_Page_166thm.jpg
d1d0cdead4b90f5258bb738589ac2cbb
8405f1dbad458638271a6347d423097f629a71b2
2314 F20101107_AAAKCC jayaraman_a_Page_030.txt
5748c81b587e02a21978dacffaf8fb10
155e75b18b6e3bd6d8f861a05b1f3c9995cb17c2
37654 F20101107_AAAKBN jayaraman_a_Page_131.QC.jpg
7e7d7a521ff5e49604547ffdf4ba308b
43671076233ce71f7e68f29e63eebbf8e663de99
46791 F20101107_AAAJWH jayaraman_a_Page_098.pro
02254d02c74f9e3fea5adb87b8a6554c
16513ce158038e8c4735e4e6d72ff56555d5bbb6
8363 F20101107_AAALEQ jayaraman_a_Page_101thm.jpg
274950564fae3decfecd19fd45f24e2e
97ccc3b80e4c1b6114cfe5ff9991d0e3b0a40386
923 F20101107_AAAKZK jayaraman_a_Page_105.txt
fe8faddd36daa59d22bec924fc6f356b
4bf84a7a1a861e194d908896c655efc7f292c5c4
2107 F20101107_AAAKYW jayaraman_a_Page_068.txt
17c7a2c0b7315435772ce067925a9da9
f2f8a4e732c1cdd83e5d417b2eed8e96bccf680d
F20101107_AAAJVU jayaraman_a_Page_115.jp2
8a0f383de4eaaa19c4819eabae9e3b19
3cefa3570e8222a6178944e58244db189e1f6dfe
34728 F20101107_AAAKBO jayaraman_a_Page_009.QC.jpg
fc3abc8b9d6853b51bb6e818d3e04195
dcd24b08339644ab426d26a6d638921a5918d075
31450 F20101107_AAAJWI jayaraman_a_Page_080.QC.jpg
b1f53f20fe7b7cc80a44252cad82f83e
893e500264985fdc881c79ae1c851da240468db6
F20101107_AAALFF jayaraman_a_Page_121thm.jpg
cc0a2cc91bbaaa5549014f089278c65b
0ad57e5a8ae1d73804d8dfb6ad408540fb70af63
35744 F20101107_AAALER jayaraman_a_Page_103.QC.jpg
5ee32035fb52119098ca0aa21763b7a6
50f5706aa5a813a21f3e5f82b79a6315fe53821b
407 F20101107_AAAKZL jayaraman_a_Page_109.txt
6af424c81310965e28ee2a1c9fe7375b
5401e36980fa2b2b281c3f8bb925c14f0fd385c5
F20101107_AAAKYX jayaraman_a_Page_071.txt
cd57270d82974eea9643fcb9523c1dde
c667d7837ffea4ff9e4ab40ffac593dfaf2dede9
18129 F20101107_AAAJVV jayaraman_a_Page_139.QC.jpg
6cfee1735913b82a11f9d488f8f8edec
bed9ede578fafc26f9d109cb0cfb7e6b357eaa54
17301 F20101107_AAAKCD jayaraman_a_Page_092.pro
21177b65d3b4b4617bb0f02ffdf2ed7e
e4439cf13bc0ac12583fd29327efc5733e138e82
67885 F20101107_AAAKBP jayaraman_a_Page_165.pro
b6e0ebafa7fee971b970d12e8dbdc311
ff659a0c4a13b92879d9c7478ff898598d208ac4
2204 F20101107_AAAJWJ jayaraman_a_Page_131.txt
4055ced511418c6d31231c8335904cc2
4624b9e7f024b2056c7e7d620a988e943434d1d0
35109 F20101107_AAALFG jayaraman_a_Page_122.QC.jpg
549bfb517dd5680ce1fec768eb71be44
6f8c2ece9c787695baaf88af0e97f5359b7aca3d
16693 F20101107_AAALES jayaraman_a_Page_105.QC.jpg
bff64505c36a809f74ac15048a49c207
a38437312d367e64a2e725bae521ca2c776eb523
F20101107_AAAKZM jayaraman_a_Page_110.txt
d39f808f1a2f112a831a76c87175f36d
d064d4dd33db4202028a7af5ca6afc245b840355
443 F20101107_AAAKYY jayaraman_a_Page_075.txt
9fa4cc9964436881e388014aab04b678
9c9f9dfa2818a6119a2ba8145bba1130cbf490ac
8561 F20101107_AAAJVW jayaraman_a_Page_065thm.jpg
0170a41f85c05a0b47c58b63338c7e1a
153861b943ae4a1afb8f5c3f19a6c71821e70b05
104531 F20101107_AAAKCE jayaraman_a_Page_113.jpg
9a1438da00d274b36525cc58a3512793
98d38c471753315f1d314767f71be1e7d1af9730
1051929 F20101107_AAAKBQ jayaraman_a_Page_170.jp2
bfbd08e04d272ed3517080e8929f2c2e
1d91a16bd467ba9d0e5d7e36025c3da2f63ddfe7
F20101107_AAAJWK jayaraman_a_Page_130.jp2
09e740b66a3e1c84678bb19373d5ee05
4d5c4c8ae57f545e7ef86ca976f19ed94ce38547
18267 F20101107_AAALFH jayaraman_a_Page_123.QC.jpg
90826dcdf618c81824693529caf68bf3
5902f4ceb15ff203b414e5c415ebb4d80f37a534
18577 F20101107_AAALET jayaraman_a_Page_107.QC.jpg
a4520946f4a36b7d5708015cd3983e27
f82c684d5a322c1983208044a2f0132019925b1b
2208 F20101107_AAAKZN jayaraman_a_Page_111.txt
cfe63e5a09bbc76f1034b215d1131bb5
32d8c5f9f7c6153d3922bd7b8e963736de7f252f
F20101107_AAAKCF jayaraman_a_Page_162.txt
aab7f316febb4e6328628e7d33116aa3
09d6a5efbf3ffa5aedecfb371a224fe0b7627580
F20101107_AAAKBR jayaraman_a_Page_073.tif
8b205cf11d620bc4278514412a846501
31a7633e75c8ab82dbacc20c78a6f0f3e710360e
56493 F20101107_AAAJWL jayaraman_a_Page_135.pro
d7b96e74903f851532093ec6c712e00a
115e90d50e435b87fd4f38a66aeb4df2685e9876
12317 F20101107_AAALFI jayaraman_a_Page_124.QC.jpg
415b84dd6e76cc34fd62c307c3fa6fc8
fcd1d324fa98512a4c061aeb99a99dc60bee3b01
6416 F20101107_AAALEU jayaraman_a_Page_107thm.jpg
7c9b4fd0a3a0092377a27db3459e50b3
329d04b84921798a483431032208bcd6579e8043
2091 F20101107_AAAKZO jayaraman_a_Page_114.txt
868e2b1570927ae283dc780b78da9e5c
a4693b8ae781759e44081add1e66fcfdc199ac6b
1797 F20101107_AAAKYZ jayaraman_a_Page_082.txt
43101d33d71d7d89d518562a4627f078
1ce0da42d99c534a8d6bf05f8489b532e7cd1b25
F20101107_AAAJVX jayaraman_a_Page_086.txt
14d680bf201a312a1789344d3b0bc540
86f38e51452a7faab3ee638294bf56bb0be4a682
2191 F20101107_AAAKCG jayaraman_a_Page_115.txt
a6aa15d7703722b00b195c44acdd445f
c1c43459449d27aba76a09a540e58eaf83e59e66
31485 F20101107_AAAJXA jayaraman_a_Page_067.QC.jpg
db7d67edc1942dcffedd63f4060c1753
ffc046ae071b6ff041e9cf593a98536b8744b87a
2088 F20101107_AAAKBS jayaraman_a_Page_001thm.jpg
673aac8f14659876adec3ce13033cdc9
363640ec9e45de483baf05143318c89b6fe7f0b8
11346 F20101107_AAAJWM jayaraman_a_Page_138.jpg
403618ed058ba746e13f2eba1d7f2de2
5feeda53139e8939d3425b96d4f46a2cb350e083
37239 F20101107_AAALFJ jayaraman_a_Page_125.QC.jpg
2605ef59c7a053855679bace9b4b991b
b233c21ff40e677a6b1bc4ddf8bc2e79bd6875bb
6417 F20101107_AAALEV jayaraman_a_Page_108thm.jpg
97cc625278ed5a47a530e87d34f979c2
0a9484c4714fe3333ac4155b03cdb7bf0ab54b71
2156 F20101107_AAAKZP jayaraman_a_Page_119.txt
e7957c9e9d376713eb1997e8b677b01c
d0f71ff8bc858a13a93943becb35c49f95fadc11
36711 F20101107_AAAJVY jayaraman_a_Page_024.QC.jpg
6438190b7b8c206e68203f9ab1a22b20
e1dbe235be294aa8eacb4eab037e128d35839d30
F20101107_AAAKCH jayaraman_a_Page_080.tif
e8b4c535b97607e37da70ce5571ab74e
bff872d25b404ab39db4ebed63c27aff84e3ab19
F20101107_AAAJXB jayaraman_a_Page_064.jp2
6f0540f31aa5b196086764b9f59a0d1d
6582a30023853afe8f89f0006ac9b14728df75ed
55810 F20101107_AAAKBT jayaraman_a_Page_053.pro
f8705f5075cb03197117b63b3f1f877e
1e2f599d61be1528099a060f645aa96658858d4f
8808 F20101107_AAAJWN jayaraman_a_Page_071thm.jpg
8b8b639cee090a67677bd6f4f40c91b6
dc71d4b36845e088fe343e8af44059e5bd168f9f
8116 F20101107_AAALFK jayaraman_a_Page_127thm.jpg
f5301e758f45fec0bc815953a0d26ab2
1c18f1bee80e939144288c744cf7b1cdff923d48
3365 F20101107_AAALEW jayaraman_a_Page_109thm.jpg
907ac73dca9719b82dd28def6d562e60
9105b94e97426ff742b18a9b8cb3a7e60c01d487
2228 F20101107_AAAKZQ jayaraman_a_Page_121.txt
d5e7cddad97c74942ed02d427bb94cc2
35c8cecc2067895d708ba7d7ec1a244f6dc28ff8
30946 F20101107_AAAJVZ jayaraman_a_Page_098.QC.jpg
c5702606ea45caaea4b4befddb7a773c
fb677b61c902c410cbd7c2b4c1e097c3545e012b
1986 F20101107_AAAKCI jayaraman_a_Page_069.txt
013a3b43eb6bb39f23ec0db4778e4535
0284ed015e1ac30eee2b7ac708c67f87b0f1a1b3
3899 F20101107_AAAJXC jayaraman_a_Page_007.txt
60b127d978130c9c846b011f4f27ada8
e9c29f41a0491f8152d40dc6c9774903d299d5d3
34236 F20101107_AAAKBU jayaraman_a_Page_127.QC.jpg
d89010968bb075d503795c8764a95574
7cadf7a567f3fb0ea56eb05bed0d28c1214414c8
113053 F20101107_AAAJWO jayaraman_a_Page_112.jpg
bb4162db820e473151625a8a054e15e2
153fc1ee3e0b488a3773e2b3a28d636575b607cb
29480 F20101107_AAALGA jayaraman_a_Page_146.QC.jpg
dfde744d7d8b247fd666794d9a1ea59c
a8d527028c0447117b1d0e23bd45f11f6af82cec
31496 F20101107_AAALFL jayaraman_a_Page_128.QC.jpg
3c41096d08c23005dbc85178adf6e802
952ee263377c5fc836b8d91dbfff78bb4d0f07b7
10995 F20101107_AAALEX jayaraman_a_Page_110.QC.jpg
9f5dc186acd538df745c9fe77703441f
64cae7a3b40997169a49a7b36be0b21e5ebdc844
1581 F20101107_AAAKZR jayaraman_a_Page_123.txt
9ff55bcecc314a3fc7cc962266e9fa9f
56d86ab61780ee603dee14b36520e62eb338f73a
32423 F20101107_AAAKCJ jayaraman_a_Page_064.QC.jpg
ed9e9b43b36b1614c68e1865d36846f7
811f48d71514f6175ef6aff7354a76e2e6d36529
113764 F20101107_AAAJXD jayaraman_a_Page_023.jpg
bb7e211ec2fd25c34b7d68922d8a70b9
254fe96ae7e720bcb0a9b7009be5edef47fc0ed9
30165 F20101107_AAAKBV jayaraman_a_Page_081.QC.jpg
bb0536c472b797a27b295b5bd305cb6c
2ddbae79bc410305bcf382db86bfebf739c4179b
7422 F20101107_AAAJWP jayaraman_a_Page_146thm.jpg
e722ed4f0a7571674891bf606e5cc8ac
ce12fd37556ba70906099bf7a9b6d31476def3ed
8686 F20101107_AAALGB jayaraman_a_Page_147thm.jpg
51d6952f3f8ebf96f42e6488a5b075a0
1646881f8c3e1d0ce28e249179e64e73184fbcb2
37339 F20101107_AAALFM jayaraman_a_Page_129.QC.jpg
5bb9aae08a6995d8318764b3866a0ed0
48fcabe3c20539a748e333a88271644bb1ed0356
8758 F20101107_AAALEY jayaraman_a_Page_111thm.jpg
8daa573fa27724acfd01aef3e7a72ce2
9580c9882ec26e81ac083517ed535e5ed1a20bf1
F20101107_AAAKZS jayaraman_a_Page_125.txt
adba7c79e7b995b66adb14e2899112bb
19dc74f6aa187519f7d2f02ce241c4b8322bef59
8964 F20101107_AAAKCK jayaraman_a_Page_050thm.jpg
3ad82c94f20a47e23161e62b2b1003e7
b9c18be518cff1a2b344e392408059e78fee5315
20545 F20101107_AAAJXE jayaraman_a_Page_106.QC.jpg
7449b9957367472316ae1db9a78fed33
519d33f00a8738d24bdf35151be373e3c1d2a683
10026 F20101107_AAAKBW jayaraman_a_Page_010.QC.jpg
727c3baad5df9a275036f6e2ed5b377d
1d165826a6fda39c70f454dbf309b0a1adae1b2a
97409 F20101107_AAAJWQ jayaraman_a_Page_080.jpg
453d6969dfcf8a83f9e144b8f89458d2
0937bd2004d10c118ae4898fdca5615fb6083126
33883 F20101107_AAALGC jayaraman_a_Page_148.QC.jpg
d574390a0fdd14827e7603dcd4912588
c35263f1559ba5efd8dfc821e1a08a055db7677c
8731 F20101107_AAALFN jayaraman_a_Page_129thm.jpg
9c9ee176e7ed6038d94f76dcea15e117
6be1f4687f991bd12a4569c610e2173c74247ab4
36558 F20101107_AAALEZ jayaraman_a_Page_112.QC.jpg
96708985fa4b7661dfee0511c2446342
a06bd3bfad7edddea38692e9e52ebd69550edd22
2066 F20101107_AAAKZT jayaraman_a_Page_126.txt
42d87b568c88c366bf82a4a5ff4b8622
9e1a3331ede398188688cd8fa93f4112dd596bb8
80767 F20101107_AAAKDA jayaraman_a_Page_063.jpg
9a80d93edad3c1489654637a0cd015b5
9b9ec297b9101c968352a29e8c66f6bc1346cd25
31165 F20101107_AAAKCL jayaraman_a_Page_099.QC.jpg
c599e63167523248ec77eccc621a86b8
25583bb36c35617ddc60081099e36bcb43c9588f
48661 F20101107_AAAJXF jayaraman_a_Page_105.jpg
8bd0459cfac9e198258e98b2b160c5c9
cab575309691e85a3b9e0662252ee62fa951efbe
10951 F20101107_AAAKBX jayaraman_a_Page_108.pro
4f5c12ecdff6e17749ec87b77d1dd7dd
d2b024af9fb38440a48c97bf1dfaeeec2b72b28a
F20101107_AAAJWR jayaraman_a_Page_109.tif
4bcd19b2e9beb9e490f573b8f32f6f42
cff86ad8198912e9d7cc56e260e3afd3fbe301de
35850 F20101107_AAALGD jayaraman_a_Page_151.QC.jpg
c8a18f75e6dabd49c883ca20bc1e39f8
e01a82a73e91447e84898638bbcb447e19166bd5
33553 F20101107_AAALFO jayaraman_a_Page_130.QC.jpg
43d3be17789dd97073cf0757f9da4ac5
4780f563d4f1a8f84235b44fd3d84edc21fefa15
2136 F20101107_AAAKZU jayaraman_a_Page_129.txt
b2458eeb69094aef48ffe63d37e30746
5d9e7bbe4363d0d48ee5934dd060bbf5e4ebf5bc
114528 F20101107_AAAKDB jayaraman_a_Page_046.jpg
d55d2783dd8fe14804a2889c9a738a27
56349ca6701f54c5e73ff1babf85f743b9fcdf46
1891 F20101107_AAAKCM jayaraman_a_Page_099.txt
6eb6a8faff22deb86f5a599a57043f31
86d6e5ff11047c9a64e31aebe4613f3007402848
533 F20101107_AAAJXG jayaraman_a_Page_181.txt
b7f07610a4aceabc352d7fa4010e3b8c
4efd2a3f47566b0ecccbad7d131f191a9ab9bef9
F20101107_AAAKBY jayaraman_a_Page_147.jp2
173c170d5f5e99761e23a4c337a4a6be
199257b7d0cdf3fc57dfbdb30ffab2e80a17e6cc
F20101107_AAAJWS jayaraman_a_Page_074.jp2
f31f0099986f2e60bef100a3e8e03fd9
7415b7a69b89828b8268af4de6b6076ffa4076c5
9177 F20101107_AAALGE jayaraman_a_Page_152thm.jpg
085d02945ac707a5bb34c77ec1e16ede
af80a6e8136cd065511a39066d76b8238f87cc66
F20101107_AAALFP jayaraman_a_Page_132.QC.jpg
42e41bf2c1f1db4fcf13a4a200b3208d
49e698ea32845544931c53ea85dd021f27966cce
1994 F20101107_AAAKZV jayaraman_a_Page_130.txt
2e0966c76b32e5c1bb93973588537073
7cc3b6e63bc9a1c6fe7bb3839c829cc74b065fc0
8913 F20101107_AAAKDC jayaraman_a_Page_131thm.jpg
7c3c8bd82294dbbb92f9c7243df8a13f
793117bc8618153f6f12fe1512e175964629a7ca
11230 F20101107_AAAKCN jayaraman_a_Page_090.QC.jpg
c52f6abf38c22e2752d601328a887bac
0b55a332e787a61a870c55881d284b4d799cdf3f
4035 F20101107_AAAJXH jayaraman_a_Page_008.txt
a53ad639a7c5f545723365613ddd9eda
ca915468580dd455aa67be03272333fca52360a8
34100 F20101107_AAAKBZ jayaraman_a_Page_087.QC.jpg
e6075d8a6f6b941a7d712356ae876e68
ca9a94e9f815c7b7e7105277727a9f889212ec28
4253 F20101107_AAAJWT jayaraman_a_Page_138.pro
6d24005ca16d3b8951d5daad7fbf803d
7ded583539ca12fb2d01972c039aab4244a9e62e
36955 F20101107_AAALGF jayaraman_a_Page_153.QC.jpg
8d485372ed08a79ea9203d3cd7f92c68
1cce37fda92747cdf83124eef2e8c0a618254139
38402 F20101107_AAALFQ jayaraman_a_Page_133.QC.jpg
88d659653d9f4528360f0dd2651020f0
04888d2d823ea08d2a5c7a84035ce1d80fe2f9f6
2173 F20101107_AAAKZW jayaraman_a_Page_132.txt
aa0aba95d3a8aab1c476c2ebf6ec4d49
933a2414439969498fc91f637d93caeb88c2894d
110149 F20101107_AAAKDD jayaraman_a_Page_100.jpg
f7c347ad408354f54f67f8e3a5e79728
9725a5087019dd9fbfc73f20b195c4f805146cec
92370 F20101107_AAAKCO jayaraman_a_Page_146.jpg
7378240210c6b09edb666f81db420c1c
8526b40681d3668ae2cdb6f34d43e04ea9495624
137128 F20101107_AAAJXI jayaraman_a_Page_179.jpg
2c3ca87a2b1aa32007d3bcf4897c1623
e41b70fc4296eab16b7f3954f0c9a83514fc6597
2140 F20101107_AAAJWU jayaraman_a_Page_028.txt
22f769d37c5115694844a7458679b32a
5e68caef2e77e34882fbb15c7723ce3d79161a35
36487 F20101107_AAALFR jayaraman_a_Page_134.QC.jpg
bd3115c7042a13d9e83e909549125c2d
444ea3ee81f48438092b6fee1e73d84bb0fd94f8
2238 F20101107_AAAKZX jayaraman_a_Page_133.txt
29bfc9596b6072d8636099956bdef755
7546d6e2d4156042051ace430dddf40c04533eaf
72814 F20101107_AAAKCP jayaraman_a_Page_175.pro
1892a29c82a043e89101d7424d68555f
3c7c6be232c364862ace63cbac796c43080fa6f2
1051919 F20101107_AAAJXJ jayaraman_a_Page_119.jp2
7d0861cfe310b40afdb8f462be5ed613
3fde64b7e5b50d72f4506d26d112ae5d1f8ba066
F20101107_AAAJWV jayaraman_a_Page_002.tif
96ec40dec04b9204febe72d00e6f96d2
fb702a76e956aa60a1492276caf476aa71494b36
9173 F20101107_AAALGG jayaraman_a_Page_153thm.jpg
ef2c3cb86ca996967159603e47432e1a
b6c5d6b4e627ecf2fe8e137078cb2d94cdf80330
9178 F20101107_AAALFS jayaraman_a_Page_135thm.jpg
1ad9b5e76d36c2aaa145e3e0ae4be6c1
98ce0c00c50d5e3ea6d00105677ac4eec152505a
F20101107_AAAKZY jayaraman_a_Page_135.txt
8a4987516e48bcfec130ceb60361684d
359dd16b832e9a154da94b87414532bc9472f420
7637 F20101107_AAAKCQ jayaraman_a_Page_076.QC.jpg
5cb9c379ab0eb5a9c6afe4c73b039c48
845bd052efa93928115670e7fda73fd573423f93
57641 F20101107_AAAJXK jayaraman_a_Page_038.pro
7dab68690a0905f38044186a09bd35b9
1847de93d24a0e03e75faf167e478a85930f37ef
8534 F20101107_AAAJWW jayaraman_a_Page_066thm.jpg
f0e5fdf2835930120ed161cc334c904e
1a62626f45f1310ac9c0041dd813e2916c239881
37530 F20101107_AAAKDE jayaraman_a_Page_167.QC.jpg
c7cd4d63c335aba0a414c13ab8d502e5
2837c0c3b2952df7acb0e449ff4135abafff13ec
9119 F20101107_AAALGH jayaraman_a_Page_154thm.jpg
8f8b067646a5bb1cadb281f443ca0445
0aa9941db15a1e70dab56bbd2ff038f57188dc9c
6208 F20101107_AAALFT jayaraman_a_Page_140thm.jpg
9a78a2dc88175dcf41a67fbbb27ab424
40cb2beabfec83ca994af75fb9ce2685411c2436
172 F20101107_AAAKZZ jayaraman_a_Page_138.txt
b3f3fb2660a357e4f933dfb2e4308821
37d68a02a5bfc5503a36a450dd392e93e15577d6
F20101107_AAAKCR jayaraman_a_Page_067.tif
f35e2fa7ba6d67ce4a7e59cfbd1cc22a
1db0594b3f32a6de5d264a9c6d8314160f2980bf
2128 F20101107_AAAJXL jayaraman_a_Page_149.txt
a1f5bc562b260eb8c76f972fce981db3
459a5943080e73b23c6012bee0f44ce615b4c697
133696 F20101107_AAAJWX jayaraman_a_Page_167.jpg
ae050b2686a7b5f709e6cbaccc1b320c
7eb8e94641049efee4fae6b8141155e40a7cedda
F20101107_AAAKDF jayaraman_a_Page_112.tif
13a08421d363ba74a99ed46557826202
06db7b5ed9ada7055cebe51e2624fece9ab7a45f
8935 F20101107_AAALGI jayaraman_a_Page_155thm.jpg
415f70ef67c58e33b30ba46450ea5c65
d9e281ab19dc7ecaa90891b1539021f0222a676b
17390 F20101107_AAALFU jayaraman_a_Page_141.QC.jpg
3efe20af5c46c1227fe000e5983aab91
db0367c9416fbfb79b7a55dc4928447025dcd278
F20101107_AAAJYA jayaraman_a_Page_164.jp2
5950ed5965023d9491d51e1b6f376833
f179ea4dfa7706c40bd640853c05c486173590ec
429 F20101107_AAAKCS jayaraman_a_Page_074.txt
b13550ddb887549765abd5ed4ce56735
0b97f1166aadf84a071ac9f85e67cace5233c448
F20101107_AAAJXM jayaraman_a_Page_105.tif
4f9d82cf5af934b40679e1977b441a71
fe48ca2ac3787a81376563d28b4bce4674490eb5
F20101107_AAAKDG jayaraman_a_Page_072.tif
cd6a9bb9c9bc2f8a288439aa76048f16
869646f9f32db89e07cef4570fbaab58e2791802
4200 F20101107_AAALGJ jayaraman_a_Page_157thm.jpg
7794d9f8d518e4149ccce5b26ce2425a
605f7adb32d124eb4aef7cc1472366255de7a2a2
5880 F20101107_AAALFV jayaraman_a_Page_141thm.jpg
32f57f5a5f1c1b8e0ee39af9ba507d1d
5e50e713ccdf61a3705984301cb48754b5ca6380
14828 F20101107_AAAJYB jayaraman_a_Page_057.QC.jpg
b12a0abc138fc9c37444163b8649e35b
18667851671cfe8390c1847a09a5ffafb2059676
65638 F20101107_AAAKCT jayaraman_a_Page_180.pro
225d3693cdca0823f5d81365c4139cee
df167f481941454eb844ee90504cec4b34582e51
F20101107_AAAJXN jayaraman_a_Page_105.pro
39d7adf447d8a8dcdc02ac705e36ccf2
c04fc81a38be99796c79d933d816c0a55a5b605d
F20101107_AAAJWY jayaraman_a_Page_029.jp2
e7b8e2783e4ec34c68aac0ca16fc22df
0bbc8927da2c7d4f03ca9285bfc8317ceaad6794
2229 F20101107_AAAKDH jayaraman_a_Page_112.txt
e044c7c2b30d14f5b4aeb2efb2367548
774ced1c258dac7e46018088d7b3e8481a951d44
15772 F20101107_AAALGK jayaraman_a_Page_159.QC.jpg
4c88ab2cb7930a64aa0513a1d41b736f
438833864d0efba5a5d2d3d250159ca1c1dff745
5977 F20101107_AAALFW jayaraman_a_Page_142thm.jpg
818828667044f66362084548dc507ee3
e2683db99db6b2f3ed09eec649b8476b3e5aae63
835 F20101107_AAAJYC jayaraman_a_Page_161.txt
6deee3a1218430c06908050f431b58a0
b7a50546b380274bd70d54950031dea722047e56
110818 F20101107_AAAKCU jayaraman_a_Page_050.jpg
79c71fac22ab773070c683aef175b141
691087696e2d1553cb17abf005c44f9bb679b388
2074 F20101107_AAAJXO jayaraman_a_Page_017.txt
e6f95177da7e9f9f7d177f5f90202e7a
85b3034e757dfe5c0a8db332d6db20f046a6d429
26168 F20101107_AAAJWZ jayaraman_a_Page_001.jpg
dc9c3370e5b8cf62e4bfea43799889eb
eae21e2ad45ec438e1e364d227b0f26d5cf00c43
56477 F20101107_AAAKDI jayaraman_a_Page_042.pro
f59c156af5fc74725f05237ba50b624e
df5d995841c7f84cf245d51f6e82366377770e6a
37178 F20101107_AAALHA jayaraman_a_Page_173.QC.jpg
f63a069b1c9abd53f80a85d8ba770b42
a5feaf93aaaaa643e2d6337942e0a29b1f3e6287
5385 F20101107_AAALGL jayaraman_a_Page_159thm.jpg
4ad8490d0a1825e9f03a3bbda4d7af39
183d3e7ad4b323e36b8b43e21a506ab55d18fcf5
38467 F20101107_AAALFX jayaraman_a_Page_143.QC.jpg
f6e50068a2db4d11ed9397983114e325
c230975e548b92b0715cf03149e8d5f120c08eb6
2685 F20101107_AAAJYD jayaraman_a_Page_180.txt
1f5d4f795a649de7146a590b95dafc4f
9f5c14dea9c95bedb175b62eb9002d63ef026c34
58571 F20101107_AAAKCV jayaraman_a_Page_011.pro
329332787ee2480c7de72f28c225a8bf
d0eae8252ef8361cc8b9b881b76915eaa8903c02
3379 F20101107_AAAJXP jayaraman_a_Page_090thm.jpg
f6bf1a5b60a44c270356098bf742df34
feb8420792384bd402c3d35b76c3829740d0a077
652 F20101107_AAAKDJ jayaraman_a_Page_010.txt
8cebda91ab6a65e6c6030daf8e8d0143
e78a0647468b990d0480d3f29964219c34b9707f
38375 F20101107_AAALHB jayaraman_a_Page_174.QC.jpg
cbcfd65a62d8957deb7fda64db8af57e
8874b63b6c8698b76ed4f588e4758f28f3c8f673
5749 F20101107_AAALGM jayaraman_a_Page_160thm.jpg
020a7605b0570df0b7f2f96b4108724a
1891ef9b735dda0e6a8bc0def7e94a1a001d71da
34597 F20101107_AAALFY jayaraman_a_Page_144.QC.jpg
63f74a109d4a0e049a2ee6150d085943
62f60052780cbc2942c1cfa03d42ffd376cb7b50
2163 F20101107_AAAJYE jayaraman_a_Page_043.txt
a5fc77ca6cf88760739f592e9b7761f2
5e7ec1078a00efcb9852399a20550d2772ab50ed
9423 F20101107_AAAKCW jayaraman_a_Page_173thm.jpg
c0a27b6f088aa2a1de1fe87e5ebdce49
1cd791ba199167f63ef5d6df337f16d55fe5a5a6
53101 F20101107_AAAJXQ jayaraman_a_Page_096.pro
5b4756d0000d54e17bfb54f5aabdc552
dcc4af2d851d193afc3642e58489a95fcb323167
F20101107_AAAKDK jayaraman_a_Page_023.jp2
eb66ebaae1e07c6bb29bc4d1a39eadf3
7400bd285b887c8bf73a82b90e76942f816aef3a
9338 F20101107_AAALHC jayaraman_a_Page_174thm.jpg
b886be0bbf696e8bff7c290859dcd59f
6c1d6509279a887b26b821cd4b4998975b3223ee
11967 F20101107_AAALGN jayaraman_a_Page_161.QC.jpg
7eb49f3684a0e7af72c308c1b9fe8e94
7437ae7f5a3afe7b050aeb43f6203ae991e809f2
8666 F20101107_AAALFZ jayaraman_a_Page_145thm.jpg
b7e078949f7e706aad7ce27c800a6b88
ec0869a3dbef4905af29d909139198a94729271f
1030330 F20101107_AAAJYF jayaraman_a_Page_067.jp2
097dd295068a35698bc5cd0f8efd8024
fc2d5493ff64542d60d521aa41685696b5618363
2200 F20101107_AAAKCX jayaraman_a_Page_137.txt
74d7e2d432b770e1650861a3f8a86c5b
7a5c2af9b19a1de8904e0a9b3770a24bbe6c2841
32684 F20101107_AAAJXR jayaraman_a_Page_078.QC.jpg
62281da7c593204b907fd432aafc5ce4
dddb843b4fb29d1b4deaacbdd4e093c028b8a580
F20101107_AAAKEA jayaraman_a_Page_066.jpg
99384cfe3e0c78a2a230568d18384955
4b83e5a51f84a3d40488f46a8d9151bf28019f43
103262 F20101107_AAAKDL jayaraman_a_Page_039.jpg
3b19ea623a2222233657318d1eeb84c5
2ba93257bda796da3efdaed2ee8cee31092b595d
40515 F20101107_AAALHD jayaraman_a_Page_175.QC.jpg
33f6daf7e89756dc3e5b0031fb2a040e
0b8edb5718e9f1f53ae8a9721fefed5b81396feb
4216 F20101107_AAALGO jayaraman_a_Page_161thm.jpg
c213416e26f20f5deb01218a2b445706
7ddec5285a2ede23cba4f8ec4d151caaa96ea14f
9167 F20101107_AAAJYG jayaraman_a_Page_085thm.jpg
4c0104d9402b1fe7c581d82d93345259
1fa796b0dc25c949e3073d5fa0a63c5aaabed5c9
F20101107_AAAKCY jayaraman_a_Page_047.txt
5ce892750970383c6dacd5127dc64cf5
9a7d8c07cddf0aaf0249b4fe5a0f0c4ecbefde62
284969 F20101107_AAAJXS jayaraman_a_Page_094.jp2
c283a75308c8aa546f2e19682429cbf4
e83796593aff0ce292d5f5908dbd3555a4a90f66
7589 F20101107_AAAKEB jayaraman_a_Page_013thm.jpg
5ad94778db29db65aac394451e4a4954
8eb5a1a0ddad54e9be2e90be1e048feaed652c1e
36822 F20101107_AAAKDM jayaraman_a_Page_085.QC.jpg
f619143a9904aa600e526132dba131c0
1ffaa55721a4d346ab8834cb144dee22813d6373
9559 F20101107_AAALHE jayaraman_a_Page_175thm.jpg
6455e1ba06d863538324cc300134f61e
5d799b94f3aa8350591a2c095310754cfc2eff9c
13193 F20101107_AAALGP jayaraman_a_Page_162.QC.jpg
3530b2aaed3c1cd60e4a2158f6ca3e72
6fe41133cea64c4382c4c7f34a624901b3309fe0
54793 F20101107_AAAJYH jayaraman_a_Page_103.pro
e9fd74189b2dd73a7e3edc437d083dd6
6feb70c9ac46ae62f2f138052853deba1803d488
F20101107_AAAKCZ jayaraman_a_Page_056.tif
957525f96406219476da8f498c9e7a4f
c9e5bf336603d1cfb4934b796fe1122ae5653b12
36859 F20101107_AAAJXT jayaraman_a_Page_053.QC.jpg
c82baa39f9d6634df603cd588d4f77fe
830e7d8acaf346a7406529f3eba4bd28bb83ee78
F20101107_AAAKEC jayaraman_a_Page_079.tif
9a81b555662519a4a20afcb4675e4d35
052cab77b576facb03575f0522e5f8f314a196c3
F20101107_AAAKDN jayaraman_a_Page_111.jp2
7a4092f01b0d2dfa48ed6952f4fd2260
03cc114e4c2deddface76700acca5642f3cba8fa
9460 F20101107_AAALHF jayaraman_a_Page_177thm.jpg
f24727cc7a3e49c1b3499e8b615ad379
7dce7552bf490fe006c43608859411a3a3d848ee
8885 F20101107_AAALGQ jayaraman_a_Page_163thm.jpg
65dc63186fe7dc43453855a2e5401d31
b1fc165503ffe3632a4d85cbf34830c7580fbe5d
F20101107_AAAJYI jayaraman_a_Page_029.tif
4fc573bf3483f8b3347755f38c9713cc
bdf09817b093c2152b2277635267fdaf6a581a41
51335 F20101107_AAAJXU jayaraman_a_Page_039.pro
979901b1393fcae9df59e4764d4ddcdf
bef9345c6d018893e9939b4888273531eaabd9d8
1794 F20101107_AAAKED jayaraman_a_Page_081.txt
2fe113167609e61670156d8ff2022eac
cf409c14ef674aa4774671a3e5559af50fbee5cd
29854 F20101107_AAAKDO jayaraman_a_Page_014.pro
79b69f0d27f764b91c0baa6e8bf85cf4
41ec45645cb90dcdf6e8376b5a356bb6b9174b20
37051 F20101107_AAALHG jayaraman_a_Page_178.QC.jpg
308ca1d25ca570545cb9c6a38ba7c54f
f8d34688ffba82ff5608390f8bcd3e5e07e5cefa
9412 F20101107_AAALGR jayaraman_a_Page_164thm.jpg
942c8ef26ae59c91fbc9a92764bd632d
f25336291e0b9a8e6e87c7f10e3ca87e753241db
31295 F20101107_AAAJYJ jayaraman_a_Page_109.jp2
a5a01f463ca12759fc56470eb1ee5a75
1ac7936c767c041c030b5324b9f1c223639972fb
120498 F20101107_AAAJXV jayaraman_a_Page_172.jpg
7a4e6461fe592c71422d72455f3c7fcd
72dc0dae95d39adec1e229fb41c5700938b5f22b
108016 F20101107_AAAKEE jayaraman_a_Page_096.jpg
745b50d9053f5a582ffddd166ccad17a
15324489bc6ba0fa1e8637047ce0843b69faa12b
37429 F20101107_AAAKDP jayaraman_a_Page_135.QC.jpg
3bccfe71238f22127abb5246d1768a11
9fc4eb6a6940e151ae21dfb6fab387f9d9907945
9422 F20101107_AAALGS jayaraman_a_Page_165thm.jpg
09ec7fa6863ba7a047b1f79b7f1c7a42
ce9a3bc76734e539482052c94fe9b46d02501632
F20101107_AAAJYK jayaraman_a_Page_093.tif
a954b0aef5317f9d23a189690c1cce6f
766f954f76ca56bbb29d8ef8accf649f4200c977
1081 F20101107_AAAJXW jayaraman_a_Page_160.txt
07cf1b5f6bafdae48abcf8d5e883cc99
1a6b8b25f81a3417d9791d75a1d0a9e211de93bd
F20101107_AAAKDQ jayaraman_a_Page_083.txt
40522ca8d1dea5e07c9d1333a8043bbd
523b8a5be88bc603607e75a42778b125ee1d33a4
9147 F20101107_AAALHH jayaraman_a_Page_178thm.jpg
f774981d50479c7c523363edef70e1e4
330cfdd279e28d2542e629aaba946edbcfae61c9
9333 F20101107_AAALGT jayaraman_a_Page_167thm.jpg
9fe96b28676055d819c77c1638e0f835
0d88039993d6fa62f0391d22ec0905b962b3ffc1
2064 F20101107_AAAJYL jayaraman_a_Page_101.txt
9a257f7c8f42acdaabbdc69c0cec1da2
acd954e9a475ae70906a43dbe4f85546d57bd7e9
6189 F20101107_AAAJXX jayaraman_a_Page_139thm.jpg
286a06a8fc4d8201c68b1fc02876820b
2c105547294ca83c2728f2f807e4631814bc2b9f
52989 F20101107_AAAKEF jayaraman_a_Page_145.pro
c4da37c94697ff65ed7ff8388b382922
357edecc40202c45e01baee80623a8836e4e310f
57022 F20101107_AAAKDR jayaraman_a_Page_143.pro
1b2e6c057964dc9e9c372ae64fd3a337
a61fc109aab3d660240876ff03cab29882c0b25b
37910 F20101107_AAALHI jayaraman_a_Page_179.QC.jpg
fa0cadc90f41884595139f34ecccfe2b
98eaef9f00bae315de21b01de726f11f4b1fec80
37526 F20101107_AAALGU jayaraman_a_Page_168.QC.jpg
863a4fdb7f304ce541badd8bd5352a92
602ac148251fdbb4419047f4f6c9ac2679c2a1fa
111034 F20101107_AAAJYM jayaraman_a_Page_111.jpg
a7b336cc96cb63cd54d453b142fd8052
c942dddca2281e11128e4a2abb91e94032794177
661 F20101107_AAAJXY jayaraman_a_Page_093.txt
e8a3fc42f191fb7fd5abe871a7298404
f81acdf11b71d40cedaad8b10471b81c80f7bb6c
F20101107_AAAKEG jayaraman_a_Page_108.tif
b210398ef06d46fedbce3ddb853f62da
97e596db2426cd4b7f7977707154ffdd56ec4e47
104526 F20101107_AAAJZA jayaraman_a_Page_101.jpg
ede36c82c2cea89d49102c98ad0496a1
1589b57d7f103810c96ad5db81fa5226819416c5
54680 F20101107_AAAKDS jayaraman_a_Page_163.pro
59af16e22416e3246ce7cde45a7a3de7
62bba52f9c749f32fdc225c99a1c5a5e7c879cfd
9092 F20101107_AAALHJ jayaraman_a_Page_179thm.jpg
a7edac39881398acaaee5606b16a4b37
41f0399db501ecb867075f3a502b856351b8e478
9341 F20101107_AAALGV jayaraman_a_Page_168thm.jpg
4af3a1931c11bfae5e6cecf802a9bc5d
ada9c14fd9aaddf788874fbe945d0648af0e9409
109992 F20101107_AAAJYN jayaraman_a_Page_104.jpg
7f32e999c411760b194b0536fda33b2f
c4df4bbd9c328046a96e605687a898fc2fd8a48b
38502 F20101107_AAAKEH jayaraman_a_Page_164.QC.jpg
69dc3a0b9f3751627141d363ed5a9cd3
b507f9b6e2ea1bc8be3bf88d9fb132af1f974cb5
14029 F20101107_AAAJZB jayaraman_a_Page_156.jpg
4a1cd6db439a1f3ef99f7a7583f6c402
9de1ba804afc8cc069e121f4ed54189d855c8422
35651 F20101107_AAAKDT jayaraman_a_Page_049.QC.jpg
e04c82a45900311e2b4c0f057a5ca671
c68057fa9411fee49c5d112564a2ac6dfb6e764e
37348 F20101107_AAALHK jayaraman_a_Page_180.QC.jpg
c50c32bd8a375978e4165c948a535345
e124170ed2c427aefd2f1c66bd22698b5ff7c23e
37116 F20101107_AAALGW jayaraman_a_Page_169.QC.jpg
db4d970fa77bf0b1601e336141407fcd
359e34d39ba0eeae0012c086fa2790c0074ea1a0
F20101107_AAAJYO jayaraman_a_Page_039.jp2
26b74a145ded3f5804774918ae3652fb
aac75fb298f959340a4245ef76cdc6672c8d2733
79151 F20101107_AAAJXZ jayaraman_a_Page_074.jpg
66923d5d5aeaaf32b8c8b6c5724b8343
e49b2460faa4f55fc222e47dc4b9da48e1880cee
2716 F20101107_AAAKEI jayaraman_a_Page_177.txt
8ebc1f9e10c621f4c7d7de133c57157d
b8d3d9fd3c04ff9af404477c041da8394f8d5097
33268 F20101107_AAAJZC jayaraman_a_Page_066.QC.jpg
a5dd05da28c459df78a42905723e315c
4d6a6633607dcbcb27be27baa9b077b22d5a923f
F20101107_AAAKDU jayaraman_a_Page_173.jp2
6864f0acaed3acf9c9a3cee3806530b8
2e9b7cdacee967e7737d81ca2cb03f9cd5385bd2
2319 F20101107_AAALHL jayaraman_a_Page_181thm.jpg
fb4dc34aea11b1ba678ce0f904d773db
d73e991c609688454923b9f33d49ded18d6b5b38
37849 F20101107_AAALGX jayaraman_a_Page_171.QC.jpg
b8f3ca3ee7dc3b75fe8c136cc5460596
c893ed4daf74cf9496e46476341c1c551ab5d9ac
497359 F20101107_AAAJYP jayaraman_a_Page_139.jp2
580612287e065653e1ec951c17c807f4
6544e7718244768aa9dc324af049048e4c31dd78
37320 F20101107_AAAKEJ jayaraman_a_Page_037.QC.jpg
71bf6e2ff7b5bbdd905682a35c2c73cb
0d39b95b5d95f20fa3575be93cac76663f0a55ef
F20101107_AAAJZD jayaraman_a_Page_088.tif
9b9ff902427a742499e8d9f0791675bf
fc34fa21906cfdadb8379da094f22c4ff64035f5
92959 F20101107_AAAKDV jayaraman_a_Page_081.jpg
05717711b3dd269615474b940217f077
b97669ee9729350642c45fcdb35b9f3e7795ee8b
F20101107_AAALHM jayaraman_a_Page_182.QC.jpg
8f113ce0159fa72dd9396e5edccca3dc
a771554ec0cae9b6bad2bbbaed11acdf0ee83ff0
F20101107_AAALGY jayaraman_a_Page_171thm.jpg
40d7a825c0f98c42086e57b1c39b9c3b
934583b116ff79d0e1e6170c3376922adf83ad73
4259 F20101107_AAAJYQ jayaraman_a_Page_138.QC.jpg
e8122f643980a25f2230c20b0a02a056
6b765551ac276a145fc258a02215ff07bfb2c393
8284 F20101107_AAAKEK jayaraman_a_Page_069thm.jpg
625f83ab5073b1bf8a3bf54291661f1a
152d7575e00fcc8d72e500428ddf2e6850b540ab
37496 F20101107_AAAJZE jayaraman_a_Page_115.QC.jpg
26edb312becd3622c9b992129c0911c1
a65e799642e923c2dafe8e9e98e5ee951ff42b18
50869 F20101107_AAAKDW jayaraman_a_Page_084.pro
62e92bffc1f84d1d1d990a47bca01b24
c63d57e5b26cb251d3d56acf196c1cb51feb022e
212359 F20101107_AAALHN UFE0022057_00001.mets
3f473a491637d40eb9823f501ddc0a52
e32683f5c76e1ed5ae105081b61c34db81688f5f
8923 F20101107_AAALGZ jayaraman_a_Page_172thm.jpg
daaf0d95728bc7a2d9af44f10c2e67b1
4f355e29e2f72b8c823fd2d253702d27b4b8b9ba
54671 F20101107_AAAJYR jayaraman_a_Page_033.pro
a9cb6cfc939c046012cc67dcc0c5fdf2
15f28ea1244e6b159709c51cec5722c15ff66aad
F20101107_AAAKFA jayaraman_a_Page_172.tif
760dbeb9a5b645d44923d727072a6fa7
fcf30f3043730904cbfcba53335b0d5f0b2030dd
9171 F20101107_AAAKEL jayaraman_a_Page_026thm.jpg
fe0dcc7644670fa451b3f98d63876430
b349f69a7f01097d57a983001ce853cd8849d8ed
132899 F20101107_AAAJZF jayaraman_a_Page_169.jpg
193638679e7c7ba1c7f5d432ce818e4f
244c96978ac82ec2f9f5c296ebdfff5948c3dfd5
33587 F20101107_AAAKDX jayaraman_a_Page_116.QC.jpg
3f4f5bcd28c6f38e2e02c1e630dd4c67
1fcb7ec23baae33c6b9fbc53c3b70f01521869b7
12136 F20101107_AAAJYS jayaraman_a_Page_157.QC.jpg
e0850c7aa0240fa336782fb392fa0d92
35f3c9b954c9c11c6f2ed04c96177cf07857d8ac
6471 F20101107_AAAKFB jayaraman_a_Page_063thm.jpg
016abd6996631b8223b256a3b439f0b5
553965e3af774ef1c9da4cf0923b0c65c91dbb65
966563 F20101107_AAAKEM jayaraman_a_Page_056.jp2
079156a089c9f06d6fdb019cb1ace278
dff64d2150b492fdef63f1b1473a3ddf84d9d599
45396 F20101107_AAAJZG jayaraman_a_Page_141.jpg
a50c85f52764c14edfd52f7ada9dc4ce
39766863c6fa34ebb9ac7dc52ec528e9f9d686a9
F20101107_AAAKDY jayaraman_a_Page_176.tif
b3d4f6aa857d527516a02f3ba10ff142
e1f50c9f91b3119a5f18b0f6986e78bb2ff46310
17470 F20101107_AAAJYT jayaraman_a_Page_160.QC.jpg
8110ad5d4f3ae2067917899d20382944
31f16620d64629be58adfbdab85f34ac7e975f61
32456 F20101107_AAAKFC jayaraman_a_Page_016.QC.jpg
0b906be78e9dbb62698e443d52bb1661
554d4b03cbb81a9f11e5dc8ee85e82f986f97f6f
2675 F20101107_AAAKEN jayaraman_a_Page_076thm.jpg
525087af4b3892ca4b7705152350a609
dd23a47990aee03cf42ad501b2b682231b398498
2164 F20101107_AAAJZH jayaraman_a_Page_097.txt
04b6f6843dcf864501a9f607f4d51a69
f75de47c7d35d8384b477a29775ce894c681961f
108914 F20101107_AAAKDZ jayaraman_a_Page_147.jpg
018f8034ed2d9eed3433e6a2f7a18d6b
b209b06c6863e3a12a1e13385223f1f6098532d3
F20101107_AAAJYU jayaraman_a_Page_173.tif
f3be9b9f7f8ed4918036bc56875bd526
f77fd849f68479ed65b35619e7f8e26dbcf28e8a
2124 F20101107_AAAKFD jayaraman_a_Page_096.txt
d3a053f104c8362926854089ef8420c0
fb0f609f1b1b13501d3045517f42440e4149feae
2186 F20101107_AAAKEO jayaraman_a_Page_072.txt
aed066f3f11b42f0b569db1b88264757
a932add4ef121867aa84c36d3723430852ae8626
527 F20101107_AAAJZI jayaraman_a_Page_107.txt
4d2d803b16a267e9c71cb151229e1cc9
40700defce992e01a46050160b1aee04bd22b44b
7293 F20101107_AAAJYV jayaraman_a_Page_079thm.jpg
ad2c50a6ca796a5d80e53974a0a609fd
32d5b103bb67ea089016b1fd2e3eef85fd4ddd5d
F20101107_AAAKFE jayaraman_a_Page_137.QC.jpg
9611b9d089ea61ab421b5dcdbbc471fa
fa420986272b4683f24292cedc4048e64dc6e226
F20101107_AAAKEP jayaraman_a_Page_166.jp2
94ccef5788227b64dcffaebbf403b7a3
0c174a74516cd1f2e401308721875229caca57fc
2269 F20101107_AAAJZJ jayaraman_a_Page_052.txt
0e54d958ba25a586469ce80923d2f602
b7b2f679563f3ad13df4b7773763142621ff7d70
70686 F20101107_AAAJYW jayaraman_a_Page_170.pro
2b6f161eb8fe7fc5c1be0313703834fb
bd805574077c1b9f523d733b17a42e7993e3b1a7
8664 F20101107_AAAKFF jayaraman_a_Page_116thm.jpg
2934831a4fb91ea80cb5e7aa4ffb15a3
601c1afe2f4c6829b1ec92d9ff4928130ac4bf0a
2261 F20101107_AAAKEQ jayaraman_a_Page_077.txt
015e3e4b9788bea3b8c0b02a333ea15b
6633590d7bff02989a90d3fb584486c982d3f926
49989 F20101107_AAAJZK jayaraman_a_Page_118.pro
4a7b86631824eb8c16dff92f56eea0a3
500cce57cd526d9ce568bfb6e4196597bd75d350
F20101107_AAAJYX jayaraman_a_Page_005.tif
171c2177d98dac20f2771b6fe9accad9
fb94879aab900747f29d2ff117214113f33e015d
8654 F20101107_AAAKER jayaraman_a_Page_031thm.jpg
ddedbd0eefdbf46e98968752a6ca54c9
c2af21c924ba4cd5a793928df225b74df1d9090b
114173 F20101107_AAAJZL jayaraman_a_Page_022.jpg
cd6a486829026c542f80f68ce72804b6
853790289a0ea56a2ded06e1984673aee9d4ad11
16625 F20101107_AAAJYY jayaraman_a_Page_142.QC.jpg
7ee98b5330f688271352619619f570ad
11e7d4cbc56bc3d259c1b99600f64ed077c5df9f
F20101107_AAAKFG jayaraman_a_Page_181.tif
7ef37f8e3979fdd7b11dcdc62182b012
1b355c2d59929071b7fa89d6e5684a39197b6e1f
85105 F20101107_AAAKES jayaraman_a_Page_060.jpg
bd216d675259669b0e2b5c01595e18fd
ef262b5f7986ed4a7e31e564e55b1636d6213395
12621 F20101107_AAAJZM jayaraman_a_Page_142.pro
fab92e78b8d704105d887e264fedffcb
8d9714e7089e59b018e198995fe51845a05c0cae
14931 F20101107_AAAJYZ jayaraman_a_Page_091.pro
5717b5cdbb606040d2179d69eb5c8dcc
d86c690374d603560e85c284d707f347ec863cc8
9130 F20101107_AAAKFH jayaraman_a_Page_052thm.jpg
f11b69a0aee399a7e18091ea8d4dbb00
54b66ff9e6487df0996adb46724ff312ebb560e6
9292 F20101107_AAAKET jayaraman_a_Page_030thm.jpg
7b2f06ece45d4deb927d49d2dcef48a3
6757f788269d905d3461a83dad712700178d6305
8118 F20101107_AAAJZN jayaraman_a_Page_074thm.jpg
01d5257574f1c8bf0cd07592a233bb67
6f0531b679848e10e06a714c5faa1a9f09941754
F20101107_AAAKFI jayaraman_a_Page_020.tif
b4754e52c5faae22a45883800fac149d
f6624c0074915e046efab68f29b9b1f63a21b30f
F20101107_AAAKEU jayaraman_a_Page_055.txt
6389627da647c1ae3dd9c594ededa4eb
c59472614090f8f574d5099e03952f487ef077b0
54133 F20101107_AAAJZO jayaraman_a_Page_049.pro
0f3a35aef48ae1a14e43f12b97e21952
d3be08f680bd62638bc00fc6222a44ec453ba44e
36471 F20101107_AAAKFJ jayaraman_a_Page_041.QC.jpg
7a16b705796a4934ff2fa84cffca1a57
f8168d45992024b9f1ed13eaafebc324b633fce7
F20101107_AAAKEV jayaraman_a_Page_004.txt
5d34e382fbc4a1bab8e98b189d0b983f
feaf9b7acd9725006967a685974de844e6720fb9
2579 F20101107_AAAJZP jayaraman_a_Page_179.txt
339d556d0b6e0bdb18e2623cc48eb6a0
2eabd751fac1059f8ad44ee860d4b2827179a7f8
7922 F20101107_AAAKFK jayaraman_a_Page_117thm.jpg
d2c23db84e8f9dafbb708901d15dcd14
32f95fbc6bf02cfb2e4da2e2f38935124325bbc8
54022 F20101107_AAAKEW jayaraman_a_Page_027.pro
f6e85331bb371b29577cbe0c0902a5a3
9df2064fc0746363d6407a05e2899cc1f1d3330f
2003 F20101107_AAAJZQ jayaraman_a_Page_066.txt
4365ed6cecab21d6879edf9d04bcae8d
a57882e541d5c2a23ec434b974c73c66c6032144







SKELETAL MUSCLE ADAPTATIONS FOLLOWING INCOMPLETE
SPINAL CORD INJURY AND EXERCISE TRAINING



















By

ARUN JAYARAMAN


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA

2008



































O 2008 Arun Jayaraman



































To my mother, wife and, the almighty









ACKNOWLEDGMENTS

I am very grateful to several individuals for their guidance and support in my dissertation

work. The greater part of this work was made possible by the guidance of my mentors and by the

love and support of my family, friends and colleagues. It is with my heartfelt gratitude that I

acknowledge each of them.

First and foremost, I would like to sincerely thank my advisor and mentor Dr.Krista

Vandenborne, for her constant guidance, support and, encouragement. Under her, not only did I

learn about conducting excellent research but also on how to be a complete academic

professional. Words cannot simply summarize my gratitude towards her. Also, I am grateful to

Dr.Walter for his remarkable wisdom and thought provoking research questions. Never has a day

gone by were I have not been amazed by his up to date knowledge on almost every research

topic. I would like to thank Dr.Behrman for her constant encouragement and infinite support

throughout my doctoral education. I would also like to express my sincere thanks and gratitude

to Dr.Rosenbek for always believing in me and encouraging me to strive harder towards by goals

and aspirations.

A special thanks to all my lab members, without whose help this dissertation would have

never been possible. I would like to thank Min, Chris, Neeti, and Jen for teaching me all the

necessary skills and techniques in the lab and for also guiding me and encouraging me through

the PhD process. I would like to thank Prithvi, Fan, Gabe, Sunita, Donovan, Rayneet, and Wendy

for constantly helping me and making work a lot of fun. A special thanks to Dr.Miles for helping

me with my document and to the Physical Therapy department for being a very important part of

my graduate student life.

Finally, I would like to express my deepest gratitude to my family. I would like to thank

my grandparents for their love and support. I would like to specially thank my mother for her









unconditional love and support throughout my life. I would like to say without her this PhD was

never ever possible. She has encouraged me all throughout and always has put my education as

her first priority. Last but not the least; I would like thank my beloved wife Sangeetha who has

been the main pillar of support in my life. I owe my success in life to her. She has always had a

smile on her face and her hand has held me through all the ups and downs in my life. Thank you

for all the sacrifices and difficulties that you have patiently endured. Finally, I would like to

thank the almighty and savior for trusting in me, loving me and being there for me. Thank you

God! You made it all possible.











TABLE OF CONTENTS


page

ACKNOWLEDGMENTS .............. ...............4.....


LIST OF TABLES ................ ...............10........... ....

LIST OF FIGURES ....___ ................ ........___.........1


AB S TRAC T ........._. ............ ..............._ 13...


CHAPTER


1 BACKGROUND ................. ...............15........_ .....

1.1 Introduction ............... .. .. .. .._ ......... ...............15.
1.2 Demographics of Spinal Cord Injury (SCI) ................ ...............15........... ..
1.3 SCI in Humans-Pathophysiology and Classification ................. .........................17
1.3.1 Definition of SCI ................. ...............17................
1.3.2 Spinal Cord Neuro-anatomy .............. ...............18....
1.3.3 SCI Pathophysiology ................. ...............19........... ....
1.3.4 Classification of SCI .............. ...............20....
1.4 SCI in the Animal M odel ................ ...............21...
1.4.1 Spinal Cord Isolation Model .............. ...............22....
1.4.2 Spinal Cord Transection Model .............. ...............22....
1.4.3 Spinal Cord Hemisection Model ................. ...............23...............
1.4.4 Spinal Cord Contusion Model ................. ............. ............... 25.....
1.5 Skeletal Muscle Adaptations in Human Following SCI ................ ..................2
1.5.1 M uscle Size ................. ...............27........... ....
1.5.2 Fiber Type Composition ............... .... ...............28.
1.5.3 Electrically Elicited Contractile Properties ................. ......... ................30
1.5.4 Voluntary Contractile Measurements ................. .......... ...........3
1.6 Skeletal Muscle Adaptations in the Animal Models Following SCI ................... .........33
1.6.1 M uscle Size ................. ...............33........... ....
1.6.2 Fiber Type Composition ............... .... ...............34.
1.6.3 Electrically Elicited Contractile Properties ................. ......... ................38
1.7 Rehabilitation Training Strategies Following SCI............... ...............39..
1.7.1 Locomotor Training .............. .. ...............39...
1.7.1.1 Locomotor training in humans............... ...............39.
1.7.1.2 Locomotor training in the animal model .............. .....................4
1.7.2 Functional Electrical Stimulation (FES) ................ ................ ......... .42
1.7.3 Resistance Training ............... .... ........ ... .. ...............45.
1.8 Skeletal Muscle Adaptations Following SCI and Locomotor training ................... ......46
1.8.1 Impact on Humans ................. ...............46................
1.8.2 Impact on the Animal Model ................................ ....... .......... .....47
1.9 Mechanisms Involved in Training Induced Muscle Plasticity and Recovery ...............49












1.9.1 Plasticity of Skeletal Muscle............... .... ..............4
1.9.2 Markers of Muscle Recovery and Regeneration ......____ ..... .....__..........5 1
1.9.2.1 Adult muscle satellite cells .....__.....___ .............._.........5
1.9.2.2 Other stem cells ............... ........ .............5
1.8.2.3 Growth factors and muscle regeneration............_ .........___......55

2 OUTLINE OF EXPERIMENTS .............. ...............59....


2.1 Experim ent 1 .............. ...............59....
2.1.1 Specific Aim .............. ...............59....
2. 1.2 Hypothesis............... ...............5
2.2 Experiment 2 ............ ..... ._ ...............59...
2.2.1 Specific Aim .............. ...............59....
2.2.2 Hypothesis............... ...............6
2.3 Experiment 3 .............. ...............60....
2.3.1 Specific Aim .............. ...............60....
2.3.2 Hypotheses ............ ..... ._ ...............60...
2.4 Experiment 4 ............ ..... ._ ...............61...
2.4.1 Specific Aim .............. ...............61....
2.4.2 Hypotheses ............ ..... ._ ...............61...
2.5 Experiment 5 .............. ...............62....
2.5.1 Specific Aim .............. ...............62....
2.5.2 Hypotheses ............ ..... ._ ...............63...

3 METHODOLOGY .............. ...............64....


3.1 Studies in People with Incomplete-SCI .............. ...............64....
3.1.1 Subj ects Description ................. ...............64....__ ....
3.1.2 Locomotor Training .....__.....___ ..........._ ...........6
3.1.3 Re si stance and Ply ometri c Trai ni ng................. ....__. ..................6
3.1.3.1 Re si stance training ....._.__._ ..... ........ ....__. ...........6
3.1.3.2 Plyometric training .............. ...............66....
3.1.4 Muscle Function Assessment .................._.__._ ...............66...
3.1.4. 1 Experimental set-up ........._.__............ .. ....__ ..........6
3.1.4.2 Voluntary contractile measurements .............. .....................6
3.1.4.3 Electrically elicited contractile measurements ................. ...............68
3.1.5 Measures of Ambulatory Function.. .........._... .....___ .......__............69
3.2 Experiments in Contusion Spinal Cord Injured Animals............... ...............70
3.2.1 Anim als .............. ...............70....
3.2.2 Contusion Injury .............. ...............71....
3.2.3 Locomotor Training .............. ........ .............7
3.2.4 In-Vitro Assay of Muscle Composition and Regeneration .............. ..............72
3.2.4.1 Immunohistochemical analysis............... ...............72
3.2.4.2 Western blot analysis............... ...............73

4 LOWER EXTREMITY SKELETAL MUSCLE FUNCTION IN PERSONS WITH
INCOMPLETE SPINAL CORD INJURY................. ...............7












4.1 Introduction ............ ..... .._ ...............77...
4.2 Methods............... ...............78
4.2.1 Subj ects .................. ...............78......___. ....
4.2.2 Experimental Set-Up ........._...... ........... ...............79.....
4.2.3 Voluntary Contractile Measurements .............. ...............80....
4.2.4 Electrically Elicited Contractile Measurements ....._.. .............. ..... ..........80
4.2.5 Voluntary Activation Defieits ................. ...............81................
4.2.6 Statistical Analyses .............. ...............81....
4.3 R results ................ ........ ........ ... .. ...............81...
4.3.1 Voluntary Contractile Measurements .............. ...............81....
4.3.2 Electrically Elicited Contractile Measurements ................. .......................83
4.3.3 Voluntary Activation Defieits ................. ...............83................
4.4 Discussion .............. ...............83....


5 LOCOMOTOR TRAINING AND MUSCLE FUNCTION AFTER INCOMPLETE
SPINAL CORD INJURY: A CASE SERIES .............. ...............95....


5.1 Introduction ................. ...............95.................
5.2 Methods............... ...............96
5.2.1 Subj ects ................. ....... ....... ...............96......
5.2.2 Locomotor Training Protocol ................. ...............97................
5.2.3 Experimental Protocol ................. ...............98........... ....
5.2.3.1 Strength assessment ................. ........... ............... 98......
5.2.3.2 Voluntary contractile measurements .............. .....................9
5.2.3.3 Voluntary activation def cits............... ...............99
5.2.4 Statistical Analyses .............. ...............99....
5.3 R results ................ ........ ....... ... .. ...............99...
5.3.1 Voluntary Contractile Measurements .............. ...............99....
5.3.2 Voluntary Activation Defieits ................. ...............100...............
5.4 Discussion ................ ...............101................


6 RESISTANCE TRAINING AND LOCOMOTOR RECOVERY AFTER
INCOMPLETE SPINAL CORD INJURY: A CASE SERIES ................. .....................111


6.1 Introduction ................. ...............111................
6.2 Methods............... ...............113
6.2.1 Subj ects ................. .... ....... ...............113......
6.2.2 Resistance Training Program ................ ...............114...............
6.2.3 Plyometric Training ................. ...............114...............
6.2.4 Dynamometry ................... ........... ...............115......
6.2.5 Voluntary Activation Defieits ................. ...............116...............
6.2.6 Locomotor Data Collection ................. ...............117...............
6.3 Results ................. ........... ...............117......
6.3.1 Dynamometry ................... ........... ...............117......
6.3.2 Voluntary Activation Defieits ................. ...............118...............
6.3.3 Locomotor Analyses ................. ...............118...............
6.4 Discussion ................ ...............118................












7 LOWER EXTREMITY SKELETAL MUSCLE MORPHOLOGY AND FIBER TYPE
COMPOSITION FOLLOWING MODERATE CONTUSION SPINAL CORD
INJURY AND LOCOMOTOR TRAINING ................. ...............125...............


7.1 Introduction ................. ...............125................
7.2 Methods............... ...............126
7.2.1 Anim als .............. .... ... ....... ...............126......
7.2.3 Locomotor Treadmill Training .............. ...............128....
7.2.4 Tissue Harvest ................. ...............128................
7.2.5 Immunohistochemical Measures............... ...............12
7.2.6 Data Analysis ................. ...............129...............
7.3 Results ................... ............. ...... .. ...... ...... ............13
7.3.1 Effects of Incomplete- SCI and Locomotor Training on Fiber
Crossectional Area (CSA) ............... .. ......... .. .. ....... .. ..........13
7.3.2 Effects of Incomplete- SCI and Locomotor Training on Fiber Type
Composition ................ ...............131....._._. .....
7.4 Discussion ................. ...............133..............


8 SKELETAL MUSCLE RECOVERY AND REGENERATION FOLLOWING
MODERATE CONTUSION SPINAL CORD INJURY AND LOCOMOTOR
TRAINING ................. ...............143................


8.1 Introduction ................. ...............143................
8.2 Methods............... ...............144
8.2.1 Anim als .............. .... ... ....... ...............144......
8.2.3 Locomotor Treadmill Training .............. ...............146....
8.2.4 Tissue Harvest ................... .. .. ....... ...... ...............146..
8.2.5 Determination of IGF-I Protein Concentration ......____ ..... .....__..........147
8.2.6 Immunohistochemistry Measurements .............. ...............147....
8.2.7 W western Blot Analysis .............. ...............148....
8.2.8 Data Analysis ................. ...............148...............
8.3 Results .................. ........... .... .... .... ..... .............4
8.3.1 Effects of Incomplete Spinal cord Injury and Locomotor Training on
Insulin-Like Growth Factor-1 (IGF-1) Expression ................. ................ ...149
8.3.2 Effects of Incomplete Spinal Cord Injury and Locomotor Training on Pax-
7................ ..... ..... .. .. .... ......... 4
8.3.3 Effects of Incomplete Spinal Cord Injury and Locomotor Training on
Myogenic Regulatory Factors (MyoD, Myf5 and Myogenin) ................... ..150
8.3.4 Effects of Incomplete Spinal Cord Injury and Locomotor Training on
Embryonic Myosin ................. ...............151.....__ .....
8.4 Discussion ............ _...... ._ ...............151...


LI ST OF REFERENCE S ............_ .......__ ...............163..


BIOGRAPHICAL SKETCH ............_...... .__ ...............182...










LIST OF TABLES


Table page

4-1 Characteristics of incomplete SCI subj ects ................. ...............88........... ..

4-2 Electrically elicited contractile measurements............... ..............8

5-1 Characteristics of incomplete SCI subj ects ................. ...............105........... ..

5-2 Values of isometric peak torque and average rate of force development ................... .....106

6-1 Pre- and post-RPT isometric torque data for the plantar flexor and knee extensor
muscle groups. ............. ...............123....











LIST OF FIGURES


Figure page

1-1 Etiology of SCI since 2000. ............. ...............56.....

1-2 Estimated lifetime costs by age at injury. ............. ...............57.....

1-3 Satellite cell number in skeletal muscle of different ages and type ................. ...............57

1-4 Satellite cell activity............... ...............58

1-5 S chemati c outline of a stem cell passing through the stages of muscle regenerati on........5 8

3-1 Set-up for locomotor training............... ...............74

3-2 Plyometric training set-up............... ...............74.

3-3 Experimental set-up on a Biodex system 3 dynamometer. ............. .....................7

3-4 Contusion injury set-up. ............. ...............75.....

3-5 anti-1VHC antibodies............... ...............7

3-6 Locomotor training in the rat model. ............ ...............76.....

4-1 Representative torque-time curve. ............. ...............89.....

4-2 Peak torque (Nm) for the knee extensor and plantar flexor muscle group s................... ....90

4-3 Torque200 (Nm) for the knee extensor and plantar flexor muscle groups ................... .......91

4-4 Average rate of torque development (ARTD)(Nm/sec) for the knee extensor and
plantar flexor muscle groups ................. ...............92................

4-5 Voluntary activation deficits (%) for the knee extensor and plantar flexor muscle
groups ................. ...............93.................

4-6 Torque trace acquired during MVIC with interpolated twitch. ............. .....................9

5-1 Torque200 (Nm) measured in the knee extensor muscle group. ............ ...................107

5-1 Torque200 (Nm) measured in the plantar flexor muscle group ................. ................. 108

5-2 Voluntary activation deficits (%) measured in the knee extensor muscle group.............1 09

5-2 Voluntary activation deficits (%) measured in the plantar flexor muscle group. ............110

6-1 Example of plyometric training device ................. ...............124..............











7-1 Average soleus muscle fiber CSA. ............. ...............139....

7-2 Average EDL muscle fiber CSA ................. ...............139........... ..

7-3 Average gastrocnemius muscle fiber CSA. ............. ...............140....

7-4 Average TA muscle fiber CSA ................. ...............140..............

7-5 MHC based fiber type composition of rat soleus.. ......___ ...... __ .......... ......141

7-6 MHC based fiber type composition of rat TA. ............ ....__ ........_........141

7-7 MHC based fiber type composition of rat EDL. ....__.................. ........._._......14

7-8 MHC based fiber type composition of rat gastrocnemius. ............ ...._.._..........142

8-1 Pax-7 positive staining. ............. ...............157....

8-2 MyoD protein levels. ............. ...............158....

8-3 M yf5 protein levels. .............. ...............159....

8-4 Myogenin protein levels. ............. ...............160....

8-5 Embryonic myosin positives ................. ...............161...............

8-6 IGF-1 levels. ............ .............162......









Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy

SKELETAL MUSCLE ADAPTATIONS FOLLOWING INCOMPLETE
SPINAL CORD INJURY AND EXERCISE TRAINING


By

Arun Jayaraman

May 2008

Chair: Krista Vandenborne
Major: Rehabilitation Science

Recovery of function after incomplete spinal cord injury (incomplete-SCI) is in an exciting

phase of research. Paralysis and paresis of lower extremity muscles following incomplete-SCI

result in persistent motor dysfunction and impaired walking. Advances in research have led to

promising exercise-training strategies in both humans and animals following SCI. However, the

mechanisms that explain the functional improvements reported following incomplete- SCI and

exercise training are not clearly understood and could possibly result from musculoskeletal

changes, neural adaptations, or a combination thereof. The primary purpose of this dissertation

was to explore the adaptations in lower extremity skeletal muscle following incomplete-SCI and

exercise training in both humans and animals. Ours findings indicate a significant loss of both

peak isometric and explosive strength in lower extremities after incomplete-SCI in humans.

Additionally, this loss in strength was attributed to a severe loss in voluntary activation of the

paretic muscles. Locomotor training and resistance training were two exercise interventions that

were tested in our study, and our findings suggest that both locomotor training and resistance

training helped in significantly improving both voluntary and explosive strength, and voluntary

activation in the lower extremity muscles of persons with incomplete-SCI. In the rat model,









incomplete-SCI resulted in significant atrophy in all four lower extremity muscles. In addition,

SCI resulted in a shift in fiber type composition measured using myosin heavy chain (MHC)

composition towards faster isoforms in all four lower extremity muscles. Locomotor training in

the rats resulted in significantly reducing the atrophy in all lower extremity muscles. In addition,

there was also a significant shift in fiber types in all hind limb muscles towards slower isoforms.

In addition, our results indicate that recovery in muscle size following SCI and locomotor

training was due to the activation of satellite cells which went to form multinucleated myotubes

which repaired or replaced damaged or lost muscle fibers. The overall findings from the present

work will provide essential feedback on deficits in muscle function following SCI and also

effects of exercise training interventions towards reducing the musculoskeletal deficits and

promoting muscle plasticity following incomplete- SCI. These findings might provide feedback

for the development and integration of these exercise interventions into the community.









CHAPTER 1
BACKGROUND

1.1 Introduction

Spinal cord injury (SCI) causes a host of physical and psychosocial problems that

interferes with an individual's personal health, feeling of well being and societal interaction. The

goal of rehabilitation after spinal cord injury is to enable the person to resume a life style which

is physically and functionally healthy and also helps the person integrate with his family,

community and society. The central role of rehabilitation requires a comprehensive

understanding of all different physiological and functional adaptations that occur with SCI. The

main focus of this dissertation is to identify adaptations in muscle function after SCI and its

response to different exercise interventions. Background literature pertaining to all chapters is

briefly discussed in chapter-1.

1.2 Demographics of Spinal Cord Injury (SCI)

It is estimated that the annual incidence of spinal cord injury (SCI), is approximately 40

cases per million population in the U. S. or approximately 11,000 new cases each year.' The

number of people in the United States who are alive as of June 2006 who have had a SCI has

been estimated to be approximately 253,000, with a range of 225,000 to 296,000 persons.l

SCI primarily affects young adults. From 1973 to 1979, the average age at injury was 28.7

years, and most injuries occurred between the ages of 16 and 30. Since 2000, the average age at

injury is 3 8.0 years since the median age of the general population of the United States has

increased by approximately 8 years since the mid-1970s, the average age at injury has also

steadily increased over time. Moreover, the percentage of persons older than 60 years of age who

had a SCI has increased from 4.7% prior to 1980 to 11.5% among injuries occurring since 2000.

1Prior to 1980, 81.8% of new spinal cord injuries occurred among males. Since 2000, 77.8% of









SCI reported to the national database have occurred among males. Over the history of the

database, there has been a slight trend toward a decreasing percentage of males sustaining SCI.I

Among those injured since 2000, 63.0% are Caucasian, 22.7% are African American, 11.8% are

Hispanic, and 2.4% are from other racial/ethnic groups.1, 2

Looking at the etiology of SCI, it is plausible that this injury by itself causes dramatic changes

in one' s lifestyle and occupational status. However, by ten years post-injury, 32.4% of persons

with paraplegia become employed, while only 24.2% of those with tetraplegia are employed

during the same year.1,2 The average yearly health care and living expenses and the estimated

lifetime costs that are directly attributable to SCI vary greatly according to severity of injury



The last section of the facts following SCI pertains to the causes of the death after SCI and

level and extent of the lesion. The most common cause of death in persons with SCI is

respiratory ailment, whereas, in the past it was renal failure. An increasing number of people

with SCI are dying of unrelated causes such as cancer or cardiovascular disease, similar to that of

the general population. Mortality rates are significantly higher during the first year after injury

than during subsequent years. Since 2000, the most frequent neurological category is incomplete

tetraplegia (34. 1%) and incomplete paraplegia (23.1%), followed by complete paraplegia

(23.0%), and complete tetraplegia (18.3%). This makes the incidence of incomplete injuries

equals to ~57% of total SCI as of 2000. Over the past few years, the percentage of persons with

incomplete tetraplegia has increased slightly while both complete paraplegia and complete

tetraplegia have decreased slightly (Fig. 1-2).1, 2









In Summary, in this section the statistics and demographics pertaining to SCI were briefly

described. A summary of the etiology, age, gender, causes of death and common types of SCI

were discussed.

1.3 SCI in Humans-Pathophysiology and Classification

The information in this section mainly pertains to the medical definition and classification

of SCI. This includes the anatomy, physiology, diagnosis and classification of SCI. This

information will help us better understand the diagnosis, extent of injury and recovery levels of

the subj ects with SCI described in following chapters.

1.3.1 Definition of SCI

SCI can be categorized into traumatic or non-traumatic injuries. The spinal cord is often

violently displaced or compressed momentarily during the injury with forceful flexion,

extension, and rotation of the spine. The vertebral body can burst and cause pressure or scatter

bone fragments into the spinal cord. SCIs are classified as concussion, contusion, laceration, or

transaction. A concussion is an injury caused by a blow or violent shake and results in temporary

loss of function.2,3 In COntusion injury, the glial tissue and spinal cord surface remain intact.

There is loss of central gray matter and white matter, which creates a cavity that, is surrounded

by a rim of intact white matter at the periphery of the spinal cord.3 Laceration of the cord occurs

with more severe injuries in which the glia is disrupted, and the spinal cord tissue may get torn.

Occasionally this can result in complete dissection of the spinal cord known as transaction

injuries. 3 Gun shot wounds, knife wounds, and puncture wounds fall into this category.

Hemorrhages caused by contusion or laceration injuries can cause further compression of the

cord.2,3

SCIs can also be classified as primary or secondary based on the modus of injury. Primary

SCIs arise from mechanical disruption, transaction, or distraction of neural elements. This injury









usually occurs with fracture and/or dislocation of the spine. However, primary SCI may occur in

the absence of spinal fracture or dislocation. Penetrating injuries due to bullets or weapons may

also cause primary SCI. More commonly, displaced bony fragments cause penetrating spinal

cord and/or segmental spinal nerve injuries. Extradural pathology may also cause primary SCI.

Spinal epidural hematomas or abscesses cause acute cord compression and injury. Spinal cord

compression from metastatic disease is a common oncologic emergency. Longitudinal

distraction with or without flexion and/or extension of the vertebral column may result in

primary SCI without spinal fracture or dislocation.1-3

Maj or causes of secondary SCI are vascular injury to the spinal cord caused by arterial

disruption, arterial thrombosis, and hypoperfusion due to shock. Anoxic or hypoxic effects

compound the extent of secondary SCI. In summary SCI can vary in nature, hence the disability

associated with it is also extremely varied based on the type, level, and extent of injury.

1.3.2 Spinal Cord Neuro-anatomy

The spinal cord is divided into 31 segments, each with a pair of anterior (motor) and dorsal

(sensory) spinal nerve roots. On each side, the anterior and dorsal nerve roots combine to form

the spinal nerve as it exits from the vertebral column through the neuro-foramina. The spinal

cord extends from the base of the skull and terminates near the lower margin of the L1 vertebral

body. Thereafter, the spinal canal contains the lumbar, sacral, and coccygeal spinal nerves that

comprise the cauda equina.4 Therefore, injuries below L1 are not considered SCIs because they

involve the segmental spinal nerves and/or cauda equina. Spinal injuries proximal to L1, above

the termination of the spinal cord, often involve a combination of spinal cord lesions and

segmental root or spinal nerve injuries.l~

Spinal tracts: The spinal cord itself is organized into a series of tracts or neuro-pathways that

carry motor (descending) and sensory (ascending) information. These tracts are organized









anatomically within the spinal cord. The corticospinal tracts are descending motor pathways

located anteriorly within the spinal cord. Axons extend from the cerebral cortex in the brain as

far as the corresponding segment, where they form synapses with motor neurons in the anterior

(ventral) horn. They decussate (cross over) in the medulla prior to entering the spinal cord. The

dorsal columns are ascending sensory tracts that transmit light touch, proprioception, and

vibration information to the sensory cortex. They do not decussate until they reach the medulla.

The lateral spinothalamic tracts transmit pain and temperature sensation. These tracts usually

decussate within 3 segments of their origin as they ascend. The anterior spinothalamic tract

transmits light touch. Autonomic function traverses within the anterior interomedial tract.

Sympathetic nervous system fibers exit the spinal cord between C7 and L1, while

parasympathetic system pathways exit between S2 and S4.4

1.3.3 SCI Pathophysiology

Trauma to the spinal cord results in primary destruction of neurons at the level of the

injury by disruption of the membrane, hemorrhage, and vascular damage. Secondary neural

damage to the spinal cord extends beyond the initial contusion. The spread of the damage is

thought to be due to the activation of biochemical events leading to necrosis and excitotoxic

damage and can continue for hours, days or weeks. Injury to the corticospinal tract or dorsal

columns, respectively, results in ipsilateral paralysis or loss of sensation of light touch,

proprioception, and vibration.1- Unlike injuries of the other tracts, injury to the lateral

spinothalamic tract causes contralateral loss of pain and temperature sensation. Because the

anterior spinothalamic tract also transmits light touch information, injury to the dorsal columns

may result in complete loss of vibration sensation and proprioception but only partial losses of

light touch sensation. Anterior cord injury causes paralysis and incomplete loss of light touch

sensation. 5









Autonomic function is transmitted in the anterior interomedial tract. The sympathetic

nervous system fibers exit from the spinal cord between C7 and Ll. The parasympathetic system

nerves exit between S2 and S4. Therefore progressively higher spinal cord lesions or injury

causes increasing degrees of autonomic dysfunction.

1.3.4 Classification of SCI

ASIA Impairment Scale: Clinicians have long used a clinical scale to grade severity of

neurological loss. First devised at Stokes Manville before World War II and popularized by

Frankel in the 1970's, the original scoring approach segregated patients into five categories, i.e.

no function (A), sensory only (B), some sensory and motor preservation (C), useful motor

function (D), and normal (E). The ASIA Impairment Scale is follows the Frankel scale but

differs from the older scale in several important respects.

The mechanism of the injury influences the type and degree of spinal cord lesion. The SCI

injuries are often classified as complete and incomplete. The difference between a complete and

incomplete injury depends on the survival of a small fractions of axons in the spinal cord.

According to the American Spinal Injury Association (ASIA), a person is a "complete" if they do

not have motor and sensory function in the anal and perineal region representing the lowest

sacral cord (S4-S5).

ASIA A is defined as a person with no motor or sensory function preserved in the sacral

segments S4-S5. This definition is clear and unambiguous. ASIA B is essentially is the

preservation of sacral S4-S5 function. It should be noted that ASIA A and B classification

depend entirely on a single observation, i.e. the preservation of motor and sensory function of

S4-5.A patient would be an ASIA C if more than half of the muscles evaluated had a grade of

less than 3/5 on a manual muscle test. If not, the person was assigned to ASIA D. ASIA E is of

interest because it implies that somebody can have spinal cord injury without having any









neurological deficits at least detectable on a neurological examination of this type. Also, the

ASIA motor and sensory scoring may not be sensitive to subtle weakness, presence of spasticity,

pain, and certain forms of dyesthesia that could be a result of spinal cord injury. Note that such a

person would be categorized as an ASIA E.

The ASIA committee has identified Hyve types of inzcomplete spinal cord injury syndromes.

A central cord syndrome is associated with greater loss of upper limb function compared to the

lower limbs. The Brown-Sequard syndrome results from a hemisection lesion of the spinal cord.

Anterior cord syndrome occurs when the injury affects the anterior spinal tracts, including the

vestibulospinal tract. Conus medullaris and cauda equina syndromes occur with damage to the

conus or spinal roots of the cord. Measures of ambulatory function are another commonly used

method to classify people with SCI. Information can be obtained from the following references.6-



In summary, in this section we briefly discussed the definition of the different types of

SCI. This was followed by a summary of the neuroanatomy and physiology of SCI. Finally we

summarized the most common classification of SCI; the ASIA scale.

1.4 SCI in the Animal Model

To obtain the necessary experimental evidence to begin clinical trials, compelling evidence

for benefit must be demonstrated in reproducible animal models of SCI. Although no single

experimental SCI animal model exactly mimics the clinical condition, animal models allow for

the rigorous study of the pathophysiology and mechanism of injury and recovery.

Appropriate cat and rodent models that are being currently investigated include the

compression, hemisection, transaction, isolation, and contusion.12-14 With each model, injury

severity and areas of damage to the spinal cord can be varied so that a spectrum of

histopathological, behavior and functional deficits can be reproduced.12-16 Rat models of SCI are









the most commonly studied because of their low cost, size factor, ease in handling and care, and

well-established SCI methods.17-2 Recently, mouse models of SCI have been developed. These

models give us the ability to enhance or delete specific genes by transgenic mechanisms. Non-

human primate models of SCI are also important in testing experimental therapeutic strategies. In

addition to various neuroanatomical considerations, the primate spinal cord more closely

resembles that of a human spinal cord and this becomes important when therapeutic and

pharmaceutical interventions are focused directly towards the injured spinal cord.11-20

1.4.1 Spinal Cord Isolation Model

Spinal cord isolation referred to as the classic silent preparation was attempted in dogs by

Tower in 1937.4 In this model; the lumbar region of the spinal cord is functionally isolated via

complete spinal cord transactions at two levels and bilateral dorsal rhizotomy between the two

transaction sites. This model eliminates supraspinal, infraspinal, and peripheral afferent input to

motoneurons located in the isolated cord segments while leaving the motoneuron skeletal muscle

fiber connections intact. Electromyographic recordings (EMG) and/or reflex testing after spinal

isolation have shown the hindlimb muscles to be virtually silent for prolonged periods.21-23

1.4.2 Spinal Cord Transection Model

In the transaction spinal cord injury model, the transmission of descending and ascending

information between the caudal cord and the brain is mechanically eliminated. In this model, SCI

is created by an incision into the spinal cord is completely transected. Following transaction

injury, there is an initial flaccid paraplegia stage in which the limbs of the animals are totally

paralyzed.12 The animals are only able to move using their forelimbs to reach for food and water.

At approximately 3 to 4 weeks following SCI, the paralyzed hind limbs of the animals change

from flaccid to spastic. After spasticity develops, the limbs are almost always held in extension

and no recovery of voluntary activity is observed. There exists a 75% decrease in the total









integrated EMG and a 66% decrease in the total duration of muscle activity in the soleus muscle,

5 to 6 months after transaction when compared to normal controls.24 Thus, in the spinal

transaction model hind limb muscles experience a significant reduction in both electrical

activation and loading. The complete transaction model has been used extensively to evaluate the

effectiveness of interventions with regard to both axonal regeneration and functional recovery.

The advantage of this model is a relative stabilization of pathological changes and subsequent

neurological outcomes.24-26Therefore, the effectiveness of particular strategies can be readily

assessed. Models in which the spinal cord is fully transected ensure the absolute completeness of

the injury, making it somewhat easier to evaluate the effectiveness of interventions with regard

to both axonal regeneration and functional recovery. The implication in studies using transaction

models is that with the ensured completeness of the lesion, anterogradely labeled axons observed

distal to the lesion have indeed regenerated from above and are responsible for the functional

recovery of the animal. While this is largely accepted, and hence remains the main advantage of

full transaction models, there is a mounting body of literature from animal studies that describes

considerable native locomotive abilities of the completely transected spinal cord (the so-called

"spinalized" animal).24-26 However, the transected spinal cord model also has some

disadvantages. First, due to the natural tension present in spinal cord, the two ends of a cut cord

will separate. Such a gap is rarely present in human SCI. In addition, in order to cut the spinal

cord, the dura has to be opened, allowing invasion by external cells leading to higher chances of

infection.""

1.4.3 Spinal Cord Hemisection Model

In hemisection models, an attempt is made to cut tracts of the spinal cord selectively.

Depending on the severity of the lesion, the resulting neurologic deficit can be relatively mild,

thus making the postoperative animal care fairly easy, particularly with regard to bladder









function.27 Hemisection models also may allow for comparison of the regenerative response in a

particular tract with its uninjured partner on the contralateral side. The rat rubrospinal system is a

useful model in this regard because the tract emerges from the red nucleus in the brain stem,

crosses over nearly completely, and descends in the dorsolateral aspect of the spinal cord, where

its lateral position makes it relatively easy to cut in a unilateral fashion while leaving the

contralateral tract uninjured. In the rat, the rubrospinal system is thought to be important for the

control of skilled limb movement, particularly of the forelimbs.18,27,28 Most of the corticospinal

tract in rats descends in the ventral aspect of the dorsal columns, just dorsally to the central

canal. In dorsal hemisection models, the lesion transects the rubrospinal and corticospinal tracts

bilaterally. In general, partial transaction models inherently raise the possibility that axons of the

particular tract in question might have escaped injury. Retrograde tracers are useful in identifying

such spared axons.27,28,18 If a tracer is applied distally to the site of partial injury, its histologic

appearance proximally in the cell body of a neuron implies that this neuron's axon was not cut

during the injury. Conversely, the absence of tracer confirms the injury's completeness. Partial

injury models also suffer from difficulties determining whether observed functional

improvement is due to true regeneration of the injured tract or to functional compensation from

other systems that are spared.27, 28, 18

Most hemisection injuries are performed on the cervical spinal cord, interrupting the

descending respiratory pathways and causing respiratory muscle paresis or paralysis. Thus, this

model has long been used to understand the mechanisms related to plasticity and recovery of the

respiratory pathways after spinal cord injury. Unfortunately, a limitation of partial injury models

is the difficulty in determining whether observed functional improvement is due to true

regeneration of the injured tract or to functional compensation from other systems that are









spared. This is one the reasons this model is not commonly used to study adaptations of the

locomotor muscles of the hind limb.

1.4.4 Spinal Cord Contusion Model

In 1911, Reginald Allen described a spinal cord injury model where he dropped a weight

onto the spinal cords of dogs exposed by laminectomy. In 1914, he reported that midline

myelotomy reduced progressive tissue damage in the contused spinal cord.29 Unfortunately,

Allen died in World War I and his work was discontinued for nearly 50 years. In 1968, Albin and

colleagues revived the contusion model when they used a primate spinal cord contusion model to

assess the efficacy of hypothermic therapy following SCI.29 After that, several investigators

started using the canine spinal cord contusion model again. Parker and colleagues assessed the

effects of dexamethasone and chlorpromazine on edema in contused dog spinal cords. At the

same time, Koozekanani and colleagues examined the causes of variability in this model, while

Collmans and others measured edema, blood flow and histopathological changes in the contused

dog spinal cord.30

Beginning with a crude weight drop model by Reginald Allen in 1911, many models in

various animals have been developed to deliver a blunt contusive force to the spinal cord, which

is more representative of what occurs in most human injuries.31 Two important aspects of human

injury warrant discussion because they are particularly relevant to injury models. The first is

observed evolution of neuropathology over time, beginning with an early phase of spreading

hemorrhagic necrosis and edema, progressing to an intermediate phase of partial repair and tissue

reorganization, and reaching a chronic phase characterized by the establishment of central cystic

cavities within atrophic parenchyma and glial scar.30-36 This temporal pattern of injury

maturation appears to be reasonably well simulated in the spinal cords of animals after a

contusion injury, thereby providing a setting for evaluating neuroprotective strategies in the









acute phase of injury.30-36 The second important observation in humans is that even in the setting

of complete paraplegia after blunt injury; the spinal cord rarely is completely transected, but

rather leaves some residual, normal-appearing cord parenchyma peripherally at the injury zone.

Contusion injury models produce a similar lesion, in which neuronal tissue remains intact along

a peripheral rim, the quantity of which is correlated with residual locomotor function.30-36

In general, contusion injury models appear to induce reproducible and consistent

neurologic injuries, thereby providing a good setting for the functional and histologic evaluation

of SCI and new treatment interventions. However, due to the incomplete nature of injury and the

complexity of the tracts, it is very difficult to verify exact changes in pathophysiology in these

models.

Common devices used to create contusion injuries: The Georgetown University device

Wrathall, is a free falling weight down a guide tube onto a footplate resting on the cord. The

NYU or MASCIS device was developed at the NYU Neurosurgery Laboratory and first

described by Gruner in 1992. In this model, a 10-g rod is dropped from different heights onto the

exposed dorsal surface of the spinal cord producing more severe neurologic injuries with

increasing height. The ESCID device (Ohio State University device displacement driven) is

somewhat different rat cord contusion model that use a computer feedback-controlled

electromechanical impactor rather than a weight drop. The Infinite Horizon device (University of

Kentucky device-force driven) is an instrument that enables the application of standard-force

injuries to the spinal cords of mice and rats. Force levels are user-selectable between 30 and 200

kDynes. A "clip compression" model of spinal cord injury in rats was introduced by Rivlin and

Tator in 1978, in which the spinal cord was compressed for variable durations between the arms

of a modified aneurysmal clip. The devices commonly used currently are the NYU impactor and









the Infinite Horizon impactor device. Overall, all these devices provide consistent, reliable spinal

cord injuries. However, based on the experimental requirement or type of injury, one device

might be more suitable than the other.

To summarize, in this section the different types of SCI in animals was briefly described

with emphasis given to the contusion SCI which is the model of SCI pertaining to this

dissertation. In the last section we saw the different types of injury devices pertaining to the

contusion injury. In the following sections the NYU impactor device will be used extensively to

cause moderate contusion SCI in the rat model. The moderate contusion injury was used in all

the animal experiments as it closely resembles the histopathologic sequela and mechanism of an

incomplete SCI in the humans, helping us to relate our animal experiments to our human studies.

1.5 Skeletal Muscle Adaptations in Human Following SCI

1.5.1 Muscle Size

Numerous studies have been conducted to study muscle atrophy after SCI.S Of the various

techniques used to measure muscle size, measures of whole muscle cross-sectional area (CSA)

have been identified to be the most accurate and reliable.37 Initially, muscle CSA was calculated

either by measuring the limb girth by a tape measure or by in-vitro measurements such as fiber

CSA. Gregory et al. 2003, quantified both human and rat fiber CSA after 11 weeks-SCI. Both

the rat and human vastus lateralis muscle showed significant atrophy (~50%) with chronic SCI.

17Adams et al. 2006 and Stewart et al. 2004 reported significant atrophy in the vastus lateralis

muscle using muscle fiber size measures following chronic SCI.38,39

Recently, muscle crossectional area (CSA) has been extensively measured by means of

Magnetic Resonance Imaging (MRI) and other non-invasive measuring tools like

ultrasonography and computed tomography.14,16,18,40-47 Not only is MRI non-invasive, it is

without harmful radiation, and has a unique ability to visualize non-muscle tissue like fat,









connective tissue and bone. It has greater tissue sensitivity and contrast resolution with multi-

planar and 3D capabilities than ultrasonography and CT. 40-47 Moreover, MRI has the advantage

of visualizing the entire length of a muscle compared to a biopsy or ultrasound. Numerous

studies have utilized MRI to study CSA after SCI in both animals and humans. Castro et al. 1999

used MRI to show that the average maximal CSA of gastrocnemius and soleus decreased by 24%

and 12% within six months of SCI, while the tibialis anterior CSA showed no change.47 The

average CSA of the quadriceps femoris, the hamstring muscle group and the adductor muscle

group decreased by 16%, 14% and 16%, respectively. 47 The average CSA of atrophied skeletal

muscle in the patients was 45-80% of that of age- and weight-matched able-bodied controls 24

weeks after the injury. The incomplete-SCI model in humans also showed significant skeletal

muscle atrophy measured using MRI.37 Individuals with chronic incomplete-SCI showed a

~28%-33% change in their muscle size as compared to able bodied controls. Maximum

difference was seen in the plantarfiexor muscles (32%) followed by knee extensors (31%),

dorsiflexors (28%) and the knee flexors (22%).37 Skeletal muscle atrophy following SCI is a

result of injury to motor neurons in the spinal cord and concurrent inactivation of affected

skeletal muscle along with subsequent changes in muscle length and mechanical loading

conditions. Fractional presence of neural inputs to the muscle allows for variable activation of

lower limb musculature after an incomplete-SCI, thus resulting in more modest atrophy in this

population after injury and also better changes for positive prognosis compared complete-SCI
37-47
group.

1.5.2 Fiber Type Composition

The type of MHC expressed in human skeletal muscle also determines the characteristics

of the muscle. Generally, muscle fibers in humans do not express more than one distinct MHC

type.' However; the atrophic response in skeletal muscle following spinal cord injury









demonstrates a number of hybrid fibers co-expressing different MHC types. In general, MHC

type transforms towards a faster type by the first year of injury with significant increases in

MHC-IIx. Histochemical fiber-typing studies also support the fact that there are dramatic

increases in faster (type II) fibers after SCI. Talmadge et al. (2002), measured the effects of SCI

on the expression of sarcoplasmic reticulum, calcium-ATPase (SERCA) and MHC isoforms in

the vastus lateralis (VL) muscle.48 SCI resulted in significant increases in fibers with MHC IIx

with~-14% and~-16% increases at six weeks and 24 weeks after SCI. 48 In addition, SCI resulted

in high proportions of MHC I and MHC IIa fibers with both SERCA isoforms (~29% and ~16%

at six weeks and ~54% and ~28% at 24 weeks for MHC I and MHC-IIa fibers respectively).48

The appearance of faster isoforms of MHC after SCI suggests that the muscle will have faster

contractile properties, ultimately making it highly fatigable. These changes seen in the muscle

are anticipated to contribute towards the functional limitations observed in this patient

population.4

Studies have used different muscle fiber classification schemes such as myofibrillar

ATPase activity, and activity (or concentration) of specific enzymes including succinate

dehydrogenase (SDH), and alpha-glycerol-phosphate dehydrogenase (GPDH), to identify and

quantify skeletal muscle adaptations after SCI.S1 These measurement techniques visualize the

activity of enzymes which are specific to each fiber type. When the enzyme reacts with an

energy source a reactive product is formed. Thus the product from the assay is used to determine

if the muscle fibers are fast or slow (mATPase), oxidative or non-oxidative (SDH), or generate

ATP aerobically or anaerobically (GPDH). "1 Gregory et al. (2003), quantified both human and

rat VL fiber adaptations 11 weeks following SCI. The VL was sectioned and fibers were

analyzed for type (I, IIa, IIb/x), SDH, GPDH, and actomyosin adenosine triphosphatase









(qATPase) activities." The IIa to IIB shift was the maj or phenotypic adaptation that occurred in

VL after SCI in both humans and rats. Rat fibers had 1.5- to 2-fold greater SDH and GPDH

activity compared to humans." The most striking differences, however, were the absence of slow

fibers in the rat and its four-fold greater proportion of IIb/x fibers compared to humans which

could be viewed as the rat' s ability to counter the greater decline in SDH activity with regard to

resistance to fatigue. SCI decreased SDH activity more in rats whereas IIa to IIb/x fiber shift

occurred to a greater extent in humans." Thus fiber type adaptations are species specific and

each species has their own mechanism of countering an insult to its neuromuscular framework.

1.5.3 Electrically Elicited Contractile Properties

Contractile properties in the humans have been studied in muscles, like the quadriceps and

soleus muscles after SCI. 42,49-53 Gerrits et al. 1999 indicated that muscles after SCI demonstrated

faster rates of contraction and relaxation than normal control muscles and also had extremely

large force oscillation amplitudes at the 10-Hz signal frequency (~65 % in SCI versus ~23% in

controls).5o~51 In addition, force loss and slowing of relaxation following repeated fatiguing

contractions were greater in SCI muscles compared with controls. The faster contractile

properties and greater fatigability of the SCI muscles are in agreement with a characteristic

predominance of fast glycolytic muscle fibers.5o~51 Within the SCI population, the chronically

paralyzed soleus on average has a 20ms shorter time to peak twitch torque and a 25% shorter

twitch half-relaxation time when compared to individuals with acute paralysis. This indicates

that the muscle functioning gets faster with the progression of the diseased state. Fast fatigable

motor units show progressive slowing during fatigue induced by repetitive activation.52

Consistent with properties of faster muscle and motor units, the soleus after SCI demonstrates a

near doubling of the half-relaxation time during fatigue.52 This indicates that as the muscle

fatigues, the calcium uptake becomes compromised or the cross-bridge cycling rate is impaired.









Normally, as the frequency of an electrical stimulus increases, muscle contraction becomes

progressively more fused and the muscle generates greater torque. A muscle that has a slower

contractile speed will fuse at a lower frequency when compared to a faster contractile speed. 52

The torque-frequency curve for a slow muscle will be shifted to the left of the torque-frequency

curve of a fast muscle. Thus a torque-frequency curve for a muscle after SCI is shifted to the

right of the torque-frequency curve for a normal muscle. Another measure that is specific to

adaptations after SCI is low-frequency fatigue, which refers to repetitive activation of a

chronically paralyzed muscle at low frequencies. 52 The preferential loss of force at low

frequency can be recovered at higher frequencies. Impairments in excitation-contraction

coupling (E-C coupling) is associated with low-frequency fatigue and likely represents an

internal safety mechanism in skeletal muscle to prevent ATP depletion.52 Low-frequency fatigue

is characterized by being delayed in onset as well as being long lasting. This type of fatigue is

found to be most prominent in fast-intermediate or fast fatigable motor units.52

1.5.4 Voluntary Contractile Measurements

Paralysis of the voluntary musculature is the most obvious effect of SCI in humans.

Damage to the descending motor tracts, anterior horn cells, and/or nerve roots leads to an

impaired capacity to voluntarily contract the skeletal muscles innervated at or below the level of

the lesion.49-5 In patients with SCI, the maximal voluntary contractions of the affected muscles

are extremely weak compared to the range of absolute forces typically produced by non-injured

individuals .49-52This may relate to reduced voluntary activation of the muscle, failure of

neuromuscular transmission, problems within the muscle itself, or some combination of these

possibilities. For example, if voluntary drive does not recruit all of the motoneurons that supply a

muscle, the voluntary force produced will be reduced. Failure to activate each motor unit at its

maximal firing frequency will also reduce force production. Similarly, the force contributed by









each motor unit will be lower if fiber size decreases (muscle fiber atrophy) from altered use of

muscle. The sensory deficits that accompany these injuries may also exacerbate the ability of

subjects to contract their muscles maximally.4-5

Few studies have measured voluntary muscle strength obj ectively after human SCI 54-56 Of

have delineated the factors that contribute to the weakness. The manual muscle test (MMT) has

been used to measure strength historically in the field of physical therapy. The face and content

validity of MMT in SCI is high, however manual muscle tests are subj ect to a ceiling effect, lack

sensitivity to change and have a relatively poor inter-rater reliability, especially at scores greater

than 3.54-56 Studies have compared different methods to assess strength after SCI (the manual

muscle test (MMT), the hand-held myometry and isokinetic dynamometry (Cybex, KinCom,

Biodex). These studies suggest that the MMT method does not seem to be sufficiently sensitive

to assess muscle strength, at least for grade 3 and higher and to detect small or moderate

increases of strength over the course of rehabilitation. Further, it has been concluded that

myometry and dynamometry measurements detect increases in strength over time, which are not

reflected by changes in MMT scores." Thus, dynamometry is currently considered a more

sensitive measure of voluntary strength in human SCI population.

In summary, following SCI, there is significant atrophy quantified using muscle and fiber

crossectional, a slow to fast muscle fiber transformation. Changes in contractile properties are

more dramatic in fibers which have a larger proportion of slow fibers. There exists a reduction is

muscle strength and voluntary muscle control. However, depending on the type of injury being

incomplete or complete, the neuromuscular architecture and function are not necessarily

compromised.









1.6 Skeletal Muscle Adaptations in the Animal Models Following SCI

A decrease in neuromuscular activity as a result of spinal cord injury (SCI) results in

significant changes in morphological, mechanical and metabolic properties of skeletal muscles

below the level of injury. However, the relationship between the injury and the muscle

adaptations is confounded by the variability among injuries, and the type of injury. Below is a

description of various adaptations that occur in skeletal muscle following SCI.

1.6.1 Muscle Size

This section covers atrophy measured using muscle wet weight and fiber size. Reduced

muscle activity and loading or inactivity results in a significant reduction of skeletal muscle mass

and muscle fiber size following SCI.26,57 Specifically, muscle atrophy is more pronounced in

single joint muscles which are involved in weight bearing and postural control.26, 57 For example,

the soleus muscle, a postural muscle crossing over the ankle joint, undergoes significant muscle

atrophy following SCI.5,26 In COntrast, the TA or EDL are known to show relatively less atrophy

compared to the soleus. The medial gastrocnemius muscle, which crosses both the knee and

ankle joints, also undergoes less atrophy than the soleus muscle even though it serves as a

synergist to the soleus muscle during plantar flexion.5,26 Degree of atrophy is fiber-type specific,

with the slow twitch muscles being more affected than the fast twitch muscles, and extensors

atrophy more than flexors. For example, following spinal transaction injury in adult cats, the

morphological adaptations in the medial gastrocnemius (slow muscle) are higher than that seen

in the tibialis anterior (fast muscle).5,26,58 Similar to fiber size, absolute wet weight also decreases

with SCI. Hutchinson et al. 2001 reported a 20-25% significant decrease in absolute wet weight

in the soleus, while there was a 6% decrease in EDL wet weight when compared to matched

controls.20 While it is clear that atrophied muscles produce less contractile force, there appears to









be dissociation between the percent loss of muscle mass and percent decline in contractile

tension indicating a loss in muscle specific force.20

1.6.2 Fiber Type Composition

Numerous methods have been used to understand the differences between fiber types. In

early 1800s fibers were grossly differentiated to red and white based on their appearance.' With

sophistication of experimental techniques different classification of fiber type have come to

existence. The histochemical assay of myofibrillar ATPase activity is one of the few

experimental techniques used to distinguish between fast and slow-contracting muscle fibers.'

Myosin ATPase activity is positively correlated with muscle contraction velocity. Basically, fast

contracting fibers hydrolyze ATP faster than slow-contracting fibers.' For example, cross-

sections of a normal soleus stained for myofibrillar ATPase show a composition with a minimal

number of fast fibers, where as a transaction SCI-soleus is composed entirely of fast fibers.' The

transaction SCI-soleus represents a dramatic slow to fast muscle fiber type transformation. In

addition, the average area of slow fibers in the soleus decreased by about 50% following SCI.

There were no changes in fiber area for the EDL after SCI.1 The soleus however generated the

same absolute force in spite of its smaller muscle fibers, indicating an increase in its specific

tension, and a significant conversion of its slow fibers to the fast type.5,24,59 So, the first

adaptation indicated after SCI is the reduction in fiber area and fiber type transformation from

slow to fast muscle. This methodology was however used starting about 20 years ago and now

more sensitive measures have been developed to substantiate this fiber type conversion after

SCI.24,59

Myosin, the molecular motor of the skeletal muscle, is a protein comprised of two myosin

heavy chains (MHC). The heavy chains determine the rate of cross-bridge reactions with actin

filaments and hence help determine the speed of muscle contraction.59 To date, four different









myosin heavy chain (MHC) isoforms have been identified in varying proportions in the hindlimb

muscles of rats. These have been identified as a slow isoform called MHC-I and three fast

isoforms called MHC-IIa, MHC-IIx, and MHC-IIb.59 A number of studies have closely linked

the MHC isoform composition of the individual muscle fibers with their velocities of unloaded

shortening, such that there is a gradation in the contractile speed of fibers containing a given

isoform in the order of (fastest to slowest) IIb > IIx > IIa > I. Antibodies specific to these

proteins identify fiber types based on these MHC's. Animal soleus muscles stained after

transaction SCI for MHC composition analysis indicate differences in the distributions of fiber

types with a greater percentage of hybrid muscle fibers which coexpress different MHCs in SCI

animals and a greater shift in MHC composition towards faster isoforms.60,61 The control normal

soleus primarily contains fibers reacting exclusively with type I myosin antibody (slow, 86.1 %)

and a small percentage of fibers reacting exclusively with type IIa myosin antibody (fast,

13.9%).60 One-week after SCI transaction (ST), the proportion of pure type I fibers decreased to

~75%.9 The remaining difference in the MHC composition in SCI animals was accounted for by

an increase in hybrid fibers, with ~15% of fibers reacting to I & IIa myosin antibody and ~10%

reacting to type IIa & IIx myosin antibody.60

Interestingly, the reduction in the proportion of fibers containing MHC-I after spinal

isolation (SI) is greater than that observed for spinal transaction. Talmadge et al. (1996) with

MHC-specific antibodies demonstrated that the soleus from control cats contained 99% type I,

1% IIa. Following ST 67% of the fibers were positive for type I, 17% IIa, 3% IIb, and 13%

hybrid fibers. After SI, 48% of the fibers were positive for type I, 1 1% were IIa, 1% was IIb,

25% were hybrid, and 15% contained embryonic MHC.62 Roy et al. (1999) also showed that cat

fast muscle tibialiss anterior) shows an ~4% increase in the fast fiber proportion and MHC-IIx









expression after 6 months of ST, while there is ~4% decrease in MHC-I fibers. Overall compared

to control values, the percent composition of MHCs in the TA was unaffected by ST with or

without training. Talmadge (1995) demonstrated that ST results in dramatic shifts in the

expression of MHC isoforms of the rat soleus (normally approx. 90% MHC-I, approx. 10%

MHC-IIa), such that 1 month after ST approx. 33% of the total MHC was MHC-IIx.48,59,61,62

Rodents show a higher degree of MHC isoform transformation after ST than cats. The

proportion of MHC-I in the rat soleus is reduced from ~90% in controls to ~25% only 3 months

following a complete mid-thoracic ST. The MHC-IIx, which is normally not found in the rat

soleus, increased to nearly 50% and that of MHC-IIa to ~30% 6 months after ST.

Immunohistochemical analyses revealed that MHC-I was progressively decreased after ST, to

only approx. 12% 1 year after ST. The reductions in the proportion of MHC-I were countered by

increases in MHC-IIa and MHC-IIx with the increase in MHC-IIx preceding the increase in

MHC-IIa. Curiously, MHC-IIb, was expressed only at very low levels. Thus, a complete

transformation from predominantly MHC-I to MHC-IIb, did not occur. Many fibers (up to

approx. 80%) contained multiple MHCs (hybrid fibers) after ST. The proportion of hybrid fibers

was maintained at a high level (approx. 50%) 1 year after ST.

Zhong and colleagues (2005) studied the effects of short-term (4 days) and long-term (60

days) SI on the rat soleus.63 The control and SI-4d groups were ~90% pure type I and ~0.5 to 5%

types I+IIa, I+IIa+IIx and IIa fibers in both groups. The SI-60d rats showed seven MHC

combinations: pure type I (37%), I+IIa (32%), I+IIa+IIx (10%), I+IIx (16%), IIa (2%), IIa+IIx (~

1%), and IIx (-2%) fibers. Thus the most dramatic adaptations in the SI-60d soleus muscles were

a marked decrease in pure type I fibers, an increase in I+IIa, and appearance of fibers containing

only IIx MHC. All of the hybrid fibers (fibers co expressing type I and II MHC isoforms) in









control and SI-4d rats contained >50% type I MHC. In the SI-60d group, however, 21% of the

hybrid fibers contained <50% type I MHC. Similarly in the medial gastrocnemius (MG) and

tibialis anterior (TA) muscles were also studied after short-term (4 days) and long-term (60 days)

spinal isolation. Pure type I fibers were rare: 3%, 5%, and 0% in the control, SI-4d, and SI-60-d

rats, respectively.63 Approximately 90% of the fibers in all groups contained only types IIx

and/or IIb MHC. Fibers containing type I plus some type II MHCs were more prevalent in the SI

than control rats. There was a significant shift towards the fastest MHC isoform with inactivity:

pure IIb fibers comprised 13%, 3 8%, and 41% of the population of the control, 4-day, and 60-

day SI rats, respectively. In addition, there was a concomitant decrease in fibers containing only

type IIx+Inb after 4 (trend) and 60 days of SI. Thus, it appears that type IIb MHC was the default

MHC isoform in the inactive MG. TA muscles from control and 4-day SI rats contained ~5%

pure type I fibers and ~15% pure type IIa fibers. In contrast, there were no pure type I or pure

type IIa fibers in the 60-day SI rats. Approximately 70%, 80%, and 95% of the fibers expressed

only types IIx and/or IIb MHC in the control, 4-day, and 60-day SI rats, respectively. Compared

to control and SI-4d rats, there was a significant decrease in type IIb fibers and increases in type

IIx and IIx+Inb fibers in the SI-60d rats. Thus, it appears that type IIx MHC was the default

MHC isoform in the inactive TA. Thus, the magnitude of the adaptations observed following

spinal isolation is more severe than after spinal transaction. This suggests that the residual

amount of electrical activation in the cat soleus after spinal transaction plays a role in

maintaining the levels of MHC-I expression.63

Hutchinson and colleagues (2001) measured adaptations in muscle size using muscle wet

weight and MHC composition in the soleus (85% slow) and EDL (90% fast) muscles following

moderate contusion SCI. They reported a 20-25% decrease in soleus wet weight after 1-week of









contusion, while the EDL showed a non-significant 6% decrease in wet weight.20 Three weeks

post contusion both the soleus and EDL wet weights returned to normal levels. Analysis of the

MHC composition showed no change in fiber type composition at 1-week after spinal contusion

in either muscle, while after three-weeks there was an upregulation of IIx MHC in both the

soleus and EDL. 20 Interestingly, the soleus muscle showed a downward trend in IIa fibers, while

the EDL demonstrated an increase in IIb fibers. Preliminary results in our lab indicate ~13%

decrease in Type I fibers in the soleus and EDL two-weeks following moderate contusion SCI.

The soleus also shows a ~ 9% increases in hybrid fibers of both myosin type I and IIa. The EDL

shows a ~20% decrease in type IIx fibers and ~9% increase in Inb fibers following contusion

SCI. 24,25,60-62 In the current study, we propose to use immunohistochemistry for MHC staining to

study fiber type transformation in four important locomotor muscles with different fiber type

composition and functional roles, specifically the soleus, gastrocnemius, EDL and the TA.

1.6.3 Electrically Elicited Contractile Properties

The physiological measurements such as muscle contractile speed, and force potentiation,

delayed onset of fatigue, force frequency relationship, doublet potentiation, sarcolemmal

membrane properties, and motoneuronal pool suppression are useful methodologies in assessing

the mechanical adaptations in skeletal muscle after SCI. 20,48,62,64

SCI results in faster twitch properties as evidenced by shorter time to peak tension and

half-relaxation time; however the maximal isometric force generated is significantly reduced.l

Maximum shortening velocity is significantly increased in SCI rats whether measured by

extrapolation from the force-velocity curve or by slack-test measurements.20,48,62,64 At a

minimum 10Hz of electrical-stim, the soleus of the SCI animal develops greater force and is less

fused than the normal soleus, implying faster contraction and relaxation times. 5 Unfused tetani

of the EDL stimulated at different frequencies did not show significant difference between the









normal EDL and SCI-EDL.S Time to peak tension was decreased by ~50% in the SCI-soleus. In

the cat soleus muscle there was ~3 8% reduction in isometric tetanic force 10-months post-SCI,

while their time to peak tension was ~41% and half-relaxation time ~50% shorter than control

cats.2, 3 These changes in twitch and tetanic properties suggest a change in the properties of the

sarcoplasmic reticulum (SR). The change in time to peak tension suggests an increase in the

calcium transportability of the SR.26,58

In the spinal transaction model, reduction in maximal tetanic force is significant. Talmadge

et al. 2002 found ~44% reduction is maximal tetanic force three months post- spinal transaction.

In addition, the time to peak tension and half-relaxation time were ~45% and ~55% shorter

respectively. 20,48,62,64 In the contusion model, our model of interest, one week post-SCI showed a

~20% decrease in both peak twitch and tetanic tension compared to controls and by three weeks

they further decreased to ~41-51% respectively.20,65 However, no significant changes existed

between the controls and injured rats for the time to peak tension and half-relaxation times.

Overall, the decline in contractile force seen in most animal models of SCI is related to the

decrease in mechanical load and neural activation associated with the injury.

In summary, with SCI there is no direct damage to the muscle and innervation of muscles

is not physically disrupted. Therefore, the interruption of transfer of electrical activity through

the motoneurons can stimulate the skeletal muscle changes that were observed in the above

sessions.

1.7 Rehabilitation Training Strategies Following SCI

1.7.1 Locomotor Training

1.7.1.1 Locomotor training in humans

Studies about locomotor training in people with SCI were first reported by Barbeau and

colleagues (1987, 1993) where they assessed the feasibility of locomotor training on a treadmill









using body-weight support.66,67 Currently, there is a significant increase in the use of locomotor

training to retrain people to walk following numerous neurological conditions in the clinical

setting. As described previously, this therapeutic intervention was derived from the elaborate

models of locomotor training in animals' models of SCI which showed consistent positive

findings.14,68-74 Several studies in people with SCI have suggested that locomotor training may

increase the likelihood that persons with upper motor neuron injuries will learn to walk over

ground independently.757

Locomotor training guidelines compiled by Behrman and Harkema (2000)79 were derived

from basic and applied science findings and include the following principles: a) maximize

weight-bearing through the legs and minimize or eliminate weight-bearing through the arms, b)

provide sensory input consistent with the motor task; specifically standing or walking, c)

promote postural control and optimize the trunk, upper and lower extremities, and hip kinematics

for walking and associated motor tasks, and d) maximize the recovery and use of normal walking

patterns and minimize the use of compensatory movement strategies. These strategies can be

applied both in the clinical setting and in community settings.

Locomotor training also focuses on achieving independent community ambulation at

normal walking speeds without assistive devices, bracing, or use of compensatory movements.

Locomotor training consists of training people on a treadmill with their body-weight partially

supported. Therapists manually assist in step training at joint angles and timing of stance and

swing phases typical of normal gait. This is followed by overground step training which consists

of evaluating factors which are limiting this individual from walking independently in the

community at normal walking speeds without an assistive device, brace, or compensatory

movements. Once these are evaluated then the person is trained to ambulate in the community.









Thus locomotor training is the combination of different gait training techniques to help get a

person with incomplete-SCI to ambulate in the community independent of assistive devices.

However, the largest clinical trial comparing the efficacy of locomotor training with over-

ground practice to defined over-ground mobility therapy in persons with SCI reported that

physical therapy strategies of body weight support on a treadmill and defined overground

mobility therapy did not produce different outcomes. It was suggested that the finding was partly

due to the unexpectedly high percentage of ASIA C subj ects who achieved functional walking

speeds, irrespective of treatment.so Interestingly, there is still a lot of controversy surrounding

the methodology and implementation of this clinical trial.so

1.7.1.2 Locomotor training in the animal model

SCI results in the loss of motor function due to the lack of supraspinal input. The concept

that locomotor movements can be initiated even in the absence of supraspinal input was studied

as early as the end of the 19th century (Freusberg 1874; Philippson 1905; Sherrington 1899,

1910; Naunyn, Dentan, and Eichorst 1874). During the 1940s and 1950s, several researchers

suggested that spinally injured animals (i.e., cats and dogs) could not only produce stepping as

described in earlier work, but these animals could use all four of their limbs for walking

overground (Freemanl952; Kellogg et al. 1946; Shurrager 1955; Shurrager and Dykman 1951;

Ten Cate 1939, 1962). In 1951, Shurrager and Dykman first reported that training could restore

locomotion after spinal cord transaction in cats. s Later, Sten Grillner's laboratory in the 1980's

(Forssberg 1979; Forssberg and Grillner 1973; Forssberg et al. 1974, 1976, 1980a Grillner 1973)

clearly showed that thoracic SCI cats could walk with their hind limbs on the treadmill while the

forelimbs stood on a fixed platform. s These animals had good coordination between their hind

limbs, placed their fore paws properly on the plantar surface during the stance phase, and









supported the weight of the hindquarters. Not only did the kinematics of the spinal cat resemble

those found in the normal cat, but so did the muscular activity.8

However, it is only in the past 20 years that this phenomenon of locomotor training has

been vigorously explored, in concert with the growing recognition of the spinal cord' s

considerable capacities for plasticity and of other new possibilities for restoring function after

spinal cord injury.68-71 Today several studies have shown that recovery of motor function

following spinal cord injury can be enhanced or accelerated by repetitive locomotor treadmill

training. The underlying principle of locomotor training relates to rhythmic loading and

repetitive motor training that provides sufficient stimulation of specific neural pathways to

facilitate functional reorganization within the spinal cord leading to improved motor output.

Furthermore, appropriate sensory input provided during training helps to achieve the optimal

motor output of the spinal neuronal circuitry. 68-80

1.7.2 Functional Electrical Stimulation (FES)

Functional electrical stimulation (FES) has been used as a therapeutic resistance exercise

strategy to assist patients in strengthening as well as executing functional movements after SCI.

82-84 FES has been used in both the complete and incomplete SCI population to help reverse

atrophic changes, reduce muscle fatigability and increase bone density after SCI. s5 FES used in

combination with treadmill walking, cycling, external bracing holds considerable promise in

assisting persons with SCI execute functional movements.82-8 In the SCI population FES and

FES in combination with other exercise interventions have been the main resistance or strength

training protocols used in people with both complete and incomplete-SCI. In the following

sections, we will review some the muscle adaptations following FES and FES in combination

with other exercise interventions. In the current study, we will propose to investigate the effect of

resistance training on muscle function following incomplete-SCI. However, our resistance









training protocol will be non-FES based and will include regular gym based resistance exercise

training.

FES consists of a variety of stimulation parameters. It can be used at contraction times

ranging from approximately 1 to 20 seconds, frequencies of 10Hz to 80Hz and voltages from

30V- 135V. 82-85 Gerrits et al. 2002 compared the effects of two types of FES (high-frequency

and low-frequency) on neuromuscular activity after a motor complete SCI. Twelve weeks of FES

resulted in ~20% increase in quadriceps tetanic force with no differences between the two

stimulation frequencies. Neither training intervention had a significant effect on the contractile

properties (maximal isometric force, maximal rate of force rise, half-relaxation time, and force-

frequency amplitude) of SCI muscles.86 Crameri et al. 2000 looked at effects of 16-weeks of FES

(3 5Hz, 70V, 60min/day) after acute SCI. They found that FES helped in controlling the

phenotype expression of the VL towards faster isoforms and prevented fiber atrophy after acute-

SCI. s7 Dudley et al. 1999 in the sub-acute SCI population, showed that 8-weeks of FES resulted

in substantial increases in the quadriceps cross-sectional area. In a similar study, 24 weeks of

FES resulted in a significant strength gain with increased bone density in the quadriceps muscle

of persons with chronic SCI when compared to untrained controls.45 Long-term FES (two years)

has also proven to yield significant differences in torque, fatigue index, bone mineral density and

twitch properties in persons with SCI. when compared to their untrained leg.88 FES has been the

predominant means of resistance training people with SCI. 88,89 It would be interesting to identify

the effects of regular exercise based resistance training on people with SCI.

FES is generally not a very favorable therapeutic intervention with the incomplete-SCI

population as they have the ability to voluntarily activate their muscles to a certain extent. Bajd

et al. 2000 conducted a two month FES training study on persons with incomplete-SCI. He









concluded that long term FES resulted in a significant improvement in knee extensor strength

and also improved the ability to activate the dorsi and plantar flexors muscle groups in the

incomplete-SCI group.90 Modlin et at. 2005 performed a FES clinical trial on 40 persons with

either a conus medullaris or cauda equina lesion. One year of FES resulted in significant

increases in quadriceps muscle CSA compared to pre-training CSA levels. 91 Overall, FES has

shown considerable promise in improving muscle function in all different models of SCI ranging

from complete injuries to cauda equina injuries. Hence, FES on its own can be used as a

resistance training therapeutic modality in the SCI population. However, the point of interest is

that FES in the incomplete takes longer periods of time to cause significant improvements in the

incomplete-SCI population. 90-91 This can be attributed to higher levels of function and voluntary

control in this population. FES stimulated cycle ergometer training (FES-CE) has been used to

improve whole muscle girth and muscle mass with persons with chronic SCI.92,93 Balldi et at.

1998 examined if FES-CE was able to prevent atrophy after acute SCI. The study concluded that

FES-CE prevents lower extremity muscle atrophy in acute SCI after 3 months of training, and

also causes significant hypertrophy after 6 months.93 In a similar study by Crameri et at. (2002),

10weeks of FES-CE resulted in significant increases in muscle fiber cross-sectional area,

reduction in percentage oflIIx fibers and increase in the citrate synthase activity, indicating a

greater oxidative capacity of muscle, in persons with chronic SCI.94

Interestingly, numerous other studies have reported significant improvements after FES-

CE on muscle morphometric and histochemical characteristics in the chronic complete SCI

population.95-9 These changes include increases in whole muscle and fiber cross-sectional area,

muscle to adipose tissue ratio, fatigue resistance, maximal rate of force rise and speed of

relaxation, and switch in MHC from fast to slower isoforms, doubling of enzymatic activity of









citrate synthase, and Einally an increase in over-ground walking speed and endurance.95-99

Overall, FES-CE has been able to provide a resistance exercise program without the potential of

over-use injury in the complete-SCI population. The actual benefit of this training intervention in

improving the functional capabilities in the complete-SCI population remains speculative.

Similarly, the functional implications of FES-CE on the incomplete SCI population are yet to be

studied.

1.7.3 Resistance Training

There is always curiosity regarding the effect of exercise on the functional well being of

people with SCI. 100-102 In the above section we saw the effect of electrically stimulated

resistance training either using weights or using CE. However, various impediments exist in the

SCI population to complete successful regular resistance exercise training protocols.

Specifically, only the incomplete-SCI population with limited voluntary muscle control can

perform regular non-electrically induced resistance training. Although they have certain degree

of voluntary control, they are still structurally and functionally ill-suited for strong propulsive

and weight-bearing exercises. One has to be aware of inducing overuse bone and muscle injury,

nociceptive and neuropathic pain, reflex sympathetic dystrophy, and some cases, cardiovascular

complicationS.103,104 Few studies have looked at the effects of resistance or strength training

protocols on the SCI population. Nilsson et al. 1975 was the first to report significant

improvement in the triceps muscles in persons with incomplete-SCI following resistance

training. Cooney et al. 1986 used a hydraulic device in a nine-week training program which

improved upper extremity power output in the chronic SCI population.105106

Persons with SCI as we know exhibit deficits in voluntary control and sensation that limit

not only the performance of daily tasks but also the overall functional and social activity. This

leads to extremely sedentary lifestyle with an increased incidence of secondary complications









including diabetes mellitus, hypertension and lipid profies. As the daily lifestyle of the average

person with SCI is without adequate activity, structured exercise activities must be added if the

individual is to reduce the likelihood of secondary complications and/or to enhance their physical

capacity. The acute exercise responses and the capacity for exercise conditioning are related to

the level and completeness of the SCI. Appropriate exercise testing and training of persons with

SCI should be based on the individual's exercise capacity as determined by accurate assessment

of the spinal lesion. Other issues that need to be taken into consideration before resistance

training can be incorporated as a therapeutic activity. For example, the scientific basis for the

exercises needs to be identified, training parameters; like dosage refinement, safety instructions,

and inclusion-exclusion criteria need to be postulated. Overall, clinicians involved in SCI

rehabilitation need to consider resistance training as a therapeutic intervention rather than

concentrate on compensation as their modus operandi. Wheelchair strength training has shown

considerable promise in improving muscle power and strength in the SCI population. However,

these studies are either limited to the upper extremity or are for wheelchair athletes only.107,10s To

conclude, important strides need to be taken in the research Hield on studying the effects of

resistance training on improving skeletal muscle function after SCI. In current study, we will

examine the effect of gym based resistance exercise training on muscle function on people with

chronic incomplete-SCI. The current study will be one of the first studies which will look at

strength training lower extremity locomotor muscles in persons with incomplete-SCI.

1.8 Skeletal Muscle Adaptations Following SCI and Locomotor training

1.8.1 Impact on Humans

Current rehabilitation research has described loss of skeletal muscle function as one of the

significant problems impacting the health care and quality of life of persons after SCI.1,2 A

significant portion of the SCI related costs can be attributed to degradation of the









musculoskeletal system resulting in decreased skeletal muscle function.1,2 Even though

locomotor training is not considered a therapeutic intervention designed to induce muscle

hypertrophy; previous studies have shown that in the incomplete-SCI population the training

stimulus and loading can be of sufficient magnitude to induce muscle plasticity. Giangregorio et

at. 2006 reported increases in whole-body lean mass, from ~45.kg to ~47kg and increases in

muscle CSAs by an average of 4.9% and 8.2% at the thigh and lower leg after 144 sessions of

locomotor training in persons with chronic incomplete-SCI.109 In a similar study performed in

the acute-SCI population, 48 sessions of locomotor training resulted in increases in muscle CSAs

ranging between ~4% to ~58%.110 The study concluded that twice-weekly locomotor training

appeared to partially reverse muscle atrophy after SCI, but failed to prevent bone lOSS.109,110

These findings are supported by research examining changes at the muscle fiber level. Stewart et

at. 2004 reported a 25% increase in the mean muscle fiber area of type I and IIa fibers in the

vastus lateralis following 6 months of body weight supported treadmill training in chronic

incomplete-SCI subj ects.39 Adams et at. 2006 in a single case study (chronic ASIA B) reported

that the vastus lateralis mean fiber area increased by 27.1% and type I fiber % distribution

increased to 24.6%, whereas type IIa and type IIx fiber % distributions both decreased following

48 sessions of locomotor training.38

1.8.2 Impact on the Animal Model

The effects of locomotor training on SCI-induced muscle adaptations have been studied to

a limited extent over the past two decades. Roy et at. as early as 198626 identified that spinalized

adult cats who exercised on a treadmill for a week showed less atrophy and fiber type

adaptations, especially in the postural muscles (slow extensors).26 In a similar study, the same

group identified that only 30 min of daily step training emphasizing weight support on a

treadmill ameliorated, and in some cases prevented, the contractile and morphological









adaptations in the soleus muscle associated with a complete low thoracic spinal cord transaction

in adult cats.ll

Similarly, a few studies have also been conducted in the rodent model, identifying muscle

adaptations after SCI and locomotor training. Versteegden et al. (1999, 2000) reported that

locomotor training resulted in an increase in muscle fiber size, myonuclear number, satellite cell

count and a decrease in the apoptotic nuclei in the soleus muscle after spinal transaction in the rat

model.112113 A unique Einding in these studies was that satellite cell fusion and restoration of

myofiber nuclear number contributed to increased muscle size in the soleus after locomotor

training.113 Stevens et al. 2006 reported that locomotor training following contusion SCI resulted

in a significant improvement in overall locomotor function (32% improvement in BBB scores)

when compared to no training group. Also, the injured animals that trained for one week had

38% greater peak soleus tetanic forces, a 9% decrease in muscle fatigue, 23% larger muscle fiber

CSA, and decreased expression of fast myosin heavy chain fiber types compared to rats

receiving no training.65,114 Overall, locomotor training has shown significant promise in

attenuating the adaptations in skeletal muscle seen after SCI. This includes prevention of atrophy

following SCI, reduced fatigability, improved muscle force production and transformation of

fiber type towards slower isoforms. However, further investigation is required to identify the

training effects on specific models of SCI.

In conclusion, locomotor training has shown to induce positive alterations in skeletal

muscle function in both humans and animals. However, most of the current data still revolve

around the complete SCI model. Further investigation in the incomplete SCI model in both

humans and animals is warranted. In this dissertation we will to answer some of the questions









regarding skeletal muscle adaptations following locomotor training in both the animal and

human model.

1.9 Mechanisms Involved in Training Induced Muscle Plasticity and Recovery

The primary functions of skeletal muscle are production of movement, posture control, and

respiration. Interestingly, skeletal muscle is susceptible to injury from direct trauma (e.g.,

intensive activity, stab wounds, gun shots etc.) or resulting from indirect causes such as

neurological disease or genetic complications."' Direct or indirect injuries may lead to loss of

muscle mass and strength leading to a functional limitation. The maintenance of a working

skeletal muscle is conferred by its remarkable ability to regenerate."' Indeed, upon muscle injury

a finely orchestrated set of cellular and molecular responses is activated, resulting in the

regeneration of a well-innervated, fully vascularized muscle apparatus.'"

Muscle fibers are the single cells that form skeletal muscles. They are individually

surrounded by a connective tissue layer (endomysium) and grouped into bundles surrounded by

the perimysium, and these bundles are surrounded by the epimysium to form a skeletal

muscle."' As the muscle fiber or myofiber matures, it is contacted by a single motor neuron and

expresses molecules for contractile function, principally different MHC isoforms and metabolic

enzymes. Both the origin of the myoblast and the motor neuron play an important role in

specifying the contractile properties of their myofiber. Nevertheless, adult skeletal muscles are

composed of a mixture of myofibers with different physiological properties, ranging from a

slow/fatigue-resi stant type to a fast-/non-fatigue-resistant type. The proportion of each fiber type

within a muscle determines its overall contractile property."

1.9.1 Plasticity of Skeletal Muscle

Adult skeletal muscle is a very stable tissue with little turnover of nuclei. Minimal damage

inflicted by daily wear and tear elicits only a slow turnover of its multinucleated muscle fibers. It









is estimated that in an adult rat muscle, no more than 1-2% of myonuclei are replaced every

week.116 Nonetheless, mammalian skeletal muscle has the ability to complete rapid and extensive

regeneration in response to severe injury or damage. The maj ority of this regeneration is carried

out by the activation, proliferation and differentiation of a resident population of myogenic cells

called satellite cells. Under normal conditions, satellite cells are quiescent but become activated

in response to injury giving rise to proliferating myogenic precursor cells that eventually

differentiate and fuse to form multinucleated myotubes. Quiescent satellite cells and their

descendant myogenic precursors are the key effectors of muscle regeneration.116-11

The early phase of muscle injury is usually accompanied by the activation of

mononucleated cells, principally inflammatory cells and myogenic cells. The factors released by

the injured muscle activate inflammatory cells within the muscle. Neutrophils are the first

inflammatory cells to invade the injured muscle. After neutrophil infiltration, macrophages

infiltrate the injured site to phagocytose cellular debris and initiate muscle regeneration by

activating myogenic cells. Thus muscle fiber necrosis and/or increased number of non-muscle

mononucleated cells within the damaged site are the main histopathological characteristics of the

initial activity following muscle injury.ll~1

Muscle degeneration after injury is followed by the activation of a muscle repair process.

The myogenic cells provide an ample source of new myonuclei for muscle repair. On cross-

section, classic characteristics of muscle regeneration are small newly formed myofibers with

centrally located myonuclei. 120,121 Newly formed myofibers are often basophilic and express

embryonic/developmental forms of MHC which reflect new fiber formation. 120,121 Fiber splitting

or branching is also a characteristic feature of muscle regeneration and is likely due to the

incomplete fusion of fibers regenerating within the same basal lamina. Once fusion of myogenic









cells is completed, newly formed myofibers increase in size, and myonuclei move to the

periphery of the muscle fiber. Under normal conditions, the regenerated muscle is

morphologically and functionally indistinguishable from undamaged muscle.120,121

1.9.2 Markers of Muscle Recovery and Regeneration

1.9.2.1 Adult muscle satellite cells

Muscle satellite cells are a population of undifferentiated, mononuclear myogenic cells

found in skeletal muscles including muscle spindles. Even though the temporal appearance of

satellite cells follows the appearance of both embryonic and fetal myoblasts, satellite cells

display specific characteristics in culture allowing their distinction from embryonic and fetal

myoblasts. 116,122,123 Satellite cells are situated between the plasma membrane and the basal

lamina of the muscle fiber. These cells are further identifiable by their relatively minute amount

of cytoplasm, sparse organelles, and high ratio of heterochromatin to euchromatin, indicative of

the inactive state of these cells. Satellite cells are present in different types of skeletal muscles

and are associated with all fiber types, although the distribution might be unequal. For instance,

the percentage of satellite cells in adult slow soleus muscle is two- to threefold higher than in the

adult fast tibialis anterior or extensor digitorum longus muscle.124 Similarly, high numbers of

satellite cells are found associated with slow muscle fibers compared with fast fibers within the

same muscle.116-122-124 Increased density of satellite cells have been observed at the motor neuron

junctions and adj acent to capillaries, suggesting that some factors associated with these structures

may play a role in homing satellite cells to specific locations or in regulating the satellite cell

pool by other means. The regulation of satellite cell density at the single fiber level is also

suggestive of a role for the muscle fiber in regulating the satellite cell pool (Fig. 1-3).116,124

Satellite cells are activated upon muscle injury, resulting from mechanical stress, direct

injury to the muscle or in course of a disease to help in muscle regeneration. In the initiation of









muscle regeneration, satellite cells first change from their quiescent state to a highly proliferating

stage. After proliferating several times, the maj ority of satellite cells fuse to form new myofibers

or j oin and repair the damaged one. During proliferation, a certain percentage of satellite cells

are restored underneath the basal lamina for subsequent rounds of regeneration (Fig. 1-4).122

The gene or the marker responsible for specification of muscle progenitor cells to the

satellite cell lineage is pax-7.125 The Pax7 gene is a member of the paired box containing gene

family of transcription factors implicated in development of the skeletal muscle of the trunk and

limbs, as well as elements of the central nervous system. 125,126 The number of Pax7 expressing

cells corresponds well with the expected number of satellite cells. Pax7 expression is upregulated

in proliferating satellite cell-derived myoblasts and a rapid down regulation of Pax7 transcripts is

seen upon myogenic differentiation. Pax7 is not expressed at detectable levels in a variety of

non-muscle cell lines. In addition, analysis of RNA from selected mouse tissues revealed only a

low level expression of Pax7 in adult skeletal muscles. 125 Normally Pax7 mRNA and protein are

found in less than 5% of satellite cells in undamaged skeletal muscle. However, the number of

Pax7-positive cells increases in muscles undergoing regeneration such as in MyoD /, mdx, and

mdx: MyoD- skeletal muscles. 125,126 Centrally located nuclei within newly regenerated muscle

fibers are also associated with Pax7 expression, suggesting that recently activated and fusing

satellite cells express Pax7. Together, these data demonstrate the specific expression ofPax7 in

quiescent and activated muscle satellite cells.124,125,126

The analysis of Pax7' skeletal muscles demonstrates the important role this gene has in

satellite cell development.125-128 Pax7-/ mice appear normal at birth but fail to grow post-natally,

leading to a 50% decrease in body weight by 7 days of age compared with wild-type littermates.

Pax7 mutant animals fail to thrive and usually die within 2 weeks after birth. These animals are









also characterized by a decreased skeletal muscle mass resulting from a fiber size decrease rather

than a decrease in fiber number.125-128Pax7' skeletal muscles have a striking absence of satellite

cells. Overall, the data suggest a key role for Pax7 in lineage determination, especially in the

specification of myogenic progenitors to the satellite cell lineage. Pax7 is unequivocally required

for satellite cell development (Fig. 1-5).125-128

In the next stage, proliferating satellite cells are referred to as myogenic precursor cells

(mpc). At the molecular level, activation of mpcs are characterized by the upregulation of two

muscle regulatory factors (MRF), Myf5 or MyoD. MRFs are part of a super family of basic

helix-loop-helix (bHLH) transcription factors. The MRF subfamily consists of MyoD (Myf-3),

Myf-5, myogenin (Myf-1), and MRF4 (Myf-6/Herculin).1l23,129 In general, quiescent satellite cells

do not have any detectable levels of MRFs. Upon satellite cell activation, MyoD upregulation

appears the earliest within 12 hrs of activation. Activation of MyoD and Myf5 expression

following muscle injury has also been observed in various in vivo models for muscle

regeneration and in varying muscle types.129,123 A study by Megeney et alI. indicated that MyoD-/

mice show increase in mpc population compared to normals, however they have a decrease in the

number of regenerated myotubes. Furthermore, MyoD- muscles display an increased

occurrence of branched myofibers suggestive of chronic or inefficient muscle regeneration. 129-135

In vitro cultures of MyoD-/ satellite cells demonstrate a myogenic cell population with abnormal

morphology characterized by a stellate, flattened appearance in contrast to the compact rounded

appearance displayed by normal myoblasts. Overall, these data suggest an important role for

MyoD in the process of satellite cell differentiation during muscle regeneration. 129-135

Myf5-deficient mice display a delayed epaxial (back muscle) embryonic myogenesis and a

normal hypaxial (trunk and limb muscles) embryonic myogenesis.129-131 These data combined









with the reciprocal delay in hypaxial myogenesis in MyoD-deficient mice and the mutually

exclusive expression of Myf5 and MyoD in early stages of embryonic muscle precursor cells

have led to the hypothesis that Myf5 and MyoD support distinct myogenic lineages during

embryonic muscle development. 129-131 Myf5 promotes satellite cell self-renewal, whereas MyoD

promotes satellite cell progression to terminal differentiation. There is new compelling evidence

that the satellite cell population is composed of hierarchal subpopulations of stem cells: the

Pax7 / Myf5+ satellite cells preferentially differentiate and become committed myogenic

progenitors, while the Pax7 / Myf5- satellite cells extensively contribute to the satellite cell

compartment.12-3

After the mpc proliferation phase, expression of Myogenin and MRF4 is upregulated in

cells, beginning their terminal differentiation program. This is followed by cell cycle arrest and

permanent exit from the cell cycle. The differentiation program is then completed with the

activation of muscle-specific proteins, such as MHC, and the fusion of mpc to repair damaged

muscle or form their own fibers. Overall, Myf5, MyoD, and Myogenin possibly play distinct

roles in myofiber maturation.122 Gross defects in embryonic muscle development of mutant mice

for Myogenin and MRF4 have impeded further study of these genes in muscle regeneration.

Mice lacking myogenin display a normal number of myoblasts but die at birth because of an

absence of myofibers. It has also been suggested that Myogenin helps in the conversion of

myoblasts to myotubes and helps in the maturation of myotubes.122

1.9.2.2 Other stem cells

Mammalian skeletal muscle regeneration involves the activation of the quiescent muscle

satellite cell population to proliferate, differentiate, and fuse to provide new myonuclei for

muscle repair. Pax7 is required for muscle satellite cell specification/survival, whereas MRFs are

essential in satellite cell proliferation and differentiation. Multipotential stem cells in adult









muscles (adult muscle-derived stem cells) are also capable of myogenic commitment. 122,123

Adult muscle-derived stem cells contribute to both muscle satellite cell pool and myonuclei.

Similarly, stem cells capable of myogenic commitment can be isolated from other adult tissues

(bone marrow stem cells, neuronal stem cells, and various mesenchymal stem cells)can be used

for repair following muscle damage or towards new fiber formation.122123

1.8.2.3 Growth factors and muscle regeneration

Muscle regeneration is a complex process in which growth factors play an important role.

Mechanisms that are controlled or altered by growth factors include satellite cell activation,

migration to the injury site, proliferation of satellite cell-derived mpcs and differentiation to

myotubes and myofibers. Insulin-like growth factors (IGFs) I and II are involved in almost all

stages of muscle regeneration; they promote satellite cell activation and proliferation, are

upregulated in regenerating muscle and may protect cells from apoptosis. Both IGF-I and

hepatocyte growth factor/scatter factor (HGF) are upregulated during muscle regeneration, HGF

being crucial during the initial stages and IGF during the initial to mid stages of regeneration. A

significant increase in muscle regeneration was observed when human mpcs were cultured with

IGF-I prior to their implantation into damaged muscle.122 IGF-I has a significant effect on

proliferate arrest and hypertrophy of myotubes derived from human fetal mpcs in culture and

causes an increase in myosin heavy chain content. HGF is known to increase the chemotaxis of

mps122,132-134

C2C12 myoblasts treated with HGF reorganized their actin cytoskeleton and developed a

polarized cell shape.136 HGF appears to increase the mpc population by means of mitogenic and

chemotactic activities, possibly resulting in an optimal my oblast density. IGFs most certainly

promote muscle repair by signaling to both the satellite cells and the myofibers. Whether distinct









roles are played by different IGFs is possible, since IGF-II appears to be upregulated later during

the process of muscle regeneration. 122,132-134

Fibroblast growth factors (FGF) are also involved with satellite cell activation,

proliferation and differentiation. FGF-2 acts as a regulator of satellite cell activity and FGF-6

expression is upregulated during muscle regeneration. Expression of FGF-6 in C2C12 cells

induces morphological changes; reduces cell adhesion and differentiation.137 A greater

proportion of the cells which expresses FGF-6 were side population cells, suggesting that FGF-6

may be involved in the maintenance of the reserve pool of progenitor cells in skeletal muscle.

Also the role of FGF in muscle regeneration may reside in the revascularization process during

regeneration through their recognized angiogenic properties. 122,132-134

In summary, mammalian skeletal muscle has little turn over of satellite cells under normal

conditions. However, upon injury, skeletal muscle activates satellite cells to both repair and

regenerate muscle fibers to prevent atrophy and damage. There are certain key phases in the

regeneration process and these processes are supported by different growth factors.



Other/~
Unkn.
6.8%
8.9%










Figure 1-1. Etiology of SCI since 2000 modified from www. spinalcord.uab.edu.












Severity of Injury 125 years old 150 years old

High Tetraplegia (C1-C4) 1$2,924,513 1$1,721,677

Low Tetraplegia (C5-C8) 1$1,653,607 $1,047, 189

Paraplegia 1$977,142 1$666,473

Incomplete Motor Functional at any Level $651,827 1$472,392

Figure 1-2. Estimated lifetime costs by age at injury modified from www.spinalcord.uab. edu.


Satel lite
Cell
ALge. Nuclei
Muscle mo %


~Animal Model


Mou~E cross-sectiCons (291,)

Rat cross-sections (11S)





Rat cross-sections 06.0


EDL 5--7
So~e~us 5-7
EDL 1
24
Sol~eus 1

24
TA 2
Sol~eus 2


Figure 1-3. Satellite cell number in skeletal muscle of different ages and type modified from
Charge SB, Rudnicki MA. Cellular and molecular regulation of muscle regeneration.
Physiol Rev. 2004; 84:209.





















~~Tranlba grn1r4r Builr ,n


dhr
Foxki














MSC ~ My MyogMenin~~1U~'~
Par MyoD- MPF4


Stalg gMEPGrl;g1

cell



Satllte el




Fiue -. cemtcoutline of a stemit cell passingy thogtesagso muscle regeneration, )gn xrsino aelt

Guslsoani E, Kunkel LM, Huard.Sendpoeior cells ikltlmsl



develoment, aintennce, nd hrp.MlTer 07 587









CHAPTER 2
OUTLINTE OF EXPERIMENTS

2.1 Experiment 1

2.1.1 Specific Aim

To characterize and specifically quantify impairments in lower extremity skeletal muscle

function after chronic incomplete spinal cord injury (SCI).

Peak isometric torque, torque developed within the initial 200 ms of contraction

(torque200), average rate of torque development (ARTD), and voluntary activation deficits will be

calculated as indices of muscle function. Measures of muscle function and electrically elicited

contractile measurements will be quantitatively performed in the knee extensor (KE) and ankle

plantar flexor (PF) muscle groups in ten individuals with chronic incomplete SCI.

2.1.2 Hypothesis

Reduced peak and instantaneous torque production, as well as greater voluntary activation

deficits of the lower extremity muscles will be found to be characteristic of individuals with

chronic incomplete SCI compared to healthy controls. In addition, significant bilateral

asymmetries will exist between limbs, with one limb being more affected than the other.

2.2 Experiment 2

2.2.1 Specific Aim

To determine the impact of nine weeks of locomotor training on lower extremity skeletal

muscle function in persons with chronic incomplete SCI.

Five individuals with chronic incomplete SCI will undergo nine weeks (Hyve sessions per

week) of locomotor training (LMT). Indices of muscle function will be determined for the KE

and PF muscle groups before and after LMT.









2.2.2 Hypothesis

Nine weeks of locomotor training will result in positive alterations in the lower extremity

muscles that include improved voluntary activation as well as an improved ability to generate

both peak and explosive torque about the knee and ankle joints in persons with chronic

incomplete SCI.

2.3 Experiment 3

2.3.1 Specific Aim

a) To determine the impact of a 12-week resistance and plyometric training on lower

extremity skeletal muscle function in persons with chronic incomplete SCI.

b) To determine the effect of 12-weeks of resistance and plyometric training on gait speed

in persons with chronic incomplete SCI.

Three ambulatory individuals with chronic motor incomplete SCI (18.7+2.2 months post

injury) will complete 12-weeks of lower extremity resistance training combined with plyometric

training (RPT). Indices of muscle function will be determined for the KE and PF muscle groups

before and after RPT. Maximal, as well as self-selected gait speeds will also be determined pre-

and post-RPT.

2.3.2 Hypotheses

a) Twelve weeks of RPT will result in improved ability to generate peak and

instantaneous torque as well as improved voluntary activation and reduced time to peak torque in

the lower extremities of persons with chronic incomplete SCI. In addition, the magnitude of

improvements in these outcomes will be most pronounced in the PF versus the KE muscle group.

b) Twelve weeks of RPT will result in significant improvements in both self-selected and

maximal gait speed in persons with chronic incomplete SCI.









2.4 Experiment 4


2.4.1 Specific Aim

a) To determine the effect of moderate T8 contusion spinal cord injury (rat model of

incomplete SCI) on muscle fiber cross-sectional area (CSA) and fiber type composition in four

lower extremity muscles soleuss, gastrocnemius, tibialis anterior, and extensor digitorum longus)

with different fiber type compositions and functional roles.

b) To compare the effect of 1-week of locomotor training on fiber CSA and fiber type

composition in lower extremity muscles with different fiber type composition and functional

roles in rats following moderate T8 contusion spinal cord injury.

c) To determine the effect of one week of locomotor training on fiber crossectional area

and fiber type composition in four lower extremity muscles soleuss, gastrocnemius, tibialis

anterior, and extensor digitorum longus) in healthy controls.

Rats (n=6 per group) will be assigned to four groups; a SCI-treadmill training group, a

SCI-no training group, control-treadmill training group, control-no training group. Moderate

spinal cord contusion injuries will be produced using a standard NYU (New York University)

impactor. Animals assigned to the training groups will be trained continuously for week (5

days/week, 2 trials/day, 20minutes/trial), starting on post-operative day eight for the SCI training

group. Fiber CSA will be assessed at two weeks post-injury for the slow-twitch, plantarfiexor

muscle, soleus, fast-twitch plantarfiexor, gastrocnemius and fast-twitch dorsiflexor muscles

tibialiss anterior [TA] and extensor digitorum longus [EDL]).

2.4.2 Hypotheses

a) Two weeks following moderate T8 spinal contusion injury, the injured rats will

experience the maximum decrease in fiber CSA in the slow-twitch plantarfiexor extensorr)

soleus when compared to the non-injured control rats. The next largest decline in muscle fiber









CSA will be seen in the gastrocnemius, followed by the fast-twitch dorsiflexors, TA and EDL.

Similarly, following moderate T8 spinal contusion injury, all muscles in the injured rats will

show a shift in fiber type towards faster myosin isoforms. Specifically, the soleus from the

injured rats will show a fiber type shift from a slower isoform to a faster isoform (MHC-I to

MHC-IIa) compared to the soleus of controls. Similarly, the gastrocnemius, TA and EDL of the

injured rats will show a fiber type shift towards faster isoforms (MHC-IIa to MHC-IIx and

MHC-IIx- MHC-IIb) when compared to controls.

b) One week of treadmill training will attenuate the decrease in fiber CSA of the injured

rats in all the muscles. Specifically, the soleus will experience maximum gains in fiber CSA,

followed by the gastrocnemius, and then the dorsiflexors. In addition, treadmill training will

attenuate the fiber type shift observed following a moderate T8 contusion spinal cord injury.

Specifically, the soleus will show fiber type transformation towards slower isoforms (MHC- IIa

to MHC-I), while the gastrocnemius, TA and EDL will show a transformation from MHC- Inb

towards IIx or IIa.

c) Healthy control rats trained for one week will show increases in fiber CSA compared to

untrained control rats. However, we anticipate that there will be no difference in the CSA values

between the trained and the untrained group.

2.5 Experiment 5

2.5.1 Specific Aim

a) To determine the impact of moderate T8 contusion SCI on satellite cell activity on the

slow-twitch soleuss) and fast-twitch (TA) rat muscles.

b) To determine the influence of one week of locomotor training on satellite cell activity

on the slow soleuss) and fast twitch (TA) muscles on spinal cord-injured rats.










c) To determine the impact of one week of locomotor training on satellite cell activity on

the slow soleuss) and fast twitch (TA) muscles on control rats.

Rats (n=6 per group) will be assigned to either a SCI-treadmill training group, a SCI-no

training group, control-treadmill training group or control-no training group. Expression of

markers of muscle regeneration will be assessed for all four training groups. Specifically,

immunofluorescence techniques will be used on the soleus and TA to quantify for Pax-7 and

EM-MHC expression and Western blot analysis will be used to quantify for MyoD, Myf5, and

Myogenin expression.

2.5.2 Hypotheses

a) Two weeks following moderate T8 spinal contusion injury, both the slow twitch soleuss)

and the fast twitch (TA) muscles will show increase in the regulation of muscle regeneration

markers compared to controls. Specifically, the levels of the markers will be higher in the slow

twitch muscles when compared to the fast twitch muscles.

b) One week of locomotor training, will result in increased regulation of muscle

regeneration markers in both the muscles types, compared to untrained SCI rats. Specifically, the

slow twitch soleuss) will show significant elevations in regeneration markers after the training in

comparison to the fast twitch (TA) following moderate T8 contusion spinal cord injury.

c) SCI rats trained for one week will show increased regulation of regeneration markers

compared to trained control rats.









CHAPTER 3
IVETHODOLOGY

3.1 Studies in People with Incomplete-SCI

3.1.1 Subjects Description

Subj ects who participate in the first three experiments are persons with chronic upper

motor neuron lesions and motor incomplete-SCI. Criteria for inclusion include: 1) age 18-70; 2)

first time SCI (C5-T10); 3) medically stable and asymptomatic for bladder infection, decubitis,

cardiopulmonary disease or other significant medical complications prohibiting testing and/or

training; 4) if using antispasticity medication, agreement to maintain current levels throughout

study; Exclusion criteria will be: 1) participation in a rehabilitation or research protocol that

could influence outcomes of this study; 2) history of congenital SCI or other disorders that may

confound treatment, study, and/or evaluation procedures; Prior to participation, written informed

consent will be obtained from all subj ects, as approved by the Institutional Review Board at the

University of Florida.

3.1.2 Locomotor Training

The locomotor training intervention consists of 45 training sessions (5x/ week) spread over

nine weeks, with each session consisting of 30 minutes of step training on the treadmill with

body weight support (BWS) immediately followed by 20 minutes of level overground walking

and community ambulation training. Including pre-training stretching, donning/doffing the

harness, and additional time spent on the treadmill for stand training and standing rest breaks, the

total session duration will be approximately 75 to 90 minutes per day. Each subject is expected

to complete all of the training sessions. With the aid of the body weight support, treadmill and

manual trainers, the treadmill training environment will facilitate delivery of locomotor specific

practice. Trunk, lower limb, and upper limb kinematics will be consistently assisted and/ or









monitored by trainers to assure appropriateness in relation to normal walking. Speed of treadmill

stepping will be kept in a range consistent with normal walking (2.2-2.8 miles/hr). Progression of

training will be achieved by decreasing BWS, altering speed, increasing trunk control,

decreasing manual assistance for limb control and increasing the time spent walking on the

treadmill per bout. A more detailed description of the training principles, parameters and

progression has been provided by Behrman & Harkema et al. 2000. Overground training will

consist of an immediate assessment of the participant' s ability to stand and/or walk

independently overground and an evaluation of the deficits limiting achievement of this goal.

These deficits became the focus for goal setting in the next day's training session. Additionally,

overground training addressed translation of the skills from the treadmill to the home and

community identifying practical ways for the participants to incorporate new skills into everyday

activities (Figure 3-1).

3.1.3 Resistance and Plyometric Training

3.1.3.1 Resistance training

Lower extremity progressive resistance training will be 12 weeks in duration and subj ects

will complete 2 to 3 sessions/week for a total of 30 sessions. Resistance exercises will include

unilateral leg press, knee extension/flexion, hip extension/flexion and ankle plantar flexion

exercises performed on adjustable load weight machines. During the initial training session a

predicted one-repetition maximum (1-RM) will be calculated for each subj ect and for each

exercise. 1-RM will be determined using a prediction table based on a single set to volitional

failure with load that allowed between 6 and 12 repetitions. During subsequent training sessions,

subj ects will perform 2-3 sets of 6-12 repetitions at a relative intensity of ~70-85% of predicted

1-RM. Maximal strength will be evaluated weekly to assess for training-related improvements

and exercise loads will be adjusted accordingly. Specifically, if the subj ect achieved the target









number of repetitions for all prescribed sets of a given exercise, a new predicted 1-RM will be

prescribed and resistance will be increased for subsequent training sessions.

3.1.3.2 Plyometric training

Unilateral plyometric jump-training exercises will be performed in both limbs in a supine

position on a ballistic jump-training device (ShuttlePro MVP @, Contemporary Design Group,

Figure3-2). Session intensity for this exercise will be modified by changing either the resistance

or the number of ground contacts and progressed over the training period, accordingly. Briefly,

after familiarization with the training device, subj ects will complete a total of 20 unilateral

ground contacts (e.g. jumps) with each limb at a resistance of ~25% of body mass. Thereafter,

upon successful completion of at least 20 ground contacts per limb (e.g. complete clearance from

the foot plate), resistance will be increased in increments of 10 lbs. When a new resistance is set,

repetition goal will be set at 10 ground contacts per limb for the initial session. Subsequent

sessions allowed for up to 20 contacts per limb. Thus, a minimum of two sessions at a given

resistance will be required before load is increased. Resistance will be held constant between

limbs throughout the training program.

3.1.4 Muscle Function Assessment

3.1.4.1 Experimental set-up

Voluntary and electrically elicited contractile measurements are performed in the self-reported

more-involved and less-involved limbs for the knee extensor and plantarfiexor muscle groups,

using a Biodex System 3 Dynamometer. Knee extensor testing will be performed with subj ects

seated in an upright position with hips flexed to ~85 and knees flexed to ~90 The axis of

rotation of the dynamometer will be aligned with the axis of the knee j oint and the lever arm

secured against the anterior aspect of the leg, proximal to the lateral malleolus. Testing of the









plantar flexor muscle group will be performed with the hips flexed at 90- 100 the knee flexed at

~10 and the ankle at ~0 plantar flexion. The anatomical axis of the ankle will be aligned with the

axis of the dynamometer, while the foot was secured to the footplate with straps placed at the

forefoot and ankle. Proximal stabilization was achieved with straps across the chest, hips and

thigh (Figure3-3).

3.1.4.2 Voluntary contractile measurements

Prior to testing, subj ects perform three warm-up contractions to get familiarized with the

testing procedures. This was followed by three maximal voluntary isometric contractions (~5

second each with 1 minute rest intervals) while being given verbal encouragement. Peak torque

will be defined as the highest value obtained during the 3 maximal isometric contractions. In the

event that the peak torque values differed by more than 10%, additional contractions will be

performed.

In addition to peak torque we also will determine the average rate of torque development

(ARTD) and the torque200, aS indices of explosive muscle strength. The ARTD will be defined

as the average increase in torque generated in unit time, and will be calculated in the time

interval corresponding to 20% to 80% of peak amplitude, starting from muscle perturbation. This

time interval was selected to reduce the effect of errors in calculating peak amplitude. Hence

ARTD was calculated through numerical differentiation as:

1 "; 6f,
ARTD =
N ,1 t

Where, N is the total number of time slots for numerical differentiation, GJis the change

in torque in the time slot i and Gtis the unit time duration for a slot. Torque200 will be defined as

the absolute torque reached at 200ms during a maximal voluntary contraction (Nm).









3.1.4.3 Electrically elicited contractile measurements

Peak twitch torque, time to peak twitch and twitch half-relaxation time will be determined by

delivering a supra-maximal electrical stimulus (600Cls pulse duration) to the muscles at rest.

Supramaximal intensity will be determined by increasing the current voltage until twitch torque

production plateaued. Time to peak twitch and twitch half-relaxation time will be calculated

from the peak twitch contractions.

Voluntary activation deficits. are performed using self-adhesive electrodes covering the

width of the muscles with sizes ranging from 3.8 6.35 cm to 7.6 x 12.7 cm. For the KE, electrodes

will be placed across the width of the distal portion of the thigh (quadriceps muscles), just above

the knee j oint and across the proximal portion of the thigh, near the origin of the muscle group.

For the PF, electrodes will be placed across the width of the proximal portion of the calf (triceps

surae muscles) just below the knee j oint line and across the distal portion of the soleus superior

to the Achillies tendon (Figure3-3).

Voluntary activation deficits will be determined using the twitch interpolation method. A

Grass S8800 stimulator with a Grass Model SIU8T stimulus isolation unit (Grass Instruments,

West Warwick, RI) will be used to briefly deliver a single biphasic, and supra-maximal pulse

was delivered at rest and during maximal voluntary isometric contraction. The stimulator and the

dynamometer will be interfaced with a personal computer through a commercially available

hardware system (MPl50 system). The data will be sampled at 400Hz and analyzed with

commercially available software (AcqKnowledge 3.7.1). The voluntary activation deficit will be

calculated based on the ratio between the torques produced by the superimposition of a

supramaximal twitch on a peak isometric contraction (a) and the torque produced by the same

stimulus in the potentiated, resting muscle (b).

Voluntary activation deficit (%) = (a/b)*100










3.1.5 Measures of Ambulatory Function

Lower extremity motor scores (LEMS). The voluntary muscle strength of 5 key muscles

(hip flexors, knee extensors, ankle dorsiflexors, toe extensors, ankle plantar flexors) of both

lower extremities is tested in accordance with the standard neurologic assessment developed by

ASIA. Each muscle will be given a value between 0 and 5 according to the strength of voluntary

muscle contraction. Maximum and minimum LEMS are 50 and 0, respectively.

Walking index for spinal cord injury (WISCI II). Physical limitation for walking

secondary to impairment is defined at the person level and indicates the ability of a person to

walk after spinal cord injury. The development of this assessment index required a rank ordering

along a dimension of impairment, from the level of most severe impairment (0) to least severe

impairment (20) based on the use of devices, braces and physical assistance of one or more

persons. The order of the levels suggests each successive level is a less impaired level than the

former. The ranking of severity is based on the severity of the impairment and not on functional

independence in the environment.

Level description.

0. Unable to stand and/or participate in assisted walking.

1. Ambulates in parallel bars, with braces and physical assistance of two persons, less than
10Ometers

2. Ambulates in parallel bars, with braces and physical assistance of two persons, 10
meters.

3. Ambulates in parallel bars, with braces and physical assistance of one person, 10 meters.

4. Ambulates in parallel bars, no braces and physical assistance of one person, 10 meters.

5. Ambulates in parallel bars, with braces and no physical assistance, 10 meters.

6. Ambulates with walker, with braces and physical assistance of one person, 10 meters.

7. Ambulates with two crutches, with braces and physical assistance of one person, 10










meters.

8. Ambulates with walker, no braces and physical assistance of one person, 10 meters.

9. Ambulates with walker, with braces and no physical assistance, 10 meters.

10. Ambulates with one cane/crutch, with braces and physical assistance of one person, 10
meters.

11. Ambulates with two crutches, no braces and physical assistance of one person, 10
meters.

12. Ambulates with two crutches, with braces and no physical assistance, 10 meters.

13. Ambulates with walker, no braces and no physical assistance, 10 meters.

14. Ambulates with one cane/crutch, no braces and physical assistance of one person, 10
meters.

15. Ambulates with one cane/crutch, with braces and no physical assistance, 10 meters.

16. Ambulates with two crutches, no braces and no physical assistance, 10 meters.

17. Ambulates with no devices, no braces and physical assistance of one person, 10 meters.

18. Ambulates with no devices, with braces and no physical assistance, 10 meters.

19. Ambulates with one cane/crutch, no braces and no physical assistance, 10 meters.

20. Ambulates with no devices, no braces and no physical assistance, 10 meters.

3.2 Experiments in Contusion Spinal Cord Injured Animals

3.2.1 Animals

The animal model will consist of young adult, female Sprague Dawley rats (16-20 weeks,

weighing 250-290gms). The animals will be housed in an AALAC accredited animal facility in a

temperature (22~11C), humidity (50110%) and light controlled room (12: 12 hours light: dark

cycle), and will be provided rodent chow and water ad libitum. The rats will be acclimatized for

a week prior to the start of experiments. All procedures will be performed in accordance with the









US Government Principle for the Utilization and Care of Vertebrate Animals and will be

approved by the Institutional Animal Care & Use Committee at the University of Florida.

3.2.2 Contusion Injury

Spinal cord contusion injuries will be produced using a NYU (New York University)

impactor device (Figure3-4). A 10g weight will be dropped from a 2.5-cm height onto the T8

segment of the spinal cord exposed by laminectomy under sterile conditions. Procedures will be

performed under ketamine (100mg/kg)-xylazine (6.7mg/kg) anesthesia. Subcutaneous lactated

Ringer' s solution (5 ml) and antibiotic spray will be administered after completion of the

surgery. Animals will receive two doses of Ampicillin per day for 5 days, starting at the day of

surgery. Animals will also be given Buprenorphine (0.05mg/Kg IM) and Ketoprofen (5.0 mg/Kg

SC) for pain and inflammation over the first 36hrs after SCI. The animals will be kept under

vigilant postoperative care, including daily examination for signs of distress, weight loss,

dehydration, and bladder dysfunction. Manual expression of bladders will be performed 2 to 3

times daily, as required, and animals will be monitored for the possibility of urinary tract

infection. Animals will be housed in pairs with the exception of the first few hours following

surgery. At post-operative day 7, open field locomotion will be assessed using the Basso-Beattie-

Bresnahan (BBB) locomotor scale and animals that do not fall within a preset range (1-4) will be

excluded from the locomotor training study.

3.2.3 Locomotor Training

Animals that will receive locomotor treadmill training will be trained for five consecutive

days (1 week of training), 2 trials/day, 20 minutes/trial, starting on post-operative day eight.

Training will consist of a quadrapedal treadmill stepping (Figure3-5). On the first day of training,

animals will be given five minutes to explore the treadmill and then encouraged to walk on the

moving treadmill at a speed of 11 meters per minute, for a series of four, five-minute bouts. A









minimum of five minutes rest will be provided between bouts. Body weight support will be

provided manually by the trainer. The level of body weight support will be adjusted to make sure

that the rats can bear their weight and there will be no collapse of their hind limbs. Typically, the

rats will start stepping when they have experienced some small load on their hind limbs. In

addition, during the first week of training, when all rats have profound hind limb paralysis,

assistance will be provided to place rat hind limbs appropriately for plantar stepping during

training. On the second day of training, animals will complete two 10 minute bouts, twice a day.

Starting on day three, animals will be trained continuously for 20 minutes with a minimum

interval between trials. Bodyweight support through the trunk and the base of the tail will be

provided as necessary and gradually removed as locomotor capability improved.

3.2.4 In-Vitro Assay of Muscle Composition and Regeneration

3.2.4.1 Immunohistochemical analysis

The muscles required for analysis will removed from one of the hind limbs of the animal.

The muscles were subsequently rapidly frozen in isopentane pre-cooled in liquid nitrogen

(storage at -800C) for the following immunohistological measurements.

Fiber CSA measures: Cryostat sections (10 Cpm) in a transverse plane will be prepared

from the central portion of each muscle taken from the both legs and mounted serially on gelatin-

coated glass slides. Immunocytochemical reactions will be performed on each cryostat section

with anti-laminin to outline the muscle fibers for cross-sectional area (CSA) quantification. The

fiber CSAs will be analyzed using the SCION image program. The pixels setting used for

conversion of pixels to micrometer is 1.50 pixels- 1 Clm2 for a 10 X objective. The average

maximal CSA for the soleus, gastrocnemius, TA and EDL will be quantified.

MHC Measures: Immunocytochemical reactions for quantifying fiber type transformation

will be performed on serial cryostat sections with anti-laminin and anti-MHC antibody at various









dilutions. Rabbit anti-laminin (Neomarker, Labvision, Fremont, CA) will be used to outline the

muscle fibers for cross-sectional area quantification. Four anti-MHC antibodies (BA-D5, SC-71,

BF-F3, and BF-3 5) will be selected on the basis of their reactivity toward adult MHC (figure3-

6). Sections will be incubated with rabbit anti-laminin and one of the anti-MHC antibodies (40C

over night), followed by incubation with rhodamine-conjugated anti-rabbit IgG and Fitc-

conjugated anti-mouse IgG (Nordic Immunological Laboratories). Stained sections will be

mounted in mounting medium for fluorescence (Vector Laboratories, Burlingame, CA) and kept

at 40C to diminish fading. Stained cross sections will be photographed (10X magnification) by

using a Leica fluorescence microscope with a digital camera. A region of the stained serial

sections from each muscle will be randomly selected for MHC composition analysis. The

proportions of each fiber type will be determined from a sample of 150-250 fiber across the

entire section of each muscle.

Immunohistochemical measures for Pax-7 and embryonic myosin will be performed

using similar methodologies.

3.2.4.2 Western blot analysis

Quantification and expression of MyoD, Myf5 and Myogenin will be measured using

Western blot analysis. Muscles will be homogenized in a lysis buffer with Fast-Prep

homogenizer machine at 13,000 RPM at 40C for five minutes. The supernatant will be preserved

for protein assay. Protein will be denatured by heating samples to 95-100 OC for 5 minutes.

Protein will be measured using BCA protein assay kit from Pierce. Electrophoresis will be

performed by mixing 40-50 Cpg protein with 5X loading buffer and loading it to 4-15% SDS page

gel from Bio-Rad. Protein will then be transferred from gel to nitrocellulose membrane.

Blocking will be conducting using 5% non fat dry milk in TBS/T (Tris Buffer Saline, Tween-









20). Blot with be incubated with primary antibody overnight at 40C according to manufacturer's

instruction. Blot will then be incubated with HRP-conjugate secondary antibody for 40 minutes

to one hour at room temperature. Finally protein will be detected using Western Blotting

Luminal Reagent from Santa Cruz.


Figure 3-1. Set-up for locomotor training.


Figure 3-2. Plyometric training set-up.




















































Figure 3-4. Contusion injury set-up modified from Meyer et al.2003.


11HCI isoforms
AiL~b
I Ha I~ IIb
B.--D5+--
SC-71 -+
BF-F3 ---+
BT-35S + + -+


Figure 3-3. Experimental set-up on a Biodex System 3 Dynamometer.


Figure 3-5. anti-MHC antibodies.


























Figure 3-6. Locomotor training in the rat model.









CHAPTER 4
LOWER EXTREMITY SKELETAL MUSCLE FUNCTION IN PERSONS WITH
INCOMPLETE SPINAL CORD INJURY

4.1 Introduction

Approximately 200,000 persons with spinal cord injury (SCI) live in the United States

alone; with roughly 11,000 new injuries occurring each year.' In addition, the relative number of

new injuries is rising and expected to have increased by approximately 20% by 2010, compared

to the 1994 prevalence. The reported costs associated with the care and treatment of persons after

SCI is estimated to range between $15,000- $125,000 annually, with an approximate lifetime

total of $43 5,000-$1,590,000.1 Interestingly, a significant proportion of these costs can be related

to the loss of skeletal muscle mass and associated secondary health-related complications 52, (i.e.

non-insulin dependent diabetes mellitus, cardio-vascular disease, osteoporosis). A decrease in

skeletal muscle function has been described as one of the most significant problems impacting

the health care and quality of life of persons after SCI.52, 39 COnsequently, the potential to

decrease costs and improve quality of life by maintaining or partially restoring skeletal muscle

size and function seems high.

Due in part to advancements in the quality of emergency care, the relative number of

injuries classified as incomplete has risen dramatically over the past 20 years.2 In fact, the

maj ority of new injuries occurring annually are now classified as incomplete.' Despite the rise in

the proportion of persons with incomplete-SCI; the preponderance of scientific literature

describing the effects of SCI on skeletal muscle involves persons with complete injuries.49,138-140

To date very little data exits describing muscle function in persons with chronic incomplete-SCI.

Interestingly, the innate plasticity associated with incomplete- SCI furnishes these persons with

the potential to progress functionally to a greater extent than the complete SCI population.141,142

In addition, novel intervention therapies have shown promise in promoting spinal plasticity and









motor function after spinal cord injury. However, improvements in functional capacity in persons

with I-SCI with rehabilitation vary greatly and the incidence of disability still remains high.143,144

In order to provide a foundation for the development of rehabilitation strategies targeting

neuromuscular deficits in persons with incomplete-SCI, a need exists to characterize and

obj ectively quantify existing impairments. Therefore, the purpose of this study was to quantify

lower extremity muscle function in persons with chronic incomplete-SCI compared to age-,

gender-, height- and body weight matched healthy controls. Specifically, we measured isometric

peak torque and performed measures of explosive or instantaneous muscle strength in the knee

extensor and ankle plantar flexor muscle groups and quantified voluntary activations deficits

using a combination of voluntary contractile measurements and superimposed electrical

stimulation.

4.2 Methods

4.2.1 Subjects

Ten persons with chronic, upper motor neuron lesions and motor incomplete-SCI

participated. Characteristics of the persons with incomplete-SCI are provided in Table 4-1.

Average age, height and body mass a standard deviation (SD) at the time of the study enrollment

were 45.4 & 14.8yrs, 155.9 & 9.4cm, and 79.9 & 12.2kg. Eight of the subjects were classified

ASIA D and two as ASIA C. Four subj ects were able to ambulate over ground, while six used a

wheelchair as their primary mode of locomotion. The incomplete-SCI subj ects were matched on

the basis of age, gender, height and weight with ten recreationally active controls (45.1 +

14.9yrs, 159. 1 & 9.0cm, and 78.01 11.7kg). Prior to participating in the study, written informed

consent was obtained from all subj ects, as approved by the Institutional Review Board at the

University of Florida, Gainesville.









4.2.2 Experimental Set-Up

Voluntary and electrically elicited contractile measurements were performed in the self-

reported more-involved and less-involved limbs for the knee extensor and plantar flexor muscle

groups, using a Biodex System 3 Dynamometert. Knee extensor testing was performed with

subj ects seated in an upright position with hips flexed to ~85 and knees flexed to ~90 The axis

of rotation of the dynamometer was aligned with the axis of the knee j oint and the lever arm

secured against the anterior aspect of the leg, proximal to the lateral malleolus. Testing of the

plantar flexor muscle group was performed with the hips flexed at 90- 100 the knee flexed at

~10. and the ankle at ~0. plantar flexion, as previously described.13, 14 The anatomical axis of the

ankle was aligned with the axis of the dynamometer, while the foot was secured to the footplate

with straps placed at the forefoot and ankle. Proximal stabilization was achieved with straps

across the chest, hips and thigh. Electrical stimulation was performed using a Grass S8800

Stimulator with a Grass Model SIU8T stimulus isolation unit Electrically induced contractions

were delivered through two 3.0" by 5.0" self-adhesive neuromuscular stimulation electrodes

placed over the proximal and distal portions of the muscle group being tested. The stimulator and

the dynamometer were interfaced with a personal computer through a commercially available

hardware system (MPl50 system)". The data were sampled at 400Hz and analyzed with

commercially available software (AcqKnowledge 3.7.1).



1 Grass Instruments, West Warwick, Rhode Island, USA.

"fBiodex Medical Systems, Inc., 20 Ramsay Road, Shirley, New York 11967
BIOPAC systems Inc., Goleta, CA









4.2.3 Voluntary Contractile Measurements

Prior to testing, subj ects performed three warm-up contractions to get familiarized with the

testing procedures. Subj ects then performed three maximal voluntary isometric contractions (~5

second each with 1 minute rest intervals) while being given verbal encouragement. Peak torque

was defined as the highest value obtained during the 3 maximal isometric contractions. In the

event that the peak torque values differed by more than 10%, additional contractions were

performed.

In addition to peak torque we also determined the average rate of torque development

(ARTD) and the torque200, aS indices of explosive muscle strength. The ARTD was defined as

the average increase in torque generated in unit time, and was calculated in the time interval

corresponding to 20% to 80% of peak amplitude, starting from muscle perturbation. This time

interval was selected to reduce the effect of errors in calculating peak amplitude. Hence ARTD

was calculated through numerical differentiation as

1 f,
ARTD =
N ,= 6 t

where N is the total number of time slots for numerical differentiation, GJis the change in torque

in the time slot i and Gtis the unit time duration for a slot. Torque200 was defined as the absolute

torque reached at 200ms during a maximal voluntary contraction (Nm).

4.2.4 Electrically Elicited Contractile Measurements

Peak twitch torque, time to peak twitch and twitch half-relaxation time were determined by

delivering a supra-maximal electrical stimulus (600Cls pulse duration) to the muscles at rest.

Supra-maximal intensity was determined by increasing the current voltage until twitch torque

production plateaued. Time to peak twitch and twitch half-relaxation time were calculated from

the peak twitch contractions.









4.2.5Voluntary Activation Deficits

Voluntary activation deficits were determined using the twitch interpolation method.l5

Briefly, a single biphasic, and supra-maximal pulse was delivered at rest and during maximal

voluntary isometric contraction. The voluntary activation deficit was calculated based on the

ratio between the torques produced by the superimposition of a supra-maximal twitch on a peak

isometric contraction (a) and the torque produced by the same stimulus in the potentiated, resting

muscle (b).

Voluntary activation deficit (%) = (a/b)*100.

4.2.6 Statistical Analyses

Independent sample T-tests were used to determine if differences existed between the

groups. Comparisons were made between the self-reported dominant side of the controls and

both the self-reported more involved side and less involved side of the incomplete-SCI group.

For all analyses, significance was established when P< 0.05. Data are presented as means+

standard error of mean. All statistical analyses were performed using SPSS for Windows,

Version 11.0.1.

4.3 Results

4.3.1 Voluntary Contractile Measurements

Individuals after incomplete-SCI demonstrated significant deficits in their ability to

generate peak isometric torque relative to non-injured controls in both the knee extensor and

plantar flexor muscle groups (p<0.05). A representative ankle plantar flexor torque trace

acquired during a peak isometric contraction of both incomplete-SCI and control subj ect is

provided in Figure 4-1. The peak torque deficit measured in both muscle groups was of similar

magnitude (Figure. 4-2). Specifically, persons after incomplete-SCI were able to produce 36%









and 24% of the knee extensor torque and 3 8% and 26% of the plantar flexor torque generated by

non-injured controls in the less-involved and more-involved limbs, respectively (p<0.01).

Significant bilateral asymmetries were noted in peak torque production between the self-reported

more-involved versus the less-involved limb in both the knee extensor (57118 vs. 85120 Nm)

and plantar flexor muscle groups (2616 vs. 3917 Nm; p<0.01).

Both indices of explosive muscle strength, ARTD and torque200, WeTO Significantly lower

in persons with incomplete-SCI relative to controls in both muscle groups tested (P<0.01;

Figure4-3, 4-4). Bilateral asymmetries in torque200 and ARTD were specific to the ankle plantar

flexor muscles. Of interest to note is that both indices of explosive muscle strength, showed more

pronounced deficits in the ankle plantar flexor muscles compared to the knee extensor muscles.

In particular large deficits were noted in the torque200 Of the ankle plantar flexor muscles with an

11.7 fold difference between the torque200 meaSured in the self-reported more involved limb and

a 5 fold difference in the less-involved limb compared to control muscles (Figure4-1&4-3). The

torque200 WAS 4.211.6 Nm in the plantar flexor muscles of the more-involved limb, 9.211.6 Nm

in the less-involved limb and 47.219.2 Nm in the non-injured controls, respectively. In contrast,

a 5.5 fold difference and 3.7 fold difference was noted in the torque200 meaSured in the knee

extensor muscles of the self-reported more involved (27.0113.2 Nm) and less involved limb

(39.9112.3 Nm) of incomplete-SCI persons compared to non-injured controls (148.6118.3 Nm).

Torque200 and ARTD data are summarized in Figures 4-3 & 4-4.









4.3.2 Electrically Elicited Contractile Measurements

No significant differences were found either within or between subj ect groups for measures

of peak twitch torque, time to peak twitch or half-relaxation times in either the knee extensor or

plantar flexor muscle groups (Table 4-2).

4.3.3Voluntary Activation Deficits

A significant injury related effect on the ability to voluntarily activate the plantar flexor

and knee extensor muscle groups was noted. Activation deficits in the knee extensors were 4218

% and 6619% in the less involved and more involved side, respectively, compared to only a

5+2% deficit in non-injured controls. The incomplete-SCI group also demonstrated a 5316 %

voluntary activation deficit in the less involved side and a 6418% deficit in the more involved

side for the plantar flexor muscle group, compared to a 512% deficit in non-injured controls

(Figure 4-5). Significant bilateral asymmetries existed for both muscle groups for voluntary

activation deficits (p<0.05, Figure 4-5). A representative torque trace acquired during a peak

isometric voluntary contraction with interpolated twitch is provided in Figure 4-6.

4.4 Discussion

The development of novel intervention therapies to promote the recovery of skeletal

muscle function after incomplete-SCI is one of the exciting paths of current rehabilitation

research.87,145-149 However, the translation of these experimental therapies to the SCI population

is enormously challenging given the extreme heterogeneity in presentation and response to

treatment of this population. As such, a comprehensive examination of skeletal muscle function

in this patient population might aid in the development of targeted therapies aimed at the

recovery of muscle function after incomplete-SCI. Accordingly, the present study demonstrates

that after chronic upper motor lesions and incomplete-SCI, both knee extensor and plantar flexor

skeletal muscles 1) generate ~70% less peak torque, 2) demonstrate significant bilateral










asymmetry in peak torque, which matches the hierarchy for self-reported functional deficits, 3)

experience voluntary activation deficits ranging between 42% and 66%, and 4) demonstrate large

deficits in the rate of torque development and instantaneous muscle strength. While in this study

both muscle groups demonstrated significant impairments in ARTD and torque200, mOTO

pronounced deficits were noted in the ankle plantar flexor muscles and bilateral asymmetries in

ARTD and torque200 WeTO Specific to the ankle plantar flexor muscles. Given the role of the

ankle plantar flexor muscles in propulsion during gait we put forward that the latter impairments

should be targeted in rehabilitative interventions aiming to restore or promote locomotion in this

population.

The deficits noted between persons after incomplete SCI and controls in their ability to

generate peak torque in the plantar flexor and knee extensor muscle groups may appear

somewhat intuitive. In addition, the bilateral asymmetries observed may be considered obvious

by many after this type of injury. However, no quantitative measurements of muscle function

have previously been reported in this population. Moreover, we contend that the methodologies

described here are more suitable than traditional evaluative tests in assessing impairments of

muscle function in persons with incomplete-SCI. Muscle strength assessments in persons with

incomplete-SCI are typically performed using manual muscle tests during ASIA evaluations. The

ASIA is used routinely to describe the level of injury and impairment and imply severity of

injury.lso1st However; this evaluative tool may not be adequate to direct targeted rehabilitation

interventions in persons with incomplete-SCI. Manual muscle tests are subj ect to a ceiling effect,

lack sensitivity to change and have a relatively poor inter-rater reliability, especially at scores

greater than 3.54,143









A myriad of physiological changes occur in persons after spinal cord injury. Many of these

changes are due to the direct effects of the injury (i.e. neural circuitry disruption) while others are

secondary in nature and attributable to a resultant decrease in neuromuscular activity. An

inability to voluntarily activate skeletal muscles may be a product of both primary and secondary

mechanisms. Twitch interpolation is a commonly used method to estimate the extent to which a

person can voluntarily activate a given muscle or muscle group.152-155 Our Eindings of small

activation deficits (~5%) in the quadriceps and ankle plantar flexor muscles of non-injured

controls are consistent with those from other laboratories." The activation deficits measured in

persons with incomplete-SCI (42-66%) are larger in magnitude compared to those measured in

patients early after surgery or long-term immobilization.156,157 Accordingly, persons with

incomplete-SCI may benefit from rehabilitation strategies that target voluntary activation deficits

to maximize skeletal muscle function, i.e. functional electrical stimulation or bio-feedback.l5

While these interventions may not directly impact the primary injury, they may be able to

ameliorate the loss of muscle function secondary to disuse or lack of neuromuscular activity.

Perhaps the most functionally relevant characteristics of muscle torque production for

persons with incomplete-SCI are the indices of explosive strength. ARTD is reflective of the

average rate of contractile torque development during maximum voluntary contraction while

torque200 iS the absolute torque generated within the initial 200ms of contraction and is indicative

of the magnitude of instantaneous torque. Both ARTD and torque200 WeTO Significantly reduced

in the ankle plantar flexor and quadriceps muscle groups of persons with incomplete-SCI.

However the deficit in instantaneous strength was more pronounced in the ankle plantar flexor

muscles. It is our contention that the initial rate of torque development and the instantaneous

strength may be most critical for performance of functional tasks (i. e. walking). For example,









steady state walking is characterized by repetitive, reciprocal contractions of the plantar flexor

muscles (i. e. propulsion at push off) that must be accomplished in Einite periods of time. A speed

commonly deemed necessary for persons to safely ambulate in the community is 1.2 m/s.145 At

this speed, the time it takes to complete one gait cycle (i.e. right heel strike to right heel strike) is

~1.0 seconds. Given that the plantar flexor muscles are reported to be active for ~40% of the gait

cycle and approximately V/2 Of that time is spent generating concentric torque, roughly 200ms is

available for torque generation by this muscle group.159 Given this available time, plantar flexor

muscles must generate torque of sufficient magnitude and at precise rates so as to propel the

mass of the body forward, translating to movement or walking.160 We speculate that the large

deficits in instantaneous torque in the ankle plantar flexors observed in this study (11.7 and 5

fold difference in torque200) may potentially limit locomotor function in persons with

incomplete-SCI. Thus, rehabilitative strategies must be employed that result in improved rates

of torque production and enhanced instantaneous torque to meet the imposed demands of

walking at community ambulating speeds.161 Although we chose to examine torque generation

at 200ms based on our calculations of muscular demands at a functionally minimal gait speed

(1.0 m/s), consideration should also be given to the fact that as functional improvements are

realized, the contractile demands (i.e. magnitude and rate of force production) will continue to

increase and the available time to generate torque will decrease.

An interesting finding in the present study was the lack of difference in the electrically

elicited contractile properties between persons after incomplete-SCI and non-injured controls.

Previous studies have used these properties as a means to explain molecular and histochemical

changes that occur in skeletal muscle.52 Studies using both animal and human models have

provided evidence for faster contractile properties following SCI.50,52,162,163 However, we









observed no differences in rate of rise or relaxation of electrically elicited contractions in

muscles after incomplete-SCI relative to non-injured controls. This is somewhat surprising in

that both the knee extensor and plantar flexor muscle groups have been characterized by faster

contractile speeds following SCI 50,162. These Eindings have been used to support the idea of a

fiber type transformation following SCI (slowafast). However, whether a fiber type transition

occurs after incomplete-SCI and the timeline for any potential shift are yet unclear. Thus, further

research and tissue sampling is warranted before we can make any suggestions towards the

muscle fiber type transformation based on contractile properties in this population.

In conclusion, this study characterizes the impairments in lower extremity skeletal muscle

function in persons after incomplete spinal cord injury relative to non-injured controls. The

examination of knee extensor and plantar flexor muscle groups in this study is clinically

meaningful given the anti-gravity responsibilities of each of these muscle groups and their

purported roles in standing and locomotor function.164,165 Reduced peak torque production,

ARTD and torque200, aS well as increased voluntary activation deficits were found to be

characteristic of affected muscles below the level of incomplete spinal cord injury. In addition, a

hierarchy of these impairments existed between limbs with significant bilateral asymmetries in

the plantar flexor muscle group for all variables tested. This characteristic asymmetry suggests

that recovery and response to rehabilitation may be specific to each side, with rate limiting

factors to functional performance potentially being limb rather than subj ect specific. We

speculate that the large deficit in the rate of torque development and instantaneous torque in the

ankle plantar flexors of persons with incomplete-SCI limits locomotor function.










Table 4-1. Characteristics of incomplete SCI subj ects
Level of ASIA Duration Mobility
injury Classification of injury LEMS WISCI-II Status
(mos)
S1 C6 D 20 35 19 Ambulator
S2 T4 D 7 44 19 Ambulator
S3 C4 D 16 45 13 Wheelchair
S4 C6 C 14 15 8 Wheelchair
S5 C6 D 37 40 16 Wheelchair
S6 C4 D 18 48 20 Ambulator
S7 C8 D 28 37 16 Wheelchair
S8 C4 C 22 26 9 Wheelchair
S9 C5 D 16 34 13 Wheelchair
S10 C6 D 39 38 19 Ambulator




Table 4-2. Electrically elicited contractile measurements


Incomplete-SCI
more-involved less-involved


Controls


Knee Extensors


Peak twitch force (Nm)
Time to peak twitch (ms)
Twitch-half relaxation time (ms)


29.1 & 2.3
123.3 & 5.8
73.8 & 5.7


27.3 & 2.8
135.6 & 5.3
107.1 & 20.1


31.6 &3.9
129.3 & 5.8
95.1 +11.0


Plantar Flexors


Peak twitch force (Nm)
Time to peak twitch (ms)
Twitch-half relaxation time (ms)


13.8 &1.5
143.8 & 7.2
116.2 & 7.2


14.8 & 0.8
143.9 & 7.3
125.7 & 9.2


14.5 & 0.8
144.3 & 5.2
127.3 A 11.2











120


ControlI


40 -i Incomplete-SCI





0 70 140 210 280 350 420 490
Ti me (ms)

Figure 4-1. Representative torque-time curve. Drop down arrows indicate time points at which
peak torque is reached in a representative incomplete-SCI and control subj ect. Shaded
areas indicate torque200 in both subjects.













300


250 -


E 200

-
P 150 -
O
1-


Knee Extensors Plantar Flexors


Figure 4-2. Peak torque (Nm) for the knee extensor and plantar flexor muscle groups, comparing
the dominant side of the control with the more involved (more-involved) and less
involved limb (less-involved) of the incomplete-SCI group. Significant difference
between control group and incomplete-SCI group. "f Significant difference between
the less-involved versus the more-involved (p<0.05).


Hmore-involved
O less-involved
O controls










180

160 -

140 -

120

100 -

80 -

60


r rL


I


Knee Extensors


UI
Plantar Flexors


Figure 4-3. Torque200 (Nm) (A) knee extensor and (B) plantar flexor muscle groups, comparing
the dominant side of the control with the more involved (more-involved) and less
involved limb (less-involved) of the incomplete-SCI group. Significant difference
between control group and incomplete-SCI group. "f Significant difference between
the less-involved versus the more-involved (p<0.05).


Smore-involved
O less-involved
M controls












Smore-involved
O less-involved
M controls


900 -


8 -
700 -

600 -

500 -

400 -


3 -
200 -

100


|l


o-l


Knee Extensors


300 -


250


200 -


150 -


100


Plantar Flexors


Figure 4-4. Average rate of torque development (ARTD)(Nm/sec) for the (A) knee extensor and
(B) plantar flexor muscle groups, comparing the dominant side of the control with the
more involved (more-involved) and less involved limb (less-involved) of the
incomplete-SCI group. Significant difference between control group and
incomplete-SCI group. "f Significant difference between the less-involved versus the
more-involved (p<0.05).













80% -

S70%

a 60% -
-
50% -
O
40%




10% -


Plantar Flexors


0%


Knee Extensors


Figure 4-5. Voluntary Activation Deficits (%) for the knee extensor and plantar flexor muscle
groups, comparing the dominant side of the control with the more involved (more-
involved) and less involved limb (less-involved) of the incomplete-SCI group. *
Significant difference between control group and incomplete-SCI group. ? Significant
difference between the less-involved versus the more-involved (p<0.05).


Hmore-involved
O less-involved
2 controls




























Figure 4-6. Torque trace acquired during MVIC with interpolated twitch to quantify muscle
activation deficit. A single supramaximal intensity electrical stimulus was
superimposed on a maximal voluntary isometric contraction (a), as well as on a
resting, potentiated plantar flexor muscle (b).


V olunltary activation (%) = a b)"100n


0 500 1000 1500 2000

Timne (msec)
Superimnposed twitch


0500


Resting twitch









CHAPTER 5
LOCOMOTOR TRAINING AND MUSCLE FUNCTION AFTER INCOMPLETE SPINAL
CORD INJURY: A CASE SERIES

5.1 Introduction

Traumatic spinal cord injury (SCI) is one of the most disabling health problems facing

adults today. Despite advances in treatment interventions individuals with SCI often lose the

ability to walk and are at risk to develop secondary health complications. Muscle atrophy and

reduced ability to generate force play essential roles in the development of disability after SCI.

Individuals with chronic complete spinal cord injury show 42-68% atrophy in the calf and thigh

muscles one year after injury, while subj ects with incomplete-SCI demonstrate a 25-30%

reduction in average lower extremity muscle cross-sectional area (CSA).46 Few studies have

performed a quantitative analysis of skeletal muscle strength after incomplete-SCI.37 However,

we recently demonstrated in persons with chronic upper motor lesions and incomplete-SCI, that

both knee extensor and plantar flexor skeletal muscles generate ~70% less peak torque, with

even larger reductions in measures of instantaneous or explosive peak torque.53

Repetitive locomotor training with body weight support has emerged as a potential

promising therapeutic intervention to promote motor recovery and ambulation following

incomplete-SCI. 166-168 Locomotor training has been suggested to have a positive impact on

walking ability, 168,169 functional independence and subj ective well being.170 Giangregorio et

at. 109 171 and Stewart et al. 3 have also shown that locomotor training involves sufficient

mechanical loading to induce muscle plasticity, increasing muscle size and altering the muscle

phenotype both after acute and chronic incomplete-SCI. Interestingly, studies involving animal

models of incomplete-SCI have also shown that LMT has the potential to augment the force

generating capabilities of affected lower hind limb muscles. 111,114 To our knowledge, no study

has systematically investigated the effect of locomotor training on lower extremity muscle force










production and instantaneous power in persons with incomplete-SCI. Mostly; studies rely on

manual muscle tests and ASIA motor scores to assess voluntary strength in persons with

incomplete-SCI. However, ASIA scores have been criticized to lack sensitivity and to have a

limited ability as indicators of neuromuscular recovery in chronic SCI.54,78,143,168,169

Therefore, the purpose of this study was to determine the effect of nine weeks of locomotor

training on lower extremity muscle function in persons with chronic incomplete-SCI using

isokinetic dynamometry. Specifically, we measured peak isometric torque, torque developed

within the initial 200 ms of contraction (Torque200) and the average rate of torque development

(ARTD) in the knee extensor and ankle plantar flexor muscle groups. In addition, we quantified

voluntary activations deficits using superimposed electrical stimulation. The knee extensor and

plantar flexors muscles groups were selected for study because of their purported role during

human locomotion.

5.2 Methods

5.2.1 Subjects

Five persons (one woman, four men) with chronic motor incomplete-SCI underwent nine

weeks (45 sessions, 5-times /week) of locomotor training. A summary of the subject's

demographics is provided in Table 5-1. Criteria for inclusion included: 1) age 18-70; 2) history

of SCI as defined by the American Spinal Injury Association (ASIA) Impairment Scale

categories C or D; 3) first time traumatic SCI at cervical or thoracic levels (C4-T12) resulting in

upper motor neuron lesions in the lower extremity; 4) medically stable and asymptomatic for

bladder infection, decubitis, cardiopulmonary disease or other significant medical complications

prohibiting testing and/or training; and 5) if using anti-spasticity medication, agreement to

maintain current levels throughout the study. Exclusion Criteria were as follows: 1) participation

in a rehabilitation or research protocol that could influence the outcome of this study. Prior to









participating in the study, written informed consent was obtained from all subj ects, as approved

by the Institutional Review Board at the University of Florida, Gainesville.

5.2.2 Locomotor Training Protocol

The locomotor training intervention consisted of 45 training sessions (5x/ week) spread

over nine weeks, with each session consisting of 30 minutes of step training on the treadmill with

body weight support (BWS) immediately followed by 20 minutes of level overground walking

and community ambulation training. Including pre-training stretching, donning/doffing the

harness, and additional time spent on the treadmill for stand training and standing rest breaks, the

total session duration was approximately 75 to 90 minutes per day. Each subject completed all

of the training sessions. With the aid of the body weight support, treadmill speed and manual

trainers, the treadmill training environment facilitated delivery of locomotor specific practice.

79,168 Trunk, lower limb, and upper limb kinematics were consistently assisted and/ or monitored

by trainers to assure appropriateness in relation to normal walking. Speed of treadmill stepping

was kept in a range consistent with normal walking (2.2-2.8 miles/hr). Progression of training

was achieved by decreasing BWS, altering speed, increasing trunk control, decreasing manual

assistance for limb control and increasing the time spent walking on the treadmill per bout. A

more detailed description of the training principles, parameters and progression has been

provided by Behrman & Harkema et al. 2000. 79 Overground training consisted of an immediate

assessment of the participant' s ability to stand and/or walk independently overground and an

evaluation of the deficits limiting achievement of this goal. These deficits became the focus for

goal setting in the next day's training session. Additionally, overground training addressed

translation of the skills from the treadmill to the home and community identifying practical ways

for the participants to incorporate new skills into everyday activities.









5.2.3 Experimental Protocol

5.2.3.1 Strength assessment

Voluntary contractile measurements were determined in the self-reported more-involved

and less-involved limbs for the knee extensor and plantar flexor muscle groups before and after

locomotor training, using a Biodex System 3 Dynamometer. Testing was performed with

subj ects seated with hips flexed to ~85., as previously described. 53 For knee extensor testing, the

knees were flexed to ~90 and the axis of rotation of the dynamometer was aligned with the axis

of the knee j oint and the lever arm secured against the anterior aspect of the leg, proximal to the

lateral malleolus. Plantar flexor testing was performed with the knee flexed at ~30 and the ankle

at ~0 plantar flexion. The anatomical axis of the ankle was aligned with the axis of the

dynamometer, while the foot was secured to the footplate with straps placed at the forefoot and

ankle. Proximal stabilization for all testing was achieved with straps across the chest, hips and

thigh.

5.2.3.2 Voluntary contractile measurements

Prior to testing, subj ects performed three warm-up contractions to become familiar with

the testing procedures. Subj ects then performed three maximal voluntary isometric contractions

(~5 seconds each with 1 minute rest intervals) while being given verbal encouragement. Peak

torque was defined as the highest value obtained during the 3 maximal contractions. In the event

that the peak torque values differed by more than 5%, additional contractions were performed. In

addition to peak torque we also determined the absolute torque generated during the initial

200ms of contraction (Torque200) aS well as the average rate of torque development (ARTD)

during the contractile effort, as previously described. 53









5.2.3.3 Voluntary activation deficits

Voluntary activation deficits were determined using the twitch interpolation method. 152

Briefly a single biphasic, supra-maximal electrical pulse was delivered at rest and during

maximal voluntary isometric contraction. Voluntary activation deficit was calculated using the

ratio between the torques produced by the superimposition of a supra-maximal twitch on a peak

isometric contraction (a) and the torque produced by the same stimulus in the potentiated, resting

muscle (b).

Voluntary activation deficit (%) = (a/b)*100.

Electrical stimulation was elicited using a Grass S8800 stimulator with a Grass Model SIU8T

stimulus isolation unit. Electrically induced contractions were delivered through two 3.0" by 5.0"

self-adhesive neuromuscular stimulation electrodes placed over the proximal and distal portions

of the muscle group being tested. The stimulator and the dynamometer were interfaced with a

personal computer through a commercially available hardware system (Biopac MPl50 system)

sampling at 400Hz and data were analyzed with commercially available software

(AcqKnowledge 3.7.1).

5.2.4 Statistical Analyses

A longitudinal, prospective case series was used in which participants completed nine-weeks of

locomotor training. Individual data have been summarized in tables and as plots.

5.3 Results

5.3.1 Voluntary Contractile Measurements

All individuals with chronic incomplete SCI demonstrated a significant improvement in

their ability to generate peak isometric torque following locomotor training. The most robust

increase in isometric peak torque production was observed in the ankle plantar flexor muscles

(average increase 43.9+20.0%) of the self-reported more involved limb, followed by the knee










extensor muscles of both the more involved (21.1112.3%) and less involved (19.816.3%) limb.

Individual gains in peak torque ranged from 8% to 45% in knee extensor and 14% to 98% in the

plantar flexor muscle groups. Note that four out of five subjects showed an increase in isometric

peak torque in at least three of the tested muscle groups. Individual torque data prior to and after

nine weeks of locomotor training are summarized in Table 5-2.

Both indices of explosive muscle torque generation, ARTD and Torque200, Showed large

improvements in the ankle plantar flexor and knee extensor muscles with locomotor training. In

particular, large bilateral improvements in plantar flexor Torque200 meaSures were realized, with

average relative improvements of 5871247% and 2191126% in the more-involved and less-

involved limbs, respectively. Individual increases in ankle plantar flexor Torque200 ranged from

8% to 83 5%. A more variable response was noted in the knee extensors with some subj ects

showing an enhancement in the more involved limb (subjects 2, 3 and 5) and others in the less

involved limb (subj ect 1 and 4). Torque200 data for both the knee extensors and ankle plantar

flexors are presented in Figures 5-1A-D. ARTD values showed a similar pattern with relatively

larger improvements in the ankle plantar flexor muscles compared to the knee extensors (Table

5-2). The mean ARTD in the ankle plantar flexor muscles improved from 36.3 f 16.5Nm/s to

46.9 f 13.3 Nm/s in the more involved limb and from 68.2 f 23.2 Nm/s to 102.8 f 32.7 Nm/s in

the less involved limb. The mean ARTD in the knee extensor muscles increased from

207.91112.9 Nm/s to 252. 11115.7Nm/s and from 325.51132.6Nm/s to 392.71137.0Nm/s.

5.3.2 Voluntary Activation Deficits

All subj ects showed voluntary activation deficits in both the knee extensor and ankle plantar

flexors muscles prior to LMT. Interestingly, a significant training effect was noted in the ability

to voluntarily activate the bilateral knee extensor muscle groups as well as the more-involved










plantar flexor muscles. Mean activation deficits in the knee extensors improved from 63f15% to

43f10% and from 41f16% to 31f16% in the more involved and less involved sides,

respectively. Only one subj ect (subj ect 4), the subject with the highest pre-LMT knee extensors

strength, did not show any improvement in knee extensor activation deficit after LMT. Similar to

the knee extensors, activation deficits in the more involved plantar flexors improved from

61f10% to 41f11% after nine weeks of locomotor training. Individual data are summarized in

Figure 5-2A & B.

5.4 Discussion

The results of this case series suggest that nine weeks of locomotor training in persons with

chronic motor incomplete SCI results in positive alterations in lower extremity skeletal muscles

that include an improved ability to generate both peak and instantaneous torque about the knee

and ankle joints. Interestingly, increases in force production were more pronounced in the ankle

plantar flexor muscles versus the knee extensor muscles, consistent with previous literature

suggesting that the ankle plantar flexors are critical for propulsive force generation during

locomotion and experience high loads.159,172 Superimposed electrical stimulation further showed

that improvements in muscle strength with locomotor training are accompanied with a decrease

in voluntary activation deficit.

A myriad of physiological changes occur in persons as a results of traumatic spinal cord

injury. Many of these changes are due to direct effects of the injury (i.e. neural circuitry

disruptions), while others are linked to pharmacological side effects or due to the lack of

neuromuscular activity and loading. Among the physiological changes is a dramatic loss in the

ability to voluntarily produce muscle force, leading to impaired motor function and disability.

We previously demonstrated that isometric peak torque generation in the knee extensor and










plantar flexor muscle groups is reduced by about 70% in person with chronic incomplete SCI (>1

year), compared to age- gender- and body weight- matched control subj ects.53 Individuals in the

present study demonstrated similar reduced plantar flexor and knee extensor peak torque values

prior to locomotor training. Forty-five sessions of locomotor training resulted in a robust increase

in isometric peak torque production in the ankle plantar flexor muscles (average increase

43.9120.0%) of the self-reported more involved limb and the knee extensor muscles of both the

more involved (21.1112.3%) and less involved (19.816.3%) limb. The ability to improve

peripheral muscle strength in persons with incomplete-SCI seemingly adds to the positive

attributes previously contributed to this experimental therapeutic intervention. In addition to peak

torque generation, we suggest that the functionally more relevant characteristics of muscle torque

production in person with incomplete SCI are represented by the indices of explosive or

instantaneous strength, ARTD and Torque200. ARTD represents the average rate of contractile

torque development during maximum voluntary contraction, while Torque200 meaSures the

absolute torque generated within the initial 200ms of contraction. We previously showed that

both ARTD and Torque200 are Significantly reduced in persons with incomplete SCI, with more

pronounced deficits in the ankle plantar flexor muscles compared to the knee extensor muscles.53

In particular large deficits were noted in the Torque200 Of the ankle plantar flexor muscles with an

11.7 fold difference between the Torque200 meaSured in the self-reported more involved limb and

a 5 fold difference in the less-involved limb compared to control muscles.

With nine weeks of locomotor training large improvements in both measures of

instantaneous muscle strength were noted. In particular, large bilateral improvements in plantar

flexor Torque200 meaSures were realized, with average relative improvements of ~600% and

200% in the more-involved and less-involved limbs, respectively. Smaller and less consistent









relative gains were realized in the knee extensor muscle group. The large increase in the

Torque200 Of both ankle plantar flexor muscle groups with locomotor training deserves special

attention, given these muscles' importance during bipedal walking. At a speed commonly

deemed necessary for persons to safely ambulate in the community (1.2 m/s),173 a time window

of only about 200ms is available to generate the necessary concentric torque in the plantar flexor

muscle group to produce forward propulsion.159 Data from our previous and current study

combined indicate that the torque produced by the ankle plantar flexors in this time window is

significantly reduced in persons with incomplete SCI and can be considerably improved with

intense locomotor training.53 An improved ability to generate instantaneous torque may be

critical to facilitating functional recovery and ambulation in patients with incomplete SCI. The

suggested importance of plantar flexor muscle torque generation for improving ambulation in

persons with central nervous system injuries is not new and has been reported in persons post-

stroke. 172,159

The ability to drive oc-motoneurons to elicit maximal muscle recruitment is often referred

to as maximal voluntary activation and can be estimated using superimposed electrical

stimulation, a method commonly implemented in a variety of populations.1l54,155,174 In a previous

study, we measured voluntary activation deficits ranging between 42% and 66% in the lower

extremity muscles of incomplete-SCI subj ects, whereas control subj ects showed a ~5% voluntary

activation deficit.53 Similar voluntary activation deficits were found in this study prior to

locomotor training. Interestingly, voluntary activation deficits were partially attenuated

following 45 sessions of locomotor training (30-40% post-training), even though they did not

return to normal values. In particular in the knee extensor muscles bilateral improvements in

voluntary muscle activation contributed significantly to gains in muscle force production, while









muscle cross-sectional area was relatively unchanged. Improvements in muscle activation in

persons with incomplete SCI with locomotor training have also been reported using iEMG.165 Of

interest to note is that voluntary activation deficits can also be observed following disuse or

immobilization. However, in these models the phenomena is transient and normalization in

muscle activation is typically observed after 3 to 4 weeks of rehabilitation.l7

Despite the measured increases in instantaneous and peak force production, and

improvements in voluntary activation only one of the five participants in this study showed any

change in their lower extremity motor scores (LEMS) after locomotor training. In specific,

subject 3 improved his LEMS score from 3 5 to 3 8. In all other subj ects no change in LEMS

score could be detected. These data are consistent with other locomotor training studies, which

often fail to demonstrate a change in ASIA scores with training in persons with chronic

injuries.79,168,169 We believe that the lack of change in ASIA motor scores in the present study

reflects a limitation in the measurement tool. Compared to isokinetic dynamometry, manual

muscle tests have a limited inter-rater reliability and have been criticized to lack sensitivity,

especially at scores above 3 (out of 5).54,143 Others have argued that while ASIA scores are

valuable in predicting motor recovery in acute patients, they may be less powerful as measures of

neuromuscular recovery in chronic SCI.110,176-178

In conclusion, nine weeks of locomotor training resulted in improved lower extremity

skeletal muscle function in persons after incomplete spinal cord injury. Specifically, extensor

muscles about the ankle and knee joint demonstrated an improved ability to generate both peak

and instantaneous torque. Relative gains in muscle function were greatest in the ankle plantar

flexor muscles, consistent with their critical role for propulsive force generation and high loading

during locomotion. Ankle plantar flexor muscles also showed a significant increase in maximal










CSA, while increases in knee extensor force production were mainly linked to improvements in

voluntary muscle activation. Finally, we suggest that skeletal muscle alterations contribute to the

functional improvements reported with locomotor training in person with incomplete-SCI.


Table 5-1.
Age
(yrs)

S1 44


Characteristics of incomplete SCI subj ects
Height Body ASIAR Level
(cm) Mass Impairment of
(kg) Classification Injury
154.9 74.8 C C6


Duration
of injury
(months)
20


Mobility
status

Power-
wheelchair

Bilateral-
canes

Bilateral-
crutches

Wheelchair


Wheelchair


LEMS
(pre-
LMT)
33/50


LEMS
(post-
LMT)
34/50


S2 21 185.4 68.0 D


S3 48 198.6 77.0 D


S4 58 183.0 90.7 D


S5 36 176.9 83.9 C


T4 8


C6 39


44/50O 44/50O


35/50 38/50


45/50


17/50


45/50


17/50










Table 5-2. Values of isometric peak torque and average rate of force development


Isometric Peak Torque

S1 S2 S3 S4 S5

Knee Extensors
M~ore Involved
Pre-LMT 35.8 95.8 26.8 176.5 10.8
Post-LMT 42.6 84.6 39.0 181.8 15.7

Less Involved
Pre-LMT 65.0 136.5 62.6 179.0 15.7
Post-LMT 70.3 190.5 78.0 199.9 18.0

Plantar Flexors
M~ore Involved
Pre-LMT 11.7 45.5 19.4 65.2 12.5
Post-LMT 23.3 51.9 29.1 63.3 20.0

Less Involved
Pre-LMT 24.8 52.3 42.7 90.2 21.9
Post-LMT 35.5 60.7 51.7 82.5 21.0


Average Rate of Torque Development

S1 S2 S3 S4 S5


Knee Extensors
M~ore Involved
Pre-LMT
Post-LMT

Less Involved
Pre-LMT
Post-LMT

Plantar Flexors
More Involved
Pre-LMT
Post-LMT

Less Involved
Pre-LMT
Post-LMT


92.6
182.5


270.4
325.9


281.5
337.1


338.0
588.1


62.3
81.8


233.1
239.2


572.1
613.3


727.1
746.8


30.8
46.0


58.1
63.3


17.8
30.8



36.5
101.0


27.4
44.4



47.0
82.7


20.0
45.9



103.8
105.0


94.7
91.2



130.3
203.5


21.2
22.2



22.9
21.9








140

1120 -



Ss-



1- 40


20 -


O


S3


S1 S2


160-


140-

-120-
E
z 100-

a 80-
s-


o I mI I m, I mr-
S1 S2 S3 S4 S5
Figure 5-1. Torque200 (Nm) measured in the knee extensor muscle group of the (A) more
involved and (B) less involved limb of individuals with incomplete-SCI before (pre-
LMT) and after locomotor training (post-LMT).


SPre -LTM

SPos5t-LTM


SPre -LTM

Post-LTM











30-

S25-
-

'20-
-
o

o 15-
5-

0-




50-
45-
40-
,"35-
E
E 30-
o 25-

2-
S15-
10-
5-


S3


S4


S5


S1 S2


S2


S3


S4 Ss


S1


Figure 5-2. Torque200 (Nm) measured in the plantar flexor muscle group of the (A) more
involved and (B) less involved limb of individuals with incomplete-SCI before (pre-
LMT) and after locomotor training (post-LMT).


OPre -LTM

Post-LTM


SPre -LTM

Post-LTM










-o S1
SS2
S3
SS4
S5


120%


100%


80%


60%


40%


20%


Alq


More involved


Less involved


Figure 5-3. Voluntary activation deficits (%) measured in the knee extensor muscle group, of the
more involved and less involved limb of individuals with incomplete-SCI before
(Pre) and after locomotor training (Post).










-o S1
SS2
S3
SS4
S5


120%


100%


80%


60%


40%


20%


Alq


More involved


Less involved


Figure 5-4. Voluntary activation deficits (%) measured in the plantar flexor muscle group of the
more involved and less involved limb of individuals with incomplete-SCI before
(Pre) and after locomotor training (Post).









CHAPTER 6
RESISTANCE TRAINING AND LOCOMOTOR RECOVERY AFTER INCOMPLETE
SPINAL CORD INJURY: A CASE SERIES

6.1 Introduction

The proportion of persons that suffer a spinal cord injury (SCI) resulting in an incomplete

lesion has risen dramatically over the past 20 years. As a result ~55% of the new injuries

sustained in the United States are now classified as incomplete. In addition, the life expectancy

for persons with an incomplete injury is higher than after a complete SCI and is approaching that

of non-injured persons, regardless of age at injury.' As such, the increased incidence and

prevalence of persons with this type of injury necessitates a comprehensive understanding of the

adaptations that occur and the potential for rehabilitative interventions to impact persons with

incomplete-SCI. Unfortunately, despite the proportion of persons sustaining and subsequently

living with incomplete SCI, the preponderance of scientific literature describing the

physiological and functional adaptations to SCI involves persons with complete injuries.

Accordingly, limited data are available that describe motor function and its impact on

functional ability in this large subject cohort. The ability to independently ambulate is a primary

goal of many persons after SCI. However, even though a large number of individuals with

incomplete SCI regain some ability to walk, limitations in gait speed may make this method of

mobility impractical for activities of daily living. Slow speed combined with other mobility

deficits (e.g. difficulty climbing stairs, curbs, etc...), could negate the ability to safely ambulate in

the community, resulting in a perceived disability. Interestingly, rehabilitation practice focusing

on compensatory approaches to locomotion has largely been based on the prevailing assumption

that neural as well as functional recovery is limited in persons with chronic SCI. However, recent

evidence from both animal and human studies indicates that with the appropriate training stimuli,









neural as well as muscular plasticity can be induced even years after injury 47,140 Improvements

in functional ability, however, vary greatly and the incidence of disability remains high.52,141

Previous data suggest that persons after incomplete SCI produce less voluntary torque

about the knee and ankle than non-injured controls. Perhaps more importantly, impairments in

the ability to produce torque in a timely manner as well as a reduced walking velocity is also

common to these persons.53 It is our belief that reduced muscle power generation significantly

impacts locomotor function and that functional recovery can be facilitated with rehabilitation

interventions that attenuate this impairment. Specifically, the ankle plantar flexor and knee

extensor muscle groups are of interest primarily because of their purported roles during bipedal

locomotion, with torque demands at these joints during walking representing the two highest in

the lower extremity. As such, the potential for impaired torque production about these j points to

be a limiting factor in locomotor performance seems high.

The common goal of resistance training programs is to increase maximal strength in the

trained musculature. In addition, the focus of plyometric training, which incorporates high-

velocity stretch-shortening type contractions, has been to improve performance in activities

requiring fast contractions (e.g. jumping or sprinting).179,180 The combination of these two types

of training has been shown to be effective in improving both maximal strength as well as muscle

power production and 179,181 TOSult in improved jump height and sprint speed in neurologically

healthy individuals Interestingly, the potential for rehabilitative-training induced changes in

muscle strength and power to affect functional ability after incomplete SCI is largely unstudied.

In addition, whether potential increases in muscle function in these persons identified during

strength testing are reflective of improved muscle power output during functional tasks is

unknown and of obvious value. Accordingly, the challenge is to now develop, evaluate and










implement strategies that maximize neuromuscular plasticity in individuals after incomplete SCI

with the hopes of resultant improvements in functional capacity and a subsequent decreased

disability. As such, the purpose of this study was to determine if improvements in muscle

function accompanied by improvements in locomotor ability can be realized following a

combined resistance and plyometric jump training program in persons with chronic incomplete

SCI.

6.2 Methods

6.2.1 Subjects

Three independently ambulatory males with chronic motor-incomplete SCI participated in

this study. Criteria for inclusion included 1) age 18-70; 2) first time SCI (C5-T10); 3) medically

stable and asymptomatic for bladder infection, decubitis, cardiopulmonary disease or other

significant medical complications prohibiting testing and/or training; 4) if using antispasticity

medication, agreement to maintain current levels throughout study; Exclusion criteria were 1)

participation in a rehabilitation or research protocol that could influence outcomes of this study.

2) history of congenital SCI or other disorders that may confound treatment, study, and/or

evaluation procedures; Prior to participation, written informed consent was obtained from all

subjects, as approved by the Institutional Review Board at the University of Florida.

Subject 1, a 22 year-old male (69 kg, 185 cm), suffered a traumatic SCI (T4, 17 months

post-injury) and was classified as American Spinal Injury Association (ASIA) impairment level

D, with a lower extremity motor score (LEMS) of 44/50. Prior to RPT this subj ect had a self-

selected gait speed of 0.71 m/s and a maximal gait speed of 1.01 m/s. This subj ect completed 29

sessions of RPT over the 12-week study period.

Subject 2, a 61 year-old male (93 kg, 189 cm), suffered a traumatic SCI (C5, 27 months

post-injury) and was classified as ASIA D with a LEMS of 48/50 prior to RPT. Subj ect 2 had a









self-selected gait speed of 0.82 m/s and a maximal gait speed of 1.18 m/s. Subj ect 2 completed

30 sessions of RPT over the 12-week study period.

Subject 3, a 58 year-old male (88 kg, 178 cm), suffered a traumatic SCI (C5, 24 months

post-injury) and was classified as ASIA D with a LEMS of 3 5/50. Prior to RPT this subj ect had a

self-selected gait speed of 0.78 m/s and a maximal gait speed of 1.06 m/s. This subject

completed 30 sessions of RPT over the 12-week study period.

6.2.2 Resistance Training Program

Lower extremity progressive resistance training was 12 weeks in duration and subj ects

completed 2-3 sessions/week for a total of 30 sessions. Resistance exercises included unilateral

leg press, knee extension/flexion, hip extension/flexion and ankle plantar-flexion exercises

performed on adjustable load weight machines. During the initial training session a predicted 1-

repetition maximum (1-RM) was calculated for each subj ect and for each exercise. 1-RM was

determined using a prediction table based on a single set to volitional failure with load that

allowed between 6 and 12 repetitions. During subsequent training sessions, subjects performed

2-3 sets of 6-12 repetitions at a relative intensity of ~70-85% of predicted 1-RM. Maximal

strength was evaluated weekly to assess for training-related improvements and exercise loads

were adjusted accordingly. Specifically, if the subj ect achieved the target number of repetitions

for all prescribed sets of a given exercise, a new predicted 1-RM was prescribed and resistance

was increased for subsequent training sessions.

6.2.3 Plyometric Training

Unilateral plyometric jump-training exercises were performed in both limbs in a supine

position on a ballistic jump-training device (ShuttlePro MVP @, Contemporary Design Group,

Figure 1). Session intensity for this exercise was modified by changing either the resistance or

the number of ground contacts and progressed over the training period, accordingly. Briefly,









after familiarization with the training device, subj ects completed a total of 20 unilateral =ground

contacts (e.g. jumps) with each limb at a resistance of ~25% of body mass. Thereafter, upon

successful completion of at least 20 ground contacts per limb (e.g. complete clearance from foot

plate), resistance was increased in increments of 10 lbs. When a new resistance was set,

repetition goal was set at 10 ground contacts per limb for the initial session. Subsequent sessions

allowed for up to 20 contacts per limb. Thus, a minimum of two sessions at a given resistance

was required before load was increased. Resistance was held consistent between limbs

throughout the training program.182,183

6.2.4 Dynamometry

Strength measurements were performed in the PF and KE muscle groups using a Biodex

isokinetic dynamometer (Biodex Corp., Shirley, NY). PF strength was assessed with subj ects

seated in a semi-reclined (~700 hip flexion) position, with the knee flexed ~150 and the ankle in

an anatomical neutral position (00 of plantar flexion). The axis of the dynamometer was aligned

with the lateral malleolus, and the foot was secured with straps placed at the forefoot and ankle.

Proximal stabilization was achieved with straps across the chest, hips, and knee. KE strength

assessments were performed with subj ects seated in the same position used for PF testing, with

the exception that the knee was flexed to 900. The axis of the dynamometer was aligned with the

knee joint line, and the leg was secured to the lever arm.

Peak torque (Nm) was defined as the highest isometric torque achieved during 3 maximal

contractions (~3 sec contractions separated by a minimum of 60 seconds rest). In the event that

the peak torque values during the three trials differed by more than 5%, additional contractions

were performed. In addition to peak torque, values for T20-80, torque200 and ARTD were also

determined both pre- and post-RPT. These measures were used as indices of a subj ects' ability to

produce torque in an explosive manner and account for potential differences in both the timing









and magnitude of torque production. T20-80, USed to represent the time to peak tension, was

defined as the amount of time to generate from 20% to 80% of peak isometric torque. This time

interval was chosen to minimize potential errors in the determination of the precise onset and

nadir of torque development while still representing a maj ority of the time interval for achieving

maximal torque production. Average rate of torque development (ARTD) was defined as the

average increase in torque generated in unit time (Nm/s), and was calculated over the same

interval as T20-80. Hence ARTD was calculated through numerical differentiation as


ARTD =
N ,= 6 t

where N is the total number of time slots for numerical differentiation, GJ is the change in torque

in the time slot i and Gtis the unit time duration for a slot. Torque200 WAS defined as the absolute

torque reached at 200ms during a maximal voluntary contraction (Nm).

Torque220 WaS defined as the absolute amount of torque generated during the initial 220ms

during a maximal voluntary contraction and is based on the calculated time that is available for

concentric torque generation during a typical gait cycle at a speed designated necessary for

community ambulationl5. For example, the speed commonly deemed necessary for persons to

safely ambulate in the community is 1.2 m/s 159. At this speed, the time it takes to complete one

gait cycle (i.e. right heel strike to right heel strike) is ~1.1 seconds. Given that the plantar flexor

muscles are reported to be active for ~40% of the gait cycle and approximately 1/2 of this active

time is spent generating concentric torque, roughly 200 milliseconds is available for force

generation (e.g. propulsion) by this muscle group.

6.2.5 Voluntary Activation Deficits

Voluntary activation deficits were determined using the twitch interpolation method.152,184

Briefly, a single biphasic, supra-maximal pulse (600Cpsec pulse duration) was delivered at rest










and during maximal voluntary isometric contraction. Voluntary activation deficit was calculated

using the ratio between the torques produced by the superimposition of a supra-maximal twitch

on a peak isometric contraction (a) and the torque produced by the same stimulus in the

potentiated resting muscle (b). Voluntary activation deficits were expressed as: voluntary

activation deficit (%) = (a/b)*100.

6.2.6 Locomotor Data Collection

Subj ects performed repeated 10 meter walks over a 14 ft. long mat (Gait Rite) that

measures the geometry and the applied pressure of each footfall as a function of time in order to

determine both self-selected and maximal overground walking speed (3 trials each). Gait

analyses were performed 3 months prior to training as well as at both pre- and post-RPT time

points. Multiple baseline tests were conducted to control for improvements resulting from natural

recovery .

6.3 Results

6.3.1 Dynamometry

All subj ects demonstrated improvements in peak torque production, T20-80, torque200 and

ARTD during post- versus pre-RPT dynamometric testing. On average, RPT resulted in a 35.0

f 9.1% and 28.9 f 4.4% improvements in peak isometric torque production in the PF and KE

muscle groups, respectively. Individual gains ranged from 17% to 76% in the plantar flexors and

from 22% to 45% in the knee extensors. Time to peak tension, represented by T20-80, decreased

from 470.8 f 82.2 ms to 312.0 f 65.7 ms in the PF and from 324.5 f 35.4 ms to 254.2 f 34.5 ms

in the KE muscle groups following training. In addition, both indices of muscle power

generation, ARTD and torque220, WeTO HOticeably improved following training. Of interest to

note is that both torque220 and ARTD showed more pronounced improvements in the PF










compared to the KE muscles with training. Specifically, a 62.1% and 122.2 % improvement in

torque220 and ARTD were seen in the PF muscles, with only a 33.4% improvement in torque220

and a 66.4 % improvement in ARTD in the KE muscle group. In addition, the largest relative

gains in indices of explosive muscle strength (T20-80, torque200 and ARTD) occurred in the PF

muscle group of the more-involved limb. Peak torque, torque200, T20-80 and ARTD data are

summarized in Table 6-1.

6.3.2 Voluntary Activation Deficits

Significant voluntary activation deficits were noted in both the PF and KE muscle groups

prior to training. RPT resulted in reductions in activation deficits in both the PF and KE muscle

groups in each subj ect. Individual data for activation deficits are presented in Table6-1. Although

significant bilateral asymmetries existed prior to and following the intervention, these differences

were seemingly attenuated in both muscle groups following RPT.

6.3.3 Locomotor Analyses

Values for maximum and self-selected gait speeds did not differ by more than 0.04 m/s

and 0.02 m/s, respectively, for any of the subj ects in this study when comparing tests done 3

months prior to the onset of training and immediately prior to training. Following RPT, a 36. 1 %

average increase in maximum gait speed and a 34.7% average improvement in self-selected gait

speed were realized.

6.4 Discussion

The results of this study suggest that a combination of resistance and plyometric training in

persons with motor incomplete SCI results in bilateral improvements in 1) peak torque

production, 2) time to peak torque and 3) rate of torque production in the plantar flexor and knee

extensor muscle groups. These improvements in muscle function can be attributed to both an

increase in muscle cross-sectional area as well as an increased ability to voluntarily activate









affected skeletal muscles. Interestingly, the magnitude of improvement in these outcomes was

most pronounced in the more- versus the less-involved limb and in the PF versus the KE muscle

group. In addition, improvements in both self-selected and maximum gait speeds were realized

and were explained by increased propulsion in the more-involved limb as well as increased lower

extremity joint powers, suggestive of improved task specific muscle function (i.e. during

walking).

Injury to descending spinal pathways as well as decreased activation history both has the

physiological consequence of reducing the ability to voluntarily activate affected skeletal

muscles. Although restoration or repair of the injured spinal cord is not a reasonable expectation

with training, the potential to improve deficits resulting from disuse seems likely and has been

demonstrated after periods of inactivity in other populations. 160,185,186 In this study, significant

activation deficits existed prior to RPT that are comparable to other models of disuse (i.e. cast

immobilization, limb-suspension). *' Interestingly, these deficits were partially attenuated with

training and this enhancement of neural function could serve to explain a portion of the strength

gains realized post-RPT. In addition to enhanced neural transmission, muscle hypertrophy post-

RPT cannot be ignored as a mechanism for improved muscle torque production during both

dynamometric testing as well as during walking. However, though significant skeletal muscle

hypertrophy (e.g. larger effector) might suggest improved torque generation independent of the

activation pattern, the magnitude of strength gains would suggest that the majority of these gains

were accounted for by means other than muscle hypertrophy.

In this study we chose to examine the morphological and contractile characteristics of the

ankle plantar flexor and knee extensor muscle groups primarily because of their purported roles

during bipedal locomotion. Torque demands at these j points during walking are the two highest in









the lower extremity. In addition, we have previously shown that torque generation about these

j points is limited in persons after incomplete SCI.53 Similarly, subj ects in the present study

presented with reduced PF and KE peak torque values prior to RPT, as well as a reduced gait

speeds. Interestingly, marked improvements in PF and KE isometric torque generation and gait

speed were realized following RPT. However, post-RPT measures of peak torque about these

joints as well as maximum gait speeds are still reduced relative to control values 53, thereby

suggesting the potential for further functional improvements if additional increases in torque

production by these muscle groups can be realized.

In addition to absolute torque production, a likely mechanism explaining impaired muscle

function during locomotor tasks may be an inability to produce properly graded and timed

muscle output. This impairment has been identified in this and other populations with central

nervous system dysfunction 53,188-190 and shown to relate to reduced gait speed.190 The

combination of a prolonged time to peak torque and a decreased ability to generate maximal

torque in these persons suggests that at least some of limitations in gait speed in persons with

incomplete SCI might result from impaired muscle function. However, the dramatic

improvements in muscle function demonstrated in the present study highlight the potential for

this type of training to attenuate existing deficits in neuromuscular function and facilitate

functional improvements.

Recent therapeutic interventions examining gait in persons after CNS injury have largely

focused on the task specificity of training with little focus on impairment level deficits 151,168,191

Although the rationale for task-specific training interventions to result in improvements in motor

function is quite strong and shown to be effective in producing cortical reorganization 192,193W

feel that in-vivo muscle function is also limiting in these persons and appropriate training can









also induce neuroplastic changes in these tissues that facilitate locomotor improvements by

improving the element of muscle function dictated by locomotor task performance. Accordingly,

given that few studies have attempted to examine the relationship between lower extremity

strength and gait in persons after incomplete SCI, comparisons to other populations with CNS

involvement yield valuable information. For example, data examining the relative importance of

lower extremity strength in persons after stroke demonstrate significant correlations between the

strength of the paretic hip flexors (r = .57), knee extensors (r = .41) and primarily the ankle

plantar flexors (r = .85), with maximal gait speed.172,194 In addition, previous simulation work

suggests that force production by the soleus and gastrocnemius is critical to trunk forward

progression, swing initiation and power generation during gait.15~9 Thus, one might predict

slower gait speeds if force production by these muscles is abnormal during locomotion. Indeed,

the negative impact of reduced plantar flexor function is supported by experimental data. For

example, Lamontagne et al. suggested that more than 50% of the variance in gait speed in

persons post-stroke was explained by the peak activation of the medial gastrocnemius. In

addition, Mulroy et al. demonstrated that ankle moments were substantially reduced in two

groups of hemiparetic persons compared to slow walking controls, with household walkers

having reduced moments relative to limited community walkers.172 These same investigators also

found that at two different time points, walking speed was strongly associated with plantar flexor

voluntary strength. Specifically, deficits in plantar flexor strength were pronounced, with the

slow subj ect group (~10% of normal age-matched speed) demonstrating strength equal to ~18%

of normal age-matched strength upon admission to rehabilitation. Interestingly, at six months

post stroke, plantar flexor strength increased to 22% of control value, an increase of ~20%, and

was associated with increased walking speed (~ 20%). Thus, these data provide support to










suggest a relationship may exist between changes in plantar flexor strength and gait speed, at

least at slow velocities. Interestingly, the relative gains in plantar flexor strength in the present

study (35.0%) are almost identical to the increases in fastest (36. 1%) and self-selected (34.7%)

gait speeds post- RPT.

In conclusion, the importance of the proposed work revolves around the fact that little is

known about the extent to which skeletal muscle plasticity may impact functional outcomes after

incomplete SCI. The desire "to be more normal" with respect to locomotor ability is one that

many persons after this type of injury possess. Accordingly, the development of appropriate

rehabilitation strategies that target improvements in locomotor ability with the goal of increasing

functional independence could have a tremendous impact on this population. The data in the

present study provide support for the use of physical rehabilitation interventions aimed at

attenuating neuromuscular impairments as a means for improving not only gait speed but also the

strategies utilized by these persons to ambulate. As such, we suggest that the benefits reported

following a combination of resistance and plyometric training represent a first step in the use of

these modalities to facilitate the recovery of motor function and functional ability in this

population. Although, we report significant gains in strength and gait speed following 12 weeks

of RPT, at this point we do not know if the subj ects in thus study reached a plateau in any of the

outcomes measured. Therefore, future studies examining the impact of physical rehabilitation

training programs after incomplete SCI should focus on the optimal volume (e.g. duration and

frequency) and intensity of training, as well as the potential of this type of training to serve as an

adjunctive therapy in the overall treatment of these persons. In addition, these studies need not

only focus only on gait, but other functional outcomes (e.g. stair climbing, sit to stand) as well as































Table 6-1. Pre- and post-RPT isometric torque data for the plantar flexor and knee extensor

muscle groups.


Ire-RPT


Post-RPT


KNEE Activation Activation
EXTENSORS Peak Torque ARTD Torqu4oo T20-80 Defieit (%) Peak Torque ARTD Torqu420 T20-80 Defieit (%)


More-involved
S1 99.8 282.4 67.5 283.0 39.0
S2 100.3 204.6 44.1 440.8 34.0
S3 65.1 196.1 28.1 370.4 50.0
Less-involved
S1 136.4 482.1 78.2 254.1 32.0
S2 143.9 501.8 69.7 240.7 20.0
S3 112.5 330.9 53.8 360.2 19.0
Pre-RPT


125.7 478.6 88.9 241.6 31.0
123.8 497.4 71.0 210.6 25.0
81.6 244.5 30.5 280.4 35.0

177.6 706.2 108.5 250.3 29.0
176.1 827.3 102.4 300.9 14.0
162.7 570.5 54.2 215.5 18.0
Post-RPT


PLANTAR Activation Activation
FLEXORS Peak Torque ARTD Torqu42o T20-80 Defieit (%) Peak Torque ARTD Torqu42o T20-80 Defieit (%)
More-involved


S1 45.4
S2 27.3
S3 17.0
Less-involved
S1 56.7
S2 32.7
S3 33.2


59.1 13.7 807.3 36.0
50.4 12.1 430.1 42.0
28.6 5.6 490.9 41.0

105.2 14.4 403.2 18.0
95.0 26.7 380.6 34.0
84.5 12.5 315.9 47.0


56.1 95.7 22.8 587.7 28.0
36.1 119.5 27.5 240.1 31.0
26.8 102.9 9.5 280.6 28.0

66.4 259.8 26.4 252.2 16.0
42.8 164.9 34.4 300.4 15.0
58.6 256.1 16.9 215.4 41.0


the potential psychosocial benefits (i.e. community integration) that likely parallel increased


functional capacity.

























Figure 6-1. Example of plyometric training device.









CHAPTER 7
LOWER EXTREMITY SKELETAL MUSCLE MORPHOLOGY AND FIBER TYPE
COMPOSITION FOLLOWING MODERATE CONTUSION SPINAL CORD INJURY AND
LOCOMOTOR TRAINING

7.1 Introduction

Spinal cord injury (SCI) is a devastating condition which causes severe long lasting

neurological dysfunction and morbidity in humans.12,52,196 In addition to effects directly related

to CNS dysfunction, common problems experienced with SCI are skeletal muscle atrophy and

impaired muscle function leading to walking disabilities .45,52,69, 197,198 Animal models of SCI are

commonly used to evaluate the pathology of SCI and to ensure the feasibility and efficacy of

new therapeutic interventions. Commonly used animal models of SCI include transaction,

isolation, and contusion injuries.199,200 While the transaction and isolation models successfully

reproduce complete SCI, the contusion model is a clinically more relevant model as it is known

to closely mimic the mechanism and histopathologic sequela of the maj ority of current human

SCI (>55%, incomplete-SCI), thus making it a relevant model to study.20,200 In COntrast to the

complete SCIs in which animals experience significant atrophy and a complete loss of locomotor

capabilities, animals with contusion injuryl99,201 Show some spontaneous recovery of muscle size

and regain some locomotor function without any specific therapeutic intervention.1 99,202,203

Locomotor treadmill training has recently gained momentum as a therapeutic intervention

to improve lower extremity function and walking after SCI. Locomotor training is based on the

principle that stepping can be generated by virtue of the neuromuscular system' s responsiveness

to phasic, peripheral sensory information associated with locomotion.72,79,204,205 Although

locomotor treadmill training programs promote changes in spinal cord properties, motor unit

morphology, and functional recovery40,198,206,207, the impact of this training intervention towards

ameliorating atrophy and improving muscle function after SCI are not clear. Currently, a few









studies have looked at skeletal muscle adaptations after contusion SCI and locomotor training.

Min et al.2008208 characterized the longitudinal changes in rat lower hindlimb muscle

morphology following contusion SCI and locomotor training by using magnetic resonance

imaging over a three month period. The greatest amount of atrophy was observed at 2-week post-

injury and locomotor training as early one-week post injury significantly reduced atrophy and

improved function. In a follow up study, Stevens et al. 200665 evaluated therapeutic potential of

early locomotor training in the soleus muscle. Locomotor training appeared to ameliorate soleus

muscle atrophy and attenuate the shift in myosin heavy composition (1VHC) towards faster

isoforms. However, this study was limited to the slow postural muscle soleus only and

information on other lower extremity muscles is still warranted. Since it is known that different

lower extremity muscles adapt differently to unloading conditions based on their specific

function role and phenotype, it is important to investigate the influence of locomotor training on

muscle size and fiber type distribution in muscles with different functional roles and fiber type

composition.

The obj ectives of this study were 1) to quantify changes in fiber size and fiber type

composition following incomplete SCI in lower extremity muscles with different functional roles

and fiber type composition in the rat 2) to study the therapeutic influence of one-week of

locomotor training on lower extremity muscle with different functional roles and fiber type

composition in spinal cord-injured animals.

7.2 Methods

7.2.1 Animals

Twenty-four Sprague-Dawley rats (female, 228-260 g, weighing 250-290gms; Charles

River, NJ, USA) were used in this study. Six rats per group were assigned to either a SCI-

training group, a SCI-no training group, a control group, or a control training group. Six of the









injured rats received treadmill locomotor training (TM) starting 1 week after SCI, when the

surgical staples were removed and soft tissue had healed sufficiently to tolerate training without

increasing the risk of trauma at the incision site. Training in the TM group was implemented for

5 consecutive days, 20 min/trial, 2 trials/day. The additional 8 injured rats received no exercise

intervention (no TM). The rats were housed in a temperature-controlled room at 21 OC and were

provided unrestricted access to food and water. All procedures were approved by the Institutional

Animal Care and Use Committee at the University of Florida.

7.2.2 Contusion Spinal Cord Injury

Spinal cord contusion injuries were produced using a protocol described previously. A

NYU (New York University) impactor was used to produce the injuries. Briefly, a 10g weight

was dropped from a 2.5-cm height onto the T8 segment of the spinal cord which was exposed by

laminectomy. The entire procedure was carried out under sterile conditions. All injuries were

performed under ketamine (100mg/kg)-xylazine (6.7mg/kg) anesthesia. Animals received two

doses of Ampicillin (100mg/kg) per day for 5 days starting on the day of surgery. To prevent

dehydration, subcutaneous lactated Ringer' s solution (5 ml) was administered after completion

of the surgery. Animals were given Buprenophine (0.05 mg/kg) and Ketoprofen (5.0 mg/kg s.c.)

for pain and inflammation over the first 36 hours after SCI. The animals were kept under vigilant

postoperative care, including daily examination for signs of distress, weight loss, dehydration,

and bladder dysfunction. Manual expression of bladders was performed 2-3 times daily until

spontaneous voiding returned (~2 weeks), and animals were monitored for the possibility of

urinary tract infection. Animals were housed in pairs with the exception of the first few hours

following surgery.









7.2.3 Locomotor Treadmill Training

Animals with spinal cord injury were exposed to treadmill locomotor training. Training

was started on post-operative day 7. There were two reasons for this. First, on day 8 the surgical

staples were removed and soft tissue had healed sufficiently so that trauma could be avoided at

the incision site. Second, red porphyrin expression around the eyes, a symptom associated with

stress, disappeared within a week post SCI. Therefore, animals could be trained without apparent

discomfort and stress at this time.

Animals assigned to the treadmill training group were given Hyve minutes to explore the

treadmill on the first training day and then encouraged to walk on the moving treadmill (11

meter/minute) for a series of four, Hyve-minute bouts. A minimum of five minutes rest was

provided between bouts. On the second day of training, animals completed two bouts of ten

minutes each, twice a day. Starting on day 3, animals trained continuously for 20 minutes with a

minimum interval between training sessions of 2 hours. Training consisted of quadrapedal

treadmill stepping. Body weight support was provided manually by the trainer as necessary. The

level of body weight support was adjusted to make sure that the animals' hind limbs did not

collapse and was gradually removed as locomotor capability improved. Typically, when all rats

had profound paraplegia, assistance was provided to place the rat hind paws in plantar stepping

position during training.

7.2.4 Tissue Harvest

Muscle samples will be harvested from the normal control rats, two weeks post injury on

the SCI-no training rats, and one week post- training on the trained SCI and trained control rats.

The muscles will then be dissected and snap-frozen at resting length in isopentane, pre-cooled in

liquid nitrogen and stored at -80 OC.









7.2.5 Immunohistochemical Measures

Cryostat sections (10Clm) in a transverse plane were prepared from the central portion of

the soleus, TA, EDL and Gastroc muscles taken from both legs and mounted serially on gelatin-

coated glass slides. Immunocytochemical reactions were performed on serial cryostat sections

with anti laminin and anti-MHC antibody at various dilutions. Rabbit anti-laminin (Neomarker,

Labvision, Fremont, CA) was used to outline the muscle fibers for cross-sectional area

quantification. Four anti-MHC abs (BA-D5, SC-71, BF-F3, and BF-35) were selected on the

basis of their reactivity toward adult MHC. Sections were incubated with rabbit anti-laminin and

one of the anti-MHC antibodies (40C over night), followed by incubation with rhodamine-

conjugated anti-rabbit IgG and Fitc-conjugated anti-mouse IgG (Nordic Immunological

Laboratories, Tilburg, The Netherlands). Stained sections were mounted in mounting medium for

fluorescence (Vector Laboratories, Burlingame, CA) and kept at 40C to diminish fading. Stained

cross-sections were photographed (10x magnification) by using a Leica fluorescence microscope

(Leica Microsystems, Bannockburn, IL) with a digital camera. A region of the stained serial

sections from each muscle was randomly selected for MHC composition analysis. The

proportions of each fiber type were determined from a sample of 150-250 fibers across the entire

section of each muscle. The pixels setting used for conversion of pixels to micrometers were 1.5

pixels to 1 Clm2 for a 10x obj ective. The average fiber CSA of all the circle fibers was

determined. However, in order to minimize the risk of including nonmuscle tissue, areas

consisting of less than 100 pixels were excluded from the analysis.

7.2.6 Data Analysis

All statistical analyses were performed with SPSS, Version 13.0.1. Tests for normality will

be performed on all of the measured variables before proceeding with tests of statistical

inference. Results are expressed as mean + standard error of mean. One-way ANOVA was used









to test for differences among the four experimental groups. In an effort to control for multiple

comparisons, post-hoc analysis was implemented. For all analyses, significance was established

when p< 0.05.

7.3 Results

7.3.1 Effects of Incomplete- SCI and Locomotor Training on Fiber Crossectional Area
(CSA)

The effect on SCI on fiber size was determined in four different hindlimb muscles-Soleus,

Tibialis Anterior (TA), Extensor Digitorum Longus (EDL), and Gastrocnemius (Gastroc).

Immunohistochemistry was used to quantify the changes in these lower extremity muscles's size

after contusion SCI. Measurements were made in animals two weeks after SCI (SCI group) and

in animals with one week of locomotor training one week after SCI (SCI locomotorr training

group). The degree of atrophy in this study seemed not to be muscle phenotype or functional role

specific. The slow extensor soleus showed the maximum atrophy followed by the predominantly

fast flexor EDL, then the mixed extensor gastrocnemius, and finally the fast flexor TA. Two

weeks following SCI, the soleus showed significant reduction in fiber CSA (~29%) in

comparison to un-trained controls (p <0.05, Fig. 7-1). Locomotor training lead to a significant

increase in soleus CSA in comparison to untrained SCI group. This change in CSA observed

after locomotor training was not significantly different from the control group. Interestingly, one

week of locomotor training in the control rats did not result in any change in fiber CSA.

As shown in Figure 7-2, SCI also produced a significant loss in muscle fiber CSA in the

EDL in comparison to the control group (~28%, p<0.05). Locomotor training resulted in

significant increase in fiber CSA compared to the untrained SCI group. Even though the training

intervention was only partially effective in restoring fiber CSA towards control levels, the









difference between the control and SCI + locomotor training groups were not significantly

different (p<0.05).

Gastrocnemius being an extremely large muscle, the fiber CSA was determined from a

sample of 150-250 fibers located at areas which mostly stained positive for MHC type I. This

method was chosen also to help us study fiber type transformation in the Gastroc muscle

following moderate contusion SCI. Two weeks of SCI resulted in significant reduction in

average fiber CSA in the predominantly slow Gastroc (~22%, p<0.05). Interestingly, locomotor

training resulted in no change in average fiber CSA in the Gastroc in comparison to the

untrained-SCI group (p<0.05). However, we feel our results did not substantially justify the

influence of locomotor training in restoring fiber CSA in the Gastroc because our study was

specific to areas containing only type I MHC fibers and we strongly feel that the training might

have significantly influenced the other MHC fiber types (results not reported, Fig.7-3).

Finally in the TA muscle, two weeks of contusion SCI resulted in a non-significant ~12.6%

decrease in average CSA in comparison to controls,( p<0.05, Fig.7-4). Locomotor training once

again resulted in restoring muscle CSA in the TA towards pre-injury levels. No changes in CSA

were observed in the control trained group compared to the control group. Overall, these results

indicate atrophy following incomplete-SCI is fiber type and functional role specific with slow-

extensor showing maximum atrophy while the fast-flexor showing the least amount of atrophy.

In addition, locomotor training significantly contributed in reducing the extent of atrophy in all

lower extremity muscles except the gastrocnemius in spinally contused-animals.

7.3.2 Effects of Incomplete- SCI and Locomotor Training on Fiber Type Composition

The myosin heavy chain (MHC) molecule is an actin-based protein which plays an

important role in specifying skeletal muscle contractile properties. Therefore, we used MHC

staining to identify fiber type composition in animals following SCI and locomotor training.









The soleus muscle from the control untrained animals primarily contained fibers reacting

exclusively with type I monoclonal antibody (mAB) (~85%), indicating slow MHC isoforms,

and a small percentage of fibers reacting with type IIa mAb, exclusively. Two weeks following

moderate T8 contusion SCI, the proportion of type I fibers was reduced by ~10% compared to

controls and subsequently the reduction in type I fibers was replaced by fibers that co-expressed

both MHC-I and MHC-IIa and IIa and IIx (mixed fibers). Locomotor training prevented the

appearance of fibers that co expressed both type IIa and IIx in the soleus. In addition the

proportion of fibers that were stained positively with both types I and IIa were lower in the SCI-

trained animals than the SCI-untrained animals (Fig.7-5).

The TA muscle from untrained controls primarily consisted of fast MHC isoforms (i.e.

fibers reacting exclusively with type IIb mAb [~50%], followed by the type IIx [~25%] and IIa

[~20%]), and only a small percentage of pure type I fibers (Fig.7-6). In the SCI no -training

group, the proportion of type IIb fibers were higher by ~15% compared to the controls, while the

proportion of IIx and IIa fibers were lower. In addition, the TA from SCI animals also showed

mixed fibers that co-expressed both MHC-I and MHC-IIa and IIx and IIb, which were not

present in the controls. Locomotor training one week post-SCI resulted in the MHC fiber type

distribution recovering towards phenotypes represented by control TA muscles. In addition, there

were also a higher proportion of mixed fibers which co-expressed type IIx and IIa instead of the

faster IIb and IIx as seen in the SCI-untrained group. There was no difference in fibers that

expressed only type I among the SCI trained and untrained and control groups.

The EDL muscle is a mixed fast muscle containing primarily of fast MHC

isoforms. The percentage composition of types I, IIa, IIx and IIb MHC isoforms in the EDL of

control rats was ~411, 1613, 3412, and 4513% respectively (Fig.7-7). Two-weeks of contusion









SCI shifted the MHC profile toward faster isoforms i.e. the type IIb from 45 to 48% and the type

IIx from 34-28%. The other changes include the appearance of IIa + IIx and IIx + IIb fibers.

One-week of locomotor training resulted in a significant decrease of the type IIb fibers from 45-

32% and increase in the type IIa from 16-21% in comparison to SCI untrained group.

Furthermore, in comparison to the SCI untrained group, locomotor training resulted in the

reduction of IIx + Inb fibers which seemed to be replaced by an increase in the I + IIa fibers.

Even though, there seems to be a slight shift in MHC isoforms after training towards the slower

isoforms, these data suggest that type IIb is the default MHC isoform in the EDL both after SCI

and training, while training seems to have a positive influence in causing some shift in the MHC

isoform from fast to slow just one-week following contusion SCI.

The gastrocnemius is a muscle which is significantly compartmentalized relative to fiber

type composition. In order to study changes in fiber type composition with contusion SCI, we

choose to study only areas with the Gastroc which stained mainly for type I fibers. In the control

rats, our regions of choice compromised 5416, 2813, 1514, and 311% pure type I, IIa, IIx and

Imb fibers respectively. Following of two weeks of moderate contusion SCI, the MHC isoform

distribution was nearly even across groups. There was a ~28% type I, ~20% IIa, ~31%IIx, and

~22% IIb fibers. Interestingly, there was no appearance of fibers co-expressing two MHC

isoforms. However, the trend was different following one-week of locomotor training one week

post-SCI. The type I, IIa, IIx and IIB fibers were approximately 51%, 23%, 17%, and 9%. In

summary, the early training intervention just one-week post-SCI seemed to change the

expression of MHC in the Gastroc comparable to control levels (Fig.7-8).

7.4 Discussion

One of the major problems associated with spinal cord injury (SCI) irrespective of the type

of injury is loss of muscle mass as manifested by a reduction in cross-sectional area (CSA). This









reduction in CSA has been historically accompanied by fastening of the muscle contractile

properties manifested by an increased expression of faster myosin heavy chain (1VHC)

isoforms.20,58,209-212 However, these adaptations seen vary based on the functional role or fiber

type composition of the observed skeletal muscle.20,65,213 To better understand the impact of

contusion-SCI on skeletal muscle mass and phenotype, we studied changes in fiber CSA and

1VHC composition in four lower extremity muscles with different functional roles and fiber type

compositions. In addition, the therapeutic influence of locomotor training was also examined in

restoring muscle mass and attenuating the change in IVHC composition. The Eindings of the

current study demonstrate that contusion SCI results in significant atrophy in all lower extremity

muscles soleuss, extensor digitorum longus, tibialis anterior and gastrocnemius), and this was

accompanied by a shift in MHC composition in all the muscles towards faster isoforms.

Interestingly, locomotor training was effective in restoring muscle mass and IVHC composition

to pre-injury levels.

Numerous studies have been conducted in looking at the loss of muscle mass following

SCI.63,208,213,214 The majority of these studies have been performed following spinal transaction

or spinal isolation were minimal loading or minimal muscle activity was recorded following the

injury. In a few studies similar to our study, were changes in muscle mass were quantified two-

weeks following spinal transaction and isolation they observed an atrophy of ~41-50% in the

soleus, ~36-49% in the medial Gastroc, ~45% in the TA, and ~40% in the EDL.23,63,215,216 These

studies have indicated that muscle adaptive responses following transaction and isolation SCI are

similar in the early stages of atrophy (14-15 days), however following chronic inactivity muscle-

specific atrophic response is more in the slow-twitch muscles compared to the fast-twitch, and

more in the extensors compared to the flexors. 23,63,215,216 In COmparison, only a few studies have









looked at skeletal muscle morphology following contusion SCI and results from these studies are

conflicting. In the first study by Hutchinson et al. 200120 TepOrted a decrease of 20-25% and 16-

21% in all lower extremity muscles, at 1 and 3 weeks following moderate contusion SCI. In this

study they reported that muscle atrophy occurred in flexor as well as the extensor muscle and

that the extent of atrophy was similar in the fast and the slow muscles. In contrast Min et al.

2008208, USing MRI observed at 2 weeks post contusion SCI showed a hierarchal pattern of

atrophy, with the extensor triceps surae having more atrophy than flexors muscles. In the current

study, at 2-weeks post contusion injury, atrophy quantified through fiber CSA showed the

following hierarchy of atrophy: soleus>EDL>Gastroc>TA. The overall atrophy of ~12-29% was

observed in all the lower extremity muscles. Significant difference in fiber CSA after contusion

SCI was observed only between the slow-extensor soleus and fast-flexor TA. In summary, in

this study we demonstrated that there was significant atrophy in all lower extremity muscles 2

weeks following contusion SCI and the extent of atrophy measured through fiber CSA was

maximal in the soleus but similar between a slow-twitch and fast-twitch muscle and also similar

between a extensor and flexor. We feel this muscle response may be attributed to the

spontaneous recovery and muscle activity observed following the contusion injury.

The appearance of the different MHC isoforms in a muscle plays a defining role in

regulating the contractile and histochemical characteristics of the muscle.5,24,209 The maximal

velocity of shortening of muscle at least in part is dependant on the MHC composition of the

muscle. Research over the years has identified atleast four different MHC isoforms being highly

expressed in rat muscles. 5,24,209 They have been identified as MHC-I, IIa, IIx and Inb isoforms.

Hybrid fibers which coexpress multiple MHC isoforms also exist. Reduction in loading and

neuromuscular activity following SCI leads to fastening of the muscle contractile properties









resulting from an increased expression of faster MHC isoforms.62,213,217,218 There is also an

increased expression of hybrid fibers which co-express different MHC isoforms. Findings of our

current study are consistent with other studies performed following contusion injury, like studies

by Hutchinson et al. 200120 and Stevens et al. 200465 who reported increases in faster MHC

isoforms and appearance of hybrid fibers co-expressing different MHC isoforms two weeks

following contusion SCI. Specifically in our current study, the soleus had an increased

expression of IIx fibers and also the appearance of hybrid fibers expressing faster isoforms,

while both the TA and EDL had increases in IIb MHC expression. In the Gastroc the fibers

which were predominantly type I shifted to expressing equal levels of all MHC isoforms. To

summarize, the findings of our study are consistent with those of other studies indicating that 2

weeks post-SCI there is a significant shift in MHC composition in all lower extremity muscles

irrespective of functional role or fiber type to switch to faster isoforms. We feel the influence of

injury in modulating MHC composition is similar across all muscle groups at earlier time points

(2-weeks) and this might turn muscle specific at more chronic time points.

Motor recovery following spinal cord injury can be enhanced or accelerated by locomotor

treadmill training.72'79'204'205 Locomotor training uses the principles showing that rhythmic

loading of the limbs and force feedback from the hindlimb muscles induces task appropriate

activity-dependent plasticity. Following moderate contusion in rats, locomotor training has been

shown to induce substantial hindlimb muscle and motor recovery.65,114,208 Locomotor training

using treadmill has also produced significant improvement in locomotor recovery (limb axis,

base of support, BBB locomotor scale) compared with those of untrained injured controls.

65,114,208 In the current study, we monitored the impact of one-week of locomotor treadmill

training on the lower extremity muscles one week-post mid-thoracic spinal cord contusion injury









by studying changes in fiber CSA and IVHC composition. In this context our Eindings are unique

and suggest that early locomotor training can be effective in halting the atrophic process and

improving the rate of recovery by restoring fiber CSA and phenotype of lower extremity muscles

following contusion SCI. At the end of one week of locomotor training, no significant

differences in fiber CSA were noted between the locomotor trained group and the control group

in all the lower extremity muscles, except for the gastrocnemius which showed slightly lower

CSA values. One possible explanation for the apparent smaller changes in the Gastroc is that

measures for CSA were restricted to only the type I fibers and we feel that the training could

have impacted fibers of other phenotypes which are predominant in the Gastroc and if we had

averaged CSA across fibers of all the phenotypes we would have had a significant training

impact. An interesting finding in this study is early training intervention resulted in similar rates

of recovery in fiber CSA in all lower extremity muscles irrespective of their functional role or

fiber type composition. In addition, locomotor training was effective in attenuating the shift

IVHC composition towards faster isoforms. There was a significant recovery in the proportion of

fibers expressing slower isoforms in all the four lower extremity muscles. The restoration of the

slow IVHC phenotypes following locomotor training may also reflect a potential modulatory

decrease in the velocity of shortening in the muscle.

In summary, the findings of this study are encouraging because they demonstrate that

skeletal muscle atrophy and changes in muscle phenotype following two-weeks contusion SCI

are similar across muscles with different functional roles and fiber types and early locomotor

training starting one week post-SCI is effective in restoring fiber CSA and IVHC isoform

phenotype irrespective of the muscle functional role or fiber type. Although there are limitations

in using animal models to understand human SCI recovery with locomotor training, the present









study demonstrates that early training interventions will be effective in ameliorate the

debilitating effects of SCI in all the lower extremity muscles.














3000


2500


E Control
E 200 Control+TM

o~ oSCI+TM
1500


1000


500




So leus



Figure 7-1. Average soleus muscle fiber CSA for control, control+TM, SCI no TM, and SCI +
TM groups at 2 weeks post SCI. *Significantly smaller average muscle fiber CSA in
SCI no TM compared to control, control+TM, and SCI + TM groups, p<0.05.


2600 a control
a control+TM
SSCl
2000 SCl+TM



S1600







600


O


E xten so r D ig ito rurn Lo ng us


Figure 7-2. Average EDL muscle fiber CSA for control, control+TM, SCI no TM, and SCI + TM
groups at 2 weeks post SCI. *Signifieantly smaller average muscle fiber CSA in SCI
no TM compared to control, control+TM, and SCI + TM groups, p<0.05.












control
5 control+TM
SCl
o SCl+TM


2600


2000 -



S1600







600-



0-


Jr n


Gastroonernius


Figure 7-3. Average gastrocnemius muscle fiber CSA for control, control+TM, SCI no TM, and
SCI + TM groups at 2 weeks post SCI. *Signifieantly smaller average muscle fiber
CSA in SCI no TM compared to control and control+TM groups, p<0.05.
#Significantly smaller average muscle fiber CSA in SCI + TM compared to control
and control+TM groups, p<0.05.


2500 control
Scontrol+TMI
SSCI
T __O SCI+TMI


S1500-



$ 1000



500-



0-


Tibialis Anterior


Figure 7-4. Average TA muscle fiber CSA for control, control+TM, SCI no TM, and SCI + TM
groups at 2 weeks post SCI.











100

90 III Control
80
SSCI
701 OSCI + TM
60


40

30

20



I I +H~a IIa IIa +Hx
Soleus


Figure 7-5. MHC based fiber type composition of rat soleus from control, control+TM, SCI, and
SCI+TM groups.


m control
SCI
O SCI+TMI


80

70

,S 60

'r50
-c
S40

S30

LL20

10

0


type I l+lla Ila Ila+11x Ilx
Tibialis Anterior


Ilb+11x Ilb


Figure 7-6. MHC based fiber type composition of rat TA from control, control+TM, SCI, and
SCI+TM groups.


_~ .I












m control
SCI
O SCI+TMI


60 _


50 -


.0 4 0





10 -

30


L;r


type I l+lla


Ila Ila+11x Ilx Ilb+11x


Extensor Digitorum Longus


Figure 7-7. MHC based fiber type composition of rat EDL from control, control+TM, SCI, and
SCI+TM groups.



70 a control
7H SCI


0 SCI+TMI


60


S50

S40
( 3

20

10



10


type I Ila Ilx Ilb
Gastrocnemius


Figure 7-8. MHC based fiber type composition of rat gastrocnemius from control, control+TM,
SCI, and SCI+TM groups.









CHAPTER 8
SKELETAL MUSCLE RECOVERY AND REGENERATION FOLLOWING MODERATE
CONTUSION SPINAL CORD INJURY AND LOCOMOTOR TRAINING

8.1 Introduction

Incomplete spinal cord injury (SCI) is a debilitating human condition resulting in severe

motor and sensory impairments below the level of injury.52,196,219 In addition to effects directly

related to CNS dysfunction, atrophy of skeletal muscle is a common problem associated with

incomplete SCI.45,52,163,206 Animal models of SCI have been used to characterize lesions, study

mechanisms of recovery, and to develop and test therapeutic interventions. 14,73,220 Although the

maj ority of current SCI' s are incomplete (>55%), most animal studies of skeletal muscle

adaptations after SCI have been done following complete spinal cord injuries.45,93,221 Therefore,

it may be relevant to use an animal model of incomplete SCI to study skeletal muscle adaptations

and the effects of therapeutic interventions. One such model is the contusion injury model which

mimics the mechanism and histopathologic sequela associated with human incomplete SCI.

Skeletal muscle possesses a remarkable ability to recover after damage or disuse atrophy.

One way skeletal muscle can recover involves the activation, proliferation, and differentiation of

a resident population of myogenic cells called satellite cells.122,133 These satellite cells induce

muscle plasticity by differentiating and fusing to form multinucleated myotubes which repair or

replace damaged or lost muscle fibers. Activation of satellite cells seems to require growth

factors, such as insulin-like growth factor 1 (IGF-1), which have also been shown to increase

muscle protein and DNA content.122,222,223 Once activated, the upregulation of these cells can be

identified by using various molecular markers.123,128 Activated satellite cells committed to

myogenic lineage express the transcription factor Pax-7. The myogenic regulatory factors, MyoD

and Myf5 are involved in satellite cell proliferation, and the expression of transcription factor

myogenin signals satellite cell terminal differentiation into myotubes. Finally, the appearance of










embryonic myosin signals new fiber formation. Despite the fact that SCI results in significant

muscle atrophy, only a few studies have been done to assess satellite cell activity after SCI, and

they have been done only after complete SCI. Furthermore, the results of these studies have

suggested greater satellite cell activity in slow-twitch extensor muscles than in fast-twitch flexor

muscles. Therefore, it may be relevant to study satellite cell activity after incomplete SCI and to

do this study in both slow-twitch extensor and fast-twitch flexor muscles.

Locomotor treadmill training has been used as a therapeutic intervention to improve lower

extremity function and/or walking after SCI. Although locomotor treadmill training programs

promote changes in spinal cord properties, motor unit morphology, and functional

recovery,40,198,206,207 the particular contributions of this therapy towards skeletal muscle plasticity

and function are still unclear. Numerous studies have shown that therapeutic interventions like

treadmill training, resistance exercise, and cycling training result in the activation of satellite

cells.224-226 However, to our knowledge, no studies have been done to assess the effects of short

term locomotor training after incomplete SCI on satellite cell activation and regulation.

The obj ectives of this study were 1) to investigate the effects of incomplete SCI (moderate

contusion model) on satellite cell activity in a slow-twitch extensor and a fast-twitch flexor

muscle in the rat and 2) to examine the influence of one week of locomotor training on satellite

cell activity in these muscles in spinal cord-injured animals. Satellite cell activity was monitored

by measuring IGF-1 and by using various molecular makers.


8.2 Methods

8.2.1 Animals

Twenty-four Sprague-Dawley rats (female, 228-260 g, weighing 250-290gms;, Charles

River, NJ, USA) were used in this study. Six rats per group were assigned to either a SCI-









training group, a SCI-no training group, a control group, a control training group. Six of the

injured rats received treadmill locomotor training (TM) starting 1 week after SCI, when the

surgical staples were removed and soft tissue had healed sufficiently to tolerate training without

increasing the risk of trauma at the incision site. Training in the TM group was implemented for

5 consecutive days, 20 min/trial, 2 trials/day. The additional 8 injured rats received no exercise

intervention (no TM). The rats were housed in a temperature-controlled room at 21 OC and were

provided unrestricted access to food and water. All procedures were approved by the Institutional

Animal Care and Use Committee at the University of Florida

8.2.2 Contusion Spinal Cord Injury

Spinal cord contusion injuries were produced using a protocol described previously. A

NYU (New York University) impactor was used to produce the injuries. Briefly, a 10g weight

was dropped from a 2.5-cm height onto the T8 segment of the spinal cord which was exposed by

laminectomy. The entire procedure was carried out under sterile conditions. All injuries were

performed under ketamine (100mg/kg)-xylazine (6.7mg/kg) anesthesia. Animals received two

doses of Ampicillin (100mg/kg) per day for 5 days starting on the day of surgery. To prevent

dehydration, subcutaneous lactated Ringer' s solution (5 ml) was administered after completion

of the surgery. Animals were given Buprenophine (0.05 mg/kg) and Ketoprofen (5.0 mg/kg s.c.)

for pain and inflammation over the first 36 hours after SCI. The animals were kept under vigilant

postoperative care, including daily examination for signs of distress, weight loss, dehydration,

and bladder dysfunction. Manual expression of bladders was performed 2-3 times daily until

spontaneous voiding returned (~2 weeks), and animals were monitored for the possibility of

urinary tract infection. Animals were housed in pairs with the exception of the first few hours

following surgery.












8.2.3 Locomotor Treadmill Training

Animals with spinal cord injury were exposed to treadmill locomotor training. Training

was started on post-operative day 7. There were two reasons for this. First, on day 8 the surgical

staples were removed and soft tissue had healed sufficiently so that trauma could be avoided at

the incision site. Second, red porphyrin expression around the eyes, a symptom associated with

stress, disappeared within a week post SCI. Therefore, animals could be trained without apparent

discomfort and stress at this time.

Animals assigned to the treadmill training group were given Hyve minutes to explore the

treadmill on the first training day and then encouraged to walk on the moving treadmill (11

meter/minute) for a series of four, Hyve-minute bouts. A minimum of five minutes rest was

provided between bouts. On the second day of training, animals completed two bouts of ten

minutes each, twice a day. Starting on day 3, animals trained continuously for 20 minutes with a

minimum interval between training sessions of 2 hours. Training consisted of quadrapedal

treadmill stepping. Body weight support was provided manually by the trainer as necessary. The

level of body weight support was adjusted to make sure that the animals' hind limbs did not

collapse and was gradually removed as locomotor capability improved. Typically, when all rats

had profound paraplegia, assistance was provided to place the rat hind paws in plantar stepping

position during training.

8.2.4 Tissue Harvest

At the time points indicated above, the soleus and TA muscles of both legs were dissected

and snap-frozen at resting length in isopentane, pre-cooled in liquid nitrogen and stored at -80

OC.









8.2.5 Determination of IGF-I Protein Concentration

Frozen soleus and TA muscles were rinsed with PBS to remove excess blood,

homogenized in 20 mL of PBS and stored overnight at -200C. The homogenates were then

centrifuged for 5 minutes at 5000 x g. The supernatants were utilized for measurements of total

IGF-1 in a commercially available ELISA kit specific for rodent IGF-I (R&D Systems,

Minneapolis, MN). IGF-I concentration was calculated based on a standard curve generated from

recombinant rat IGF-I. This kit detects total rodent IGF-I, and the measurements are not affected

by the presence of IGF-I binding proteins or IGF-II.227 This kit has been validated for the

determination of rat IGF-I at 30-3000 pg/ml with an intra-assay precision of ~4.3% and an inter-

assay precision of ~6.0%.227 All samples were measured on a micro-plate reader at 450nm in

duplicate.

8.2.6 Immunohistochemistry Measurements

Cryostat sections (10 Clm) in a transverse plane were prepared from the central portion of

each muscle taken from both legs and mounted serially on gelatin-coated glass slides.

Immunocytochemical reactions were performed on cryostat sections with anti-laminin and anti-

Pax-7 or anti-embryonic myosin antibody at various dilutions. Rabbit anti-laminin

(Neomarker,Labvi sion, Fremont, CA) was used to outline the muscle fibers. Sections were

incubated with rabbit anti-laminin and the anti-Pax-7 (1:300) and anti-embryonic myosin (1:10)

antibodies (40C over night), followed by incubation with rhodamine-conjugated anti-rabbit IgG

and Fitc-conjugated anti-mouse IgG (Nordic Immunological Laboratories, Tilburg, The

Netherlands). Stained sections were mounted in mounting medium for fluorescence (Vector

Laboratories, Burlingame, CA) and kept at 40C to diminish fading. Stained cross-sections were

photographed (10 x magnification) by using a Leica fluorescence microscope (Leica

Microsystems, Bannockburn, IL) with a digital camera. Regions of the stained sections from









each muscle were randomly selected for positive pax-7 and embryonic myosin. The proportions

of each fiber type were determined from a sample of 150-250 fibers across the entire section of

each muscle. The pixels setting used for conversion of pixels to micrometers was 1.5 pixels to 1

pum2 for a 10 x objective.

8.2.7 Western Blot Analysis

Quantifieation and expression of MyoD, Myf5 and Myogenin will be measured using

Western blot analysis. Muscles will be homogenized in a lysis buffer with Fast-Prep

homogenizer machine at 13,000 RPM at 40C for Hyve minutes. The supernatant will be preserved

for protein assay. Protein will be denatured by heating samples to 95-100 OC for 5 minutes.

Protein will be measured using BCA protein assay kit from Pierce. Electrophoresis will be

performed by mixing 40-50 Cpg protein with 5X loading buffer and loading it to 4-15% SDS page

gel from Bio-Rad. Protein will then be transferred from gel to nitrocellulose membrane.

Blocking will be conducting using 5% non fat dry milk in TBS/T (Tris Buffer Saline, Tween-

20). Blot with be incubated with primary antibody overnight at 40C according to manufacturer' s

instruction. Blot will then be incubated with HRP-conjugate secondary antibody for 40 minutes

to one hour at room temperature. Finally protein will be detected using Western Blotting

Luminal Reagent from Santa Cruz.

8.2.8 Data Analysis

All statistical analyses were performed with SPSS, Version 13.0.1. Tests for normality will

be performed on all of the measured variables before proceeding with tests of statistical

inference. Results are expressed as mean + standard error of mean. One-way ANOVA was used

to test for differences among the four experimental groups. In an effort to control for multiple

comparisons, post-hoc analysis was implemented. For all analyses, significance was established

when p< 0.05.









8.3 Results

8.3.1 Effects of Incomplete Spinal cord Injury and Locomotor Training on Insulin-Like
Growth Factor-1 (IGF-1) Expression

Activation and regulation of satellite cells seems to require IGF-1. Therefore, an enzyme-

linked immune sorbent assay (ELISA) was used to quantify IGF-1 levels in the slow-twitch

soleus and fast-twitch tibialis anterior (TA) muscles. Measurements were made in animals two

weeks after SCI (SCI group) and in animals with one week of locomotor training one week after

SCI (SCI locomotorr training group). The IGF-1 levels in the soleus muscle were approximately

four-fold higher in the SCI group in comparison to the control group (p<0.01) (Fig.8-6A).

Locomotor training lead to an approximately 2-3-fold increase in soleus IGF-1 levels in

comparison to the untrained SCI group. SCI or locomotor training did not affect IGF-1 levels in

the TA muscle (Fig.8-6B). These results indicate that there are significant increases in IGF-1

protein levels following SCI in the slow-twitch extensor soleus muscle. In addition, locomotor

training results in additional increases in soleus IGF-1 protein levels in spinal cord-injured

animals.

8.3.2 Effects of Incomplete Spinal Cord Injury and Locomotor Training on Pax-7

Pax-7 is a transcription factor, and its expression is upregulated in activated and

proliferating satellite cells. Therefore, immunohistochemistry was used to study the frequency of

Pax-7-positive myonuclei in transverse sections of the soleus and TA muscles (Figs 8-1 A& B).

Although the number of Pax-7-positive myonuclei seemed to be increased in the soleus and TA

muscles two weeks after SCI, these increases were not significant. However, locomotor training

lead to an approximately 2-fold increase in Pax-7 positive-myonuclei in the soleus muscle and an

approximately 50% increase in the TA muscle in comparison to the untrained SCI group. These

results indicate that locomotor training leads to significant increases in Pax-7-positive myonuclei









in both the slow- twitch extensor soleus and fast-twitch flexor TA muscles in spinal cord-injured

animals. Furthermore, it is interesting to note that the number of Pax-7-positive fibers in the

soleus muscle was almost 2-fold higher compared to the TA muscle in the SCI and locomotor

training groups (p<0.05).

8.3.3 Effects of Incomplete Spinal Cord Injury and Locomotor Training on Myogenic
Regulatory Factors (MyoD, Myf5 and Myogenin)

Myogenic regulatory factors (MRFs) are known to regulate satellite cell activity as the

cells pass through the different stages of muscle regeneration. Upon satellite cell activation,

MyoD and Myf5 are thought to be involved in promoting satellite cell proliferation and

progression toward terminal differentiation. Therefore, western blot analysis was used to

quantify MyoD and Myf5 protein levels in the slow-twitch soleus and fast-twitch TA muscles.

Two weeks after SCI, there was no significant difference in the MyoD or Myf5 protein levels in

both the soleus and TA muscles in comparison to the control group. Although we saw increases

in MyoD and Myf5 protein levels in both muscles following locomotor training in spinal cord-

injured animals, the results were not significant. (Fig.8-2 & 3).

Myogenin levels are known to be upregulated when satellite cells begin their terminal

differentiation program. Therefore, we measured myogenin protein levels using western blot

analysis. Two weeks after SCI, the myogenin levels in the soleus muscle were approximately 2-

3-fold higher in the SCI group in comparison to the control group (p<0.05) (Fig.8-4A). However,

locomotor training did not result in any change in myogenin levels in the soleus muscle in

comparison to the SCI untrained group. SCI or locomotor training did not affect myogenin

protein levels in the TA muscle (Fig.8-4B). Overall, these results show that following SCI soleus

myogenin levels were increased, but MyoD and Myf5 levels were not significantly altered. In










addition, locomotor training did not significantly impact any of the MRF protein levels in either

of the muscles.

8.3.4 Effects of Incomplete Spinal Cord Injury and Locomotor Training on Embryonic

Myosin

Expression of embryonic myosin indicates new fiber formation. Therefore we used

immunohistochemistry to study the frequency of embryonic myosin-positive muscle fibers in

transverse sections of the soleus and TA muscles (Figs 8-5 A& B). Although the number of

embryonic myosin-positive fibers seemed to be increased in the soleus muscle two weeks after

SCI, the increase was not significant. However, one week of locomotor training lead to an

approximately 3-fold increase in embryonic myosin-positive in the soleus in comparison to the

untrained SCI group (p<0.001). In contrast, SCI resulted in a significant increase in the

embryonic myosin-positive-fibers in the TA muscle, while locomotor training in spinal cord-

injured animals had no effect (p<0.01). It is also interesting to note that the numbers of

embryonic myosin positive fibers in the soleus muscle was almost 6-fold higher compared to the

TA in the SCI and locomotor training group (p<0.05). These results indicate that SCI alone

without training results in significant increases in embryonic myosin in the TA, while locomotor

training in combination with SCI results in significant embryonic myosin levels in the soleus.

8.4 Discussion

Atrophy in skeletal muscle has been shown to be associated with a loss of myonuclei

independent of the manner the atrophy was induced. Satellite cells once activated proliferate and

migrate to the site of muscle fiber atrophy and then differentiate to either form a new fiber or

help repair the damaged fiberl22,128 228-230 To better understand recovery from contusion-SCI

induced atrophy and loss of myonuclei, we studied satellite cell activity in two different muscles,

the soleus and the TA. In our current study, we found that there was an increase in satellite cell









activity following contusion SCI. In addition, locomotor training initiated one-week following

contusion-SCI further substantially increased satellite cell activity. Furthermore, we found the

increase in satellite cell activity to be different in the slow-extensor soleus compared to the fast-

flexor tibialis anterior (TA).

Satellite cell activation requires the influence of growth factors.122,231,232 In Our study, we

saw significant increases in IGF-1 levels in the soleus following contusion SCI, which was

further significantly increased with locomotor training. However, IGF-1 levels remained stable

in the TA muscle. We studied the growth factor IGF-1 as it has been shown to stimulate satellite

cell activation, proliferation and differentiation in the rat muscle and also to increase myonuclei

number and myofiber size.233,234 In addition, exercise results in elevated IGF-I levels, which

could result in an increase in satellite cell activation and a compensatory hypertrophy of skeletal

muscle, thereby making it relevant to study IGF-1 levels following SCI and locomotor

training.223,235,236 There are a few studies which involved SCI and IGF-1. In the first study by

Resnick et al 237 2004, IGF-1 levels were up-regulated within the spinal cord following contusion

injury. Even though the regions of IGF-1 measurement were different in our studies, we felt

since IGF-1 presence is systemic in nature our studies related and both studies reported increased

IGF-1 levels following contusion SCI. However, Versteegden et al. 2000238 found no changes in

IGF-1 mRNA levels 30 days following transaction SCI and cycling training which is different to

our results. In retrospect, the authors of the study felt that they waited too long to measure IGF-1

levels and transient increase in IGF could have occurred at an earlier time point. Interestingly,

based on our results we feel the increases in IGF-1 following SCI and locomotor training could

have triggered satellite cell activity in the soleus, while no significant changes in IGF-1 levels in

the TA mirrors the significant lack of satellite cell activity in this muscle.









In the present study, SCI resulted in stimulating similar increases in pax-7-positive

myonuclei in both soleus and TA muscles. In addition, locomotor training lead to a further

substantial increase in pax-7-positive myonuclei in both soleus and TA, with the soleus almost

having the twice the number of Pax-7-positive myonuclei compared to the TA. Pax-7 was

quantified in the current study because activated satellite cells express Pax7. 125,229Most activated

satellite cells then proliferate, thereby down regulating Pax7 and then differentiate. 125,239,240

Furthermore, treadmill training is known to stimulate satellite cell activity in skeletal muscle,

thereby making it relevant to study Pax-7 activity following SCI and locomotor training. In our

results, the soleus and TA had similar levels of Pax7 following contusion SCI. These results are

different from that seen in other types of SCI like isolation were they found the slow twitch

muscle to have higher satellite cell expression compared to the fast twitch.216 We however feel

the contusion SCI presenting itself with spared spinal tracts and varied activation levels could be

the reason for the similar expression of Pax-7 in both slow and fast muscle. However, locomotor

training following SCI resulted in twice the number of Pax-7 positives compared to the TA. The

reason for this may be due to differences in muscle activity, with the slow soleus being 20-times

more active and frequently recruited than the fast flexor TA muscle during locomotor tasks.241

To summarize, both SCI and locomotor training resulted in activating satellite cells in both the

soleus and TA, with the soleus having higher levels of satellite cell activation compared to the

TA due to its higher recruitment in loading conditions.

Surprisingly in this study we did not Eind any significant changes in myogenic regulatory

factor (MRF) protein levels in both muscles following SCI or SCI + locomotor training except

for myogenin in the soleus. Myogenin protein levels in the soleus were significantly elevated

following SCI and SCI + locomotor training in comparison to controls. We studied MRF









proteins as they are transcription factors that influence and modulate the proliferation and

differentiation of the satellite cells.23,1 13,122,216,238 Specifically, Myogenin is a MRF protein that

regulates the terminal differentiation of satellite cells to myoblasts. Based on our myogenin

results we suggest that following SCI and locomotor training one-week-post -SCI leads to

significant terminal differentiation of satellite cells into myoblasts.128,242 However there was no

difference in myogenin levels between the SCI trained and un-trained group. These results were

similar to a study by Versteegden et al. 1999113, who observed similar significant increases in

soleus myogenin expression 10 days after transaction SCI and cycling training 5 days post-SCI

with no differences in myogenin levels between the trained and un-trained SCI groups. We

suggest it could be because myogenin proteins levels may have reached their maximum levels

following SCI and the training does not cause any further increase. In summary, we feel that

even though there were no increases in MyoD and Myf5 protein levels, significant increases in

myogenin following SCI and SCI + locomotor training indicate terminal differentiation of

satellite cells only in the soleus.

Although after SCI there were a few embryonic myosin positive muscle fibers in the both

the soleus and TA compared to no positives in the controls, their numbers were very minimal.

However following locomotor training we noticed significant increases in embryonic myosin

numbers in the soleus muscle after locomotor training, while in the TA the numbers remained

insignificant. In the present study we quantified the developmental isoform of the myosin heavy

chains termed as embryonic myosin because it sequentially precedes the appearance of definitive

adult myosin heavy chains in rats and is an indicator of new fiber formation.63'91'213 Based on our

results we can suggest that locomotor training following SCI resulted in new fiber formation in

the soleus muscle. A point of interest in this study which is similar to a study by Yablonka-









Reuveni et al. 1994 239iS that the numbers of embryonic myosin positive cells in our study were

less than half the number of positive satellite cells, indicating that not all the satellite cell

descendants entered the phase of terminal differentiation, suggesting muscle plasticity through

regeneration does directly related to activated satellite cell numbers especially following

contusion SCI.

So in conclusion were satellite cells involved in the exercise induced maintenance of

muscle fiber size following contusion SCI? Even though there are limitations in study and

alternate theories, we suggest that satellite cell activation to form new fibers could be one of the

pathways in which the soleus recovers after contusion SCI and locomotor training. One of the

limitations of the study was that MRF levels of MyoD and Myf5 which indicate satellite cell

proliferation did not significantly change in both the SCI and locomotor training group in both

muscles. A plausible explanation we feel is that these proteins are transiently expressed, and the

time points we used only provide snapshots of satellite cells or MRF activity through their entire

cycle and hence we might have missed the expression of these proteins. We also have a few

suggestions regarding the lack of satellite cell activity in the fast TA. First, there was very less

atrophy in the TA to start of with and hence there was less muscle plasticity required to recover

from the atrophy. Also, in a fast muscle like the TA, myonuclear number is significantly high

and therefore satellite cells may not have been required to restore myonuclear levels.

In summary, atrophy following contusion SCI may be associated with myonuclear loss.

Increase in satellite cell activity and new fiber formation might be potential mechanisms to

compensate for atrophy and myonuclear loss in the soleus and locomotor training might

accelerate the recovery of the soleus muscle through muscle regeneration as a response to

increased activity, while in the TA; it might be an order of events which need further









investigation. Overall this study provides more information on the exercise induced contribution

of satellite cells towards muscle plasticity through regeneration after moderate contusion SCI.












o CON
H CON+TM
* SCI
H SCI+TM


Soleus


o CON
IllCON+TM
SSCI
H SCI+TM


Tibialis Anterior


Figure 8-1 Pax-7 staining for the (A) soleus and (B) tibialis anterior muscle. Significant
difference between SCI+TM group from the other groups (p<0.05)














Soleus o untrol
untrol+T1V
1.2 HSCI
SSCI+TM







.O 0.8




0.6



S 0.4



0.2





control control+TM SCI SCI+TM
A



TA 0 control
Control+TM
1.4 SC
SSCI+TM


1.2








.o


S0.4



0.2 -6



0.




control control+TM SCI SCI+TM
B



Figure 8-2 MyoD protein levels in the (A) soleus and (B) TA muscle.












Soleus


a control
Control+TM
SCI
SCI+TM


control control+TM


SCI


SCI+TM


control control+TM SCI SCI+TM
B


Figure 8-3 Myf5 protein levels in the (A) soleus and (B) TA muscle.


o control
5 Control+TM
SCI
H SCI+TM














Soleus 0 control
a control+T10
1.4 H SCI
5 SCI+TM









0.8






0.4



0.2




control control+TM SCI SCI+TM
A



TA o antrol
cuntrol+TM
1.6 SCI
SSCI+TM

1.4


1.2






S0.8


on 0.6
Or

0.4


0.2




control control+TM SCI SCI+TM
B



Figure 8-4 Myogenin protein in the (A) soleus and (B) TA muscle.* Significant difference
between the SCI no training group and the control groups (p<0.05). # Significant

difference between SCI+TM group from the other groups (p<0.05).














9- CON
5 CON+TM
HSCI
8 HSCI+TM

7-

6-




l4-
c*
3-

2-

1-

0
Soleus
A



1.2
6a SCl+TM
H SCI
1 O control
a control + TM

0.8



0.6
CD
c*
0.4



0.2



0

Tibilais Anterior
B


Figure 8-5 Embryonic myosin positives. (A) soleus and (B) TA muscle.* Significant difference
between the SCI group and the other groups (p<0.05).














Soleus (IGF-1 )
control
Scontrol+TM
120 MC
MSCI+TM


100



80



S60



40



20




control control+TM SCI SCI+TM
A



TA (IGF-1)
contmil
Hcontrol+TM
5u M SCI
MSCI+TM




35
















control control+TM SCI SCI+TM



B



Figure 8-6 IGF-1 levels. (A) soleus and (B) TA muscle. Significant difference between the SCI
no training group and the control groups. Significant difference between SCI+TM

group from the other groups (p<0.05).









LIST OF REFERENCES


1. www. spinalcord.uab.edu (National Spinal Cord Injury Database), accessed April 2007.

2. http:.//www.ninds.nih.gov (National Institute of Neurological Disorders and Stroke),
accessed April 2007. .

3. Fuller KS. Traumatic spinal cord injury. Pathology for Physical Therapists 2003; 1086-
1097.

4. Somers MF. Spinal cord injury: Functional Rehabilitation 2001.

5. Lieber RL. Skeletal muscle adaptability. II: Muscle properties following spinal-cord
injury. Dev M~ed ChildNeurol 1986; 28: 533-542.

6. Dittuno PL, Dittuno Jr JF, Jr. Walking index for spinal cord injury (WISCI II): scale
revision. Spinal Cord 2001; 39: 654-656.

7. Mahoney FI, Barthel DW. Functional evaluation: the barthel index. M~d State M~ed J
1965; 14: 61-65.

8. Collen FM, Wade DT, Robb GF, Bradshaw CM. The Rivermead mobility index: a
further development of the rivermead motor assessment. Int Disabil Stud 1991; 13: 50-
54.

9. Keith RA, Granger CV, Hamilton BB, Sherwin FS. The functional independence
measure: a new tool for rehabilitation. Adv Clin Rehabil 1987; 1: 6-18.

10. Catz A, Itzkovich M, Agranov E, Ring H, Tamir A. SCIM--spinal cord independence
measure: a new disability scale for patients with spinal cord lesions. Spinal Cord 1997;
35: 850-856.

11. Itzkovich M, Tamir A, Philo O, Steinberg F, Ronen J, Spasser R, Gepstein R, Ring H,
Catz A. reliability of the catz-itzkovich spinal cord independence measure assessment by
interview and comparison with observation. Am JPhys 2ed Rehabil 2003; 82: 267-272.

12. Alaimo MA, Smith JL, Roy RR, Edgerton VR. EMG activity of slow and fast ankle
extensors following spinal cord transaction. JApplPhysiol 1984; 56: 1608-1613.

13. Albin MS, White RJ, Yashon D, Massopust LC, Jr. Functional and electrophysiologic
limitations of delayed spinal cord cooling after impact injury. Surg Forum 1968; 19: 423-
424.

14. de Leon RD, Hodgson JA, Roy RR, Edgerton VR. Locomotor capacity attributable to
step training versus spontaneous recovery after spinalization in adult cats. JNeurophysiol
1998; 79: 1329-1340.









15. de Leon RD, London NJ, Roy RR, Edgerton VR. Failure analysis of stepping in adult
spinal cats. Prog Brain Res 1999; 123: 341-348.

16. Edgerton VR, Roy RR, Hodgson JA, Prober RJ, de Guzman CP, de Leon R. Potential of
adult mammalian lumbosacral spinal cord to execute and acquire improved locomotion in
the absence of supraspinal input. JNeurotrauma 1992; 9 Suppl 1: S119-128.

17. Gregory CM, Vandenborne K, Castro MJ, Dudley GA. Human and rat skeletal muscle
adaptations to spinal cord injury. Can JAppl Physiol 2003; 28: 491-500.

18. Gruner JA. A monitored contusion model of spinal cord injury in the rat. JNeurotrauma
1992; 9: 123-126; discussion 126-128.

19. Houle JD, Morris K, Skinner RD, Garcia-Rill E, Peterson CA. Effects of fetal spinal cord
tissue transplants and cycling exercise on the soleus muscle in spinalized rats. Muscle
Nerve 1999; 22: 846-856.

20. Hutchinson KJ, Linderman JK, Basso DM. Skeletal muscle adaptations following spinal
cord contusion injury in rat and the relationship to locomotor function: a time course
study. JNeurotrauma 2001; 18: 1075-1089.

21. Eldridge L, Liebhold M, Steinbach JH. Alterations in cat skeletal neuromuscular
junctions following prolonged inactivity. JPhysiol 1981; 313: 529-545.

22. Pierotti DJ, Roy RR, Bodine-Fowler SC, Hodgson JA, Edgerton VR. Mechanical and
morphological properties of chronically inactive cat tibialis anterior motor units. J
Physiol 1991; 444: 175-192.

23. Hyatt JP, Roy RR, Baldwin KM, Edgerton VR. Nerve activity-independent regulation of
skeletal muscle atrophy: role of MyoD and myogenin in satellite cells and myonuclei. Am
JPhysiol Cell Physiol 2003; 285: C1161-1173.

24. Lieber RL, Friden JO, Hargens AR, Feringa ER. Long-term effects of spinal cord
transaction on fast and slow rat skeletal muscle. II. Morphometric properties. Exp Neurol
1986; 91: 435-448.

25. Lieber RL, Johansson CB, Vahlsing HL, Hargens AR, Feringa ER. Long-term effects of
spinal cord transaction on fast and slow rat skeletal muscle. I. Contractile properties. Exp
Neurol 1986; 91: 423-434.

26. Roy RR, Acosta L, Jr. Fiber type and fiber size changes in selected thigh muscles six
months after low thoracic spinal cord transaction in adult cats: exercise effects. Exp
Neurol 1986; 92: 675-685.

27. Kwon BK, Oxland TR, Tetzlaff W. Animal models used in spinal cord regeneration
research. Spine 2002; 27: 1504-1510.

28. Taoka Y, Okajima K. Spinal cord injury in the rat. Prog Neurobiol 1998; 56: 341-3 58.










29. Albin MS, White RJ, Acosta-Rua G, Yashon D. Study of functional recovery produced
by delayed localized cooling after spinal cord injury in primates. JNeurosurg 1968; 29:
113-120.

30. Koozekanani SH, Vise WM, Hashemi RM, McGhee RB. Possible mechanisms for
observed pathophysiological variability in experimental spinal cord injury by the method
of Allen. JNeurosurg 1976; 44: 429-434.

31. Parker AJ, Smith CW. Functional recovery from spinal cord trauma following
dexamethazone and chlorpromazine therapy in dogs. Res Vet Sci 1976; 21: 246-247.

32. Basso DM, Beattie MS, Bresnahan JC, Anderson DK, Faden AI, Gruner JA, Holford TR,
Hsu CY, Noble LJ, Nockels R, Perot PL, Salzman SK, Young W. MASCIS evaluation of
open field locomotor scores: effects of experience and teamwork on reliability.
Multicenter Animal Spinal Cord Injury Study. JNeurotrauma 1996; 13: 343-359.

33. Bresnahan JC, Beattie MS, Stokes BT, Conway KM. Three-dimensional computer-
assisted analysis of graded contusion lesions in the spinal cord of the rat. JNeurotrauma
1991; 8: 91-101.

34. Guizar-Sahagun G, Grij alva I, Madrazo I, Franco-Bourland R, Salgado H, Ibarra A, Oliva
E, Zepeda A. Development of post-traumatic cysts in the spinal cord of rats-subj ected to
severe spinal cord contusion. Surg Neurol 1994; 41: 241-249.

35. Noble LJ, Wrathall JR. Spinal cord contusion in the rat: morphometric analyses of
alterations in the spinal cord. Exp Neurol 1985; 88: 135-149.

36. Osterholm JL, Mathews GJ. Treatment of severe spinal cord injuries by biochemical
norepinephrine manipulation. Surg Forum 1971; 22: 415-417.

37. Shah PK, Stevens JE, Gregory CM, Pathare NC, Jayaraman A, Bickel SC, Bowden M,
Behrman AL, Walter GA, Dudley GA, Vandenborne K. Lower-extremity muscle cross-
sectional area after incomplete spinal cord injury. Arch Phys M~edRehabil 2006; 87: 772-
778.

38. Adams MM, Ditor DS, Tarnopolsky MA, Phillips SM, McCartney N, Hicks AL. The
effect of body weight-supported treadmill training on muscle morphology in an
individual with chronic, motor-complete spinal cord injury: A case study. J Spinal Cord
M~ed 2006; 29: 167-171.

39. Stewart BG, Tarnopolsky MA, Hicks AL, McCartney N, Mahoney DJ, Staron RS,
Phillips SM. Treadmill training-induced adaptations in muscle phenotype in persons with
incomplete spinal cord injury. Muscle Nerve 2004; 30: 61-68.

40. Edgerton VR, Leon RD, Harkema SJ, Hodgson JA, London N, Reinkensmeyer DJ, Roy
RR, Talmadge RJ, Tillakaratne NJ, Timoszyk W, Tobin A. Retraining the injured spinal
cord. JPhysiol 2001; 533: 15-22.










41. Martin TP, Stein RB, Hoeppner PH, Reid DC. Influence of electrical stimulation on the
morphological and metabolic properties of paralyzed muscle. JAppl Physiol 1992; 72:
1401-1406.

42. Rochester L, Chandler CS, Johnson MA, Sutton RA, Miller S. Influence of electrical
stimulation of the tibialis anterior muscle in paraplegic subjects. 1. Contractile properties.
Paraplegia 1995; 33: 437-449.

43. Reeves ND, Maganaris CN, Narici MV. Ultrasonographic assessment of human skeletal
muscle size. Eur JAppl Physiol 2004; 91: 116-118.

44. Engstrom CM, Loeb GE, Reid JG, Forrest WJ, Avruch L. Morphometry of the human
thigh muscles. A comparison between anatomical sections and computer tomographic
and magnetic resonance images. JAnat 1991; 176: 139-156.

45. Dudley GA, Castro MJ, Rogers S, Apple DF, Jr. A simple means of increasing muscle
size after spinal cord injury: a pilot study. Eur JAppl Physiol Occup Physiol 1999; 80:
394-396.

46. Castro MJ, Apple DF, Jr., Staron RS, Campos GE, Dudley GA. Influence of complete
spinal cord injury on skeletal muscle within 6 mo of injury. JAppl Physiol 1999; 86:
350-358.

47. Mahoney ET, Bickel CS, Elder C, Black C, Slade JM, Apple D, Jr., Dudley GA. Changes
in skeletal muscle size and glucose tolerance with electrically stimulated resistance
training in subj ects with chronic spinal cord injury. Arch Phys 2edRehabil 2005; 86:
1502-1504.

48. Talmadge RJ, Castro MJ, Apple DF, Jr., Dudley GA. Phenotypic adaptations in human
muscle fibers 6 and 24 wk after spinal cord injury. JAppl Physiol 2002; 92: 147-154.

49. Castro MJ, Apple DF, Jr., Rogers S, Dudley GA. Influence of complete spinal cord injury
on skeletal muscle mechanics within the first 6 months of injury. Eur JAppl Physiol
2000; 81: 128-131.

50. Gerrits HL, De Haan A, Hopman MT, van Der Woude LH, Jones DA, Sargeant AJ.
Contractile properties of the quadriceps muscle in individuals with spinal cord injury.
Muscle Nerve 1999; 22: 1249-1256.

51. Gerrits HL, Hopman MT, Sargeant AJ, de Haan A. Reproducibility of contractile
properties of the human paralysed and non-paralysed quadriceps muscle. Clin Physiol
2001; 21: 105-113.

52. Shields RK. Muscular, skeletal, and neural adaptations following spinal cord injury. J
Orthop Sports Phys Ther 2002; 32: 65-74.









53. Jayaraman A, Gregory CM, Bowden M, Stevens JE, Shah P, Behrman AL, Vandenborne
K. Lower extremity skeletal muscle function in persons with incomplete spinal cord
injury. Spinal Cord 2006; 44: 680-687.

54. Noreau L, Vachon J. Comparison of three methods to assess muscular strength in
individuals with spinal cord injury. Spinal Cord 1998; 36: 716-723.

55. Schwartz S, Cohen ME, Herbison GJ, Shah A. Relationship between two measures of
upper extremity strength: manual muscle test compared to hand-held myometry. Arch
Phys MedRehabil 1992; 73: 1063-1068.

56. Drolet M, Noreau L, Vachon J, Moffet H. Muscle strength changes as measured by
dynamometry following functional rehabilitation in individuals with spinal cord injury.
Arch Phys Med Rehabil 1999; 80: 791-800.

57. West SP, Roy RR, Edgerton VR. Fiber type and fiber size of cat ankle, knee, and hip
extensors and flexors following low thoracic spinal cord transaction at an early age. Exp
Neurol 1986; 91: 174-182.

58. Roy RR, Pierotti DJ, Flores V, Rudolph W, Edgerton VR. Fibre size and type adaptations
to spinal isolation and cyclical passive stretch in cat hindlimb. JAnat 1992; 180 ( Pt 3):
491-499.

59. Termin A, Staron RS, Pette D. Myosin heavy chain isoforms in histochemically defined
fiber types of rat muscle. Histochemistry 1989; 92: 453-457.

60. Peterson CA, Murphy RJ, Dupont-Versteegden EE, Houle JD. Cycling exercise and fetal
spinal cord transplantation act synergistically on atrophied muscle following chronic
spinal cord injury in rats. Neurorehabil Neural Repair 2000; 14: 85-91.

61. Talmadge RJ, Roy RR, Edgerton VR. Persistence of hybrid fibers in rat soleus after
spinal cord transaction. Anat Rec 1999; 255: 188-201.

62. Talmadge RJ, Roy RR, Chalmers GR, Edgerton VR. MHC and sarcoplasmic reticulum
protein isoforms in functionally overloaded cat plantaris muscle fibers. JAppl Physiol
1996; 80: 1296-1303.

63. Zhong H, Roy RR, Woo J, Kim JA, Edgerton VR. Differential modulation of myosin
heavy chain phenotype in an inactive extensor and flexor muscle of adult rats. JAnat
2007; 210: 19-31.

64. Roy RR, Sacks RD, Baldwin KM, Short M, Edgerton VR. Interrelationships of
contraction time, Vmax, and myosin ATPase after spinal transaction. JAppl Physiol
1984; 56: 1594-1601.

65. Stevens JE, Liu M, Bose P, O'Steen WA, Thompson FJ, Anderson DK, Vandenborne K.
Changes in soleus muscle function and fiber morphology with one week of locomotor
training in spinal cord contusion injured rats. JNeurotrauma 2006; 23: 1671-1681.









66. Barbeau H, Wainberg M, Finch L. Description and application of a system for locomotor
rehabilitation. M~edBiolEng Comput 1987; 25: 341-344.

67. Visintin M, Barbeau H. The effects of parallel bars, body weight support and speed on
the modulation of the locomotor pattern of spastic paretic gait. A preliminary
communication. Paraplegia 1994; 32: 540-553.

68. Wolpaw JR, Tennissen AM. Activity-dependent spinal cord plasticity in health and
disease. Annu Rev Neurosci 2001; 24: 807-843.

69. Edgerton VR, Tillakaratne NJ, Bigbee AJ, de Leon RD, Roy RR. Plasticity of the spinal
neural circuitry after injury. Annu Rev Neurosci 2004; 27: 145-167.

70. Rossignol S, Brustein E, Bouyer L, Barthelemy D, Langlet C, Leblond H. Adaptive
changes of locomotion after central and peripheral lesions. Can JPhysiol Pharmacol
2004; 82: 617-627.

71. Dietz V, Harkema SJ. Locomotor activity in spinal cord-injured persons. JAppl Physiol
2004; 96: 1954-1960.

72. De Leon RD, Hodgson JA, Roy RR, Edgerton VR. Retention of hindlimb stepping ability
in adult spinal cats after the cessation of step training. JNeurophysiol 1999; 81: 85-94.

73. Lovely RG, Gregor RJ, Roy RR, Edgerton VR. Weight-bearing hindlimb stepping in
treadmill-exercised adult spinal cats. Brain Res 1990; 514: 206-218.

74. Chau C, Barbeau H, Rossignol S. Early locomotor training with clonidine in spinal cats. J
Neurophysiol 1998; 79: 392-409.

75. Barbeau H, Basso M, Behrman A, Harkema S. Treadmill training after spinal cord injury:
good but not better. Neurology 2006; 67: 1900-1901; author reply 1901-1902.

76. Barbeau H, McCrea DA, O'Donovan MJ, Rossignol S, Grill WM, Lemay MA. Tapping
into spinal circuits to restore motor function. Brain Res Brain Res Rev 1999; 30: 27-51.

77. Dobkin B, Barbeau H, Deforge D, Ditunno J, Elashoff R, Apple D, Basso M, Behrman
A, Harkema S, Saulino M, Scott M. The evolution of walking-related outcomes over the
first 12 weeks of rehabilitation for incomplete traumatic spinal cord injury: the
multicenter randomized Spinal Cord Injury Locomotor Trial. Neurorehabil Neural
Repair 2007; 21: 25-35.

78. Wernig A, Nanassy A, Muller S. Maintenance of locomotor abilities following Laufband
(treadmill) therapy in para- and tetraplegic persons: follow-up studies. Spinal Cord 1998;
36: 744-749.

79. Behrman AL, Harkema SJ. Locomotor training after human spinal cord injury: a series of
case studies. Phys Ther 2000; 80: 688-700.










80. Dobkin B, Apple D, Barbeau H, Basso M, Behrman A, Deforge D, Ditunno J, Dudley G,
Elashoff R, Fugate L, Harkema S, Saulino M, Scott M. Weight-supported treadmill vs
over-ground training for walking after acute incomplete SCI. Neurology 2006; 66: 484-
493.

81. Shurrager PS, Dykman RA. Walking spinal carnivores. J Comp Physiol Psychol 1951;
44: 252-262.

82. Gordon T, Mao J. Muscle atrophy and procedures for training after spinal cord injury.
Phys Ther 1994; 74: 50-60.

83. Stein RB. Functional electrical stimulation after spinal cord injury. JNeurotrauma 1999;
16: 713-717.

84. Scremin AM, Kurta L, Gentili A, Wiseman B, Perell K, Kunkel C, Scremin OU.
Increasing muscle mass in spinal cord injured persons with a functional electrical
stimulation exercise program. Arch Phys M~edRehabil 1999; 80: 153 1-1536.

85. Stein RB, Chong SL, James KB, Kido A, Bell GJ, Tubman LA, Belanger M. Electrical
stimulation for therapy and mobility after spinal cord injury. Prog Brain Res 2002; 137:
27-34.

86. Gerrits HL, Hopman MT, Sargeant AJ, Jones DA, De Haan A. Effects of training on
contractile properties of paralyzed quadriceps muscle. Muscle Nerve 2002; 25: 559-567.

87. Crameri RM, Weston AR, Rutkowski S, Middleton JW, Davis GM, Sutton JR. Effects of
electrical stimulation leg training during the acute phase of spinal cord injury: a pilot
study. Eur JAppl Physiol 2000; 83: 409-415.

88. Shields RK, Dudley-Javoroski S. Musculoskeletal plasticity after acute spinal cord injury:
effects of long-term neuromuscular electrical stimulation training. JNeurophysiol 2006;
95: 2380-2390.

89. Harridge SD, Andersen JL, Hartkopp A, Zhou S, Biering-Sorensen F, Sandri C, Kj aer M.
Training by low-frequency stimulation of tibialis anterior in spinal cord-injured men.
Muscle Nerve 2002; 25: 685-694.

90. Bajd T, Kralj A, Stefancic M, Lavrac N. Use of functional electrical stimulation in the
lower extremities of incomplete spinal cord injured patients. Artif Organs 1999; 23: 403-
409.

91. Modlin M, Forstner C, Hofer C, Mayr W, Richter W, Carraro U, Protasi F, Kern H.
Electrical stimulation of denervated muscles: first results of a clinical study. ArtifOrgans
2005; 29: 203-206.

92. Ragnarsson KT, Pollack S, O'Daniel W, Jr., Edgar R, Petrofsky J, Nash MS. Clinical
evaluation of computerized functional electrical stimulation after spinal cord injury: a
multi center pilot study. Arch Phys M~edRehabil 198 8; 69: 672-677.









93. Baldi JC, Jackson RD, Moraille R, Mysiw WJ. Muscle atrophy is prevented in patients
with acute spinal cord injury using functional electrical stimulation. Spinal Cord 1998;
36: 463-469.

94. Crameri RM, Weston A, Climstein M, Davis GM, Sutton JR. Effects of electrical
stimulation-induced leg training on skeletal muscle adaptability in spinal cord injury.
Scand J2ed Sci Sports 2002; 12: 316-322.

95. Postans NJ, Hasler JP, Granat MH, Maxwell DJ. Functional electric stimulation to
augment partial weight-bearing supported treadmill training for patients with acute
incomplete spinal cord injury: A pilot study. Arch Phys M~edRehabil 2004; 85: 604-610.

96. Mohr T, Andersen JL, Biering-Sorensen F, Galbo H, Bangsbo J, Wagner A, Kj aer M.
Long-term adaptation to electrically induced cycle training in severe spinal cord injured
individuals. Spinal Cord 1997; 35: 1-16.

97. Andersen JL, Mohr T, Biering-Sorensen F, Galbo H, Kj aer M. Myosin heavy chain
isoform transformation in single fibres from m. vastus lateralis in spinal cord injured
individuals: effects of long-term functional electrical stimulation (FES). Pflugers Arch
1996; 431: 513-518.

98. Gerrits HL, de Haan A, Sargeant AJ, Dallmeij er A, Hopman MT. Altered contractile
properties of the quadriceps muscle in people with spinal cord injury following functional
electrical stimulated cycle training. Spinal Cord 2000; 38: 214-223.

99. Chilibeck PD, Syrotuik DG, Bell GJ. The effect of strength training on estimates of
mitochondrial density and distribution throughout muscle fibres. Eur JAppl Physiol
Occup Physiol 1999; 80: 604-609.

100. Nash MS, Jacobs PL, Montalvo BM, Klose KJ, Guest RS, Needham-Shropshire BM.
Evaluation of a training program for persons with SCI paraplegia using the Parastep 1
ambulation system: part 5. Lower extremity blood flow and hyperemic responses to
occlusion are augmented by ambulation training. Arch Phys 2edRehabil 1997; 78: 808-
814.

101. van der Ploeg HP, van der Beek AJ, van der Woude LH, van Mechelen W. Physical
activity for people with a disability: a conceptual model. Sports M~ed 2004; 34: 639-649.

102. Kj aer M. Why exercise in paraplegia? Br J Sports 2ed 2000; 34: 322-323.

103. Gellman H, Sie I, Waters RL. Late complications of the weight-bearing upper extremity
in the paraplegic patient. Clin Orthop Relat Res 1988; 132-135.

104. Silfverskiold J, Waters RL. Shoulder pain and functional disability in spinal cord injury
patients. Clin Orthop Relat Res 1991; 141-145.

105. Nilsson S, Staff PH, Pruett ED. Physical work capacity and the effect of training on
subjects with long-standing paraplegia. Scand JRehabil2~ed 1975; 7: 51-56.










106. Cooney MM, Walker JB. Hydraulic resistance exercise benefits cardiovascular fitness of
spinal cord injured. M~ed Sci Sports Exerc 1986; 18: 522-525.

107. Dallmeij er AJ, Hopman MT, van As HH, van der Woude LH. Physical capacity and
physical strain in persons with tetraplegia; the role of sport activity. Spinal Cord 1996;
34: 729-735.

108. Janssen TW, van Oers CA, Hollander AP, Veeger HE, van der Woude LH. Isometric
strength, sprint power, and aerobic power in individuals with a spinal cord injury. M~ed
Sci Sports Exerc 1993; 25: 863-870.

109. Giangregorio LM, Webber CE, Phillips SM, Hicks AL, Craven BC, Bugaresti JM,
McCartney N. Can body weight supported treadmill training increase bone mass and
reverse muscle atrophy in individuals with chronic incomplete spinal cord injury? Appl
Physiol Nutr Metab 2006; 31: 283-291.

110. Giangregorio LM, Hicks AL, Webber CE, Phillips SM, Craven BC, Bugaresti JM,
McCartney N. Body weight supported treadmill training in acute spinal cord injury:
impact on muscle and bone. Spinal Cord 2005; 43: 649-657.

111. Roy RR, Talmadge RJ, Hodgson JA, Zhong H, Baldwin KM, Edgerton VR. Training
effects on soleus of cats spinal cord transected (T12-13) as adults. Muscle Nerve 1998;
21: 63-71.

112. Dupont-Versteegden EE, Houle JD, Gurley CM, Peterson CA. Early changes in muscle
fiber size and gene expression in response to spinal cord transaction and exercise. Am J
Physiol 1998; 275: C1124-1133.

113. Dupont-Versteegden EE, Murphy RJ, Houle JD, Gurley CM, Peterson CA. Activated
satellite cells fail to restore myonuclear number in spinal cord transected and exercised
rats. Am JPhysiol 1999; 277: C589-597.

114. Liu M, Bose P, Walter GA, Anderson DK, Thompson FJ, Vandenborne K. Changes in
muscle T2 relaxation properties following spinal cord injury and locomotor training. Eur
JAppl Physiol 2006; 97: 355-361.

115. Lieber RL. Skeletal muscle structure and function. Implications for rehabilitation and
sports medicine; 1992.

116. Schmalbruch H, Lewis DM. Dynamics of nuclei of muscle fibers and connective tissue
cells in normal and denervated rat muscles. Muscle Nerve 2000; 23: 617-626.

117. Robertson TA, Maley MA, Grounds MD, Papadimitriou JM. The role of macrophages in
skeletal muscle regeneration with particular reference to chemotaxis. Exp Cell Res 1993;
207: 321-331.

118. Orimo S, Hiyamuta E, Arahata K, Sugita H. Analysis of inflammatory cells and
complement C3 in bupivacaine-induced myonecrosis. Muscle Nerve 1991; 14: 515-520.










119. MacIntyre DL, Reid WD, McKenzie DC. Delayed muscle soreness. The inflammatory
response to muscle injury and its clinical implications. Sports 2ed 1995; 20: 24-40.

120. Blaveri K, Heslop L, Yu DS, Rosenblatt JD, Gross JG, Partridge TA, Morgan JE.
Patterns of repair of dystrophic mouse muscle: studies on isolated fibers. Dev Dyn 1999;
216: 244-256.

121. Bourke DL, Ontell M. Branched myofibers in long-term whole muscle transplants: a
quantitative study. Anat Rec 1984; 209: 281-288.

122. Charge SB, Rudnicki MA. Cellular and molecular regulation of muscle regeneration.
Physiol Rev 2004; 84: 209-23 8.

123. Holterman CE, Rudnicki MA. Molecular regulation of satellite cell function. Semin Cell
Dev Biol 2005; 16: 575-584.

124. Gibson MC, Schultz E. The distribution of satellite cells and their relationship to specific
fiber types in soleus and extensor digitorum longus muscles. Anat Rec 1982; 202: 329-
337.

125. Seale P, Sabourin LA, Girgis-Gabardo A, Mansouri A, Gruss P, Rudnicki MA. Pax7 is
required for the specification of myogenic satellite cells. Cell 2000; 102: 777-786.

126. Olguin HC, Olwin BB. Pax-7 up-regulation inhibits myogenesis and cell cycle
progression in satellite cells: a potential mechanism for self-renewal. Dev Biol 2004; 275:
375-388.

127. Oustanina S, Hause G, Braun T. Pax7 directs postnatal renewal and propagation of
myogenic satellite cells but not their specification. Embo J 2004; 23: 3430-3439.

128. Zammit PS, Relaix F, Nagata Y, Ruiz AP, Collins CA, Partridge TA, Beauchamp JR.
Pax7 and myogenic progression in skeletal muscle satellite cells. J Cell Sci 2006; 119:
1824-1832.

129. Sabourin LA, Girgis-Gabardo A, Seale P, Asakura A, Rudnicki MA. Reduced
differentiation potential of primary MyoD-/- myogenic cells derived from adult skeletal
muscle. JCell Biol l999; 144: 631-643.

130. Megeney LA, Kablar B, Garrett K, Anderson JE, Rudnicki MA. MyoD is required for
myogenic stem cell function in adult skeletal muscle. Genes Dev 1996; 10: 1 173-1 183.

131. Kablar B, Rudnicki MA. Skeletal muscle development in the mouse embryo. Histol
Histopathol 2000; 15: 649-656.

132. Shi X, Garry DJ. Muscle stem cells in development, regeneration, and disease. Genes
Dev 2006; 20: 1692-1708.










133. Ehrhardt J, Morgan J. Regenerative capacity of skeletal muscle. Curr Opin Neurol 2005;
18: 548-553.

134. Tomczak KK, Marinescu VD, Ramoni MF, Sanoudou D, Montanaro F, Han M, Kunkel
LM, Kohane IS, Beggs AH. Expression profiling and identification of novel genes
involved in myogenic differentiation. Faseb J 2004; 18: 403-405.

135. Peault B, Rudnicki M, Torrente Y, Cossu G, Tremblay JP, Partridge T, Gussoni E,
Kunkel LM, Huard J. Stem and progenitor cells in skeletal muscle development,
maintenance, and therapy. Mol1 Ther 2007; 15: 867-877.

136. Bischoff R. Chemotaxis of skeletal muscle satellite cells. Dev Dyn 1997; 208: 505-5 15.

137. Floss T, Arnold HH, Braun T. A role for FGF-6 in skeletal muscle regeneration. Genes
Dev 1997; 11: 2040-2051.

138. Modlesky CM, Slade JM, Bickel CS, Meyer RA, Dudley GA. Deteriorated geometric
structure and strength of the midfemur in men with complete spinal cord injury. Bone
2005; 36: 331-339.

139. Mangold S, Keller T, Curt A, Dietz V. Transcutaneous functional electrical stimulation
for grasping in subj ects with cervical spinal cord injury. Spinal Cord 2005; 43: 1-13.

140. Skold C, Harms-Ringdahl K, Seiger A. Movement-provoked muscle torque and EMG
activity in longstanding motor complete spinal cord injured individuals. JRehabil2~ed
2002; 34: 86-90.

141. Waters RL, Adkins RH, Yakura JS, Sie I. Motor and sensory recovery following
incomplete tetraplegia. Arch Phys 2ed Rehabil 1994; 75: 3 06-3 11.

142. Muslumanoglu L, Aki S, Ozturk Y, Soy D, Filiz M, Karan A, Berker E. Motor, sensory
and functional recovery in patients with spinal cord lesions. Spinal Cord 1997; 35: 386-
389.

143. Herbison GJ, Isaac Z, Cohen ME, Ditunno JF, Jr. Strength post-spinal cord injury:
myometer vs manual muscle test. Spinal Cord 1996; 34: 543-548.

144. Field-Fote EC. Combined use of body weight support, functional electric stimulation, and
treadmill training to improve walking ability in individuals with chronic incomplete
spinal cord injury. Arch Phys 2ed Rehabil 2001; 82: 8 18-824.

145. Kim CM, Eng JJ, Whittaker MW. Level walking and ambulatory capacity in persons with
incomplete spinal cord injury: relationship with muscle strength. Spinal Cord 2004; 42:
156-162.

146. Gregory CM, Vandenborne K, Huang HF, Ottenweller JE, Dudley GA. Effects of
testosterone replacement therapy on skeletal muscle after spinal cord injury. Spinal Cord
2003; 41: 23-28.










147. Chilibeck PD, Jeon J, Weiss C, Bell G, Burnham R. Histochemical changes in muscle of
individuals with spinal cord injury following functional electrical stimulated exercise
training. Spinal Cord 1999; 37: 264-268.

148. Sloan KE, Bremner LA, Byrne J, Day RE, Scull ER. Musculoskeletal effects of an
electrical stimulation induced cycling programme in the spinal injured. Parplegia~~~~PPPP~~~~PPPP 1994;
32: 407-415.

149. Donaldson N, Perkins TA, Fitzwater R, Wood DE, Middleton F. FES cycling may
promote recovery of leg function after incomplete spinal cord injury. Spinal Cord 2000;
38: 680-682.

150. Maynard FM, Jr., Bracken MB, Creasey G, Ditunno JF, Jr., Donovan WH, Ducker TB,
Garber SL, Marino RJ, Stover SL, Tator CH, Waters RL, Wilberger JE, Young W.
International Standards for Neurological and Functional Classification of Spinal Cord
Injury. American Spinal Injury Association. Spinal Cord 1997; 35: 266-274.

151. Dobkin BH, Apple D, Barbeau H, Basso M, Behrman A, Deforge D, Ditunno J, Dudley
G, Elashoff R, Fugate L, Harkema S, Saulino M, Scott M. Methods for a randomized trial
of weight-supported treadmill training versus conventional training for walking during
inpatient rehabilitation after incomplete traumatic spinal cord injury. Neurorehabil
Neural Repair 2003; 17: 153-167.

152. Shield A, Zhou S. Assessing voluntary muscle activation with the twitch interpolation
technique. Sports M~ed 2004; 34: 253-267.

153. Todd G, Gorman RB, Gandevia SC. Measurement and reproducibility of strength and
voluntary activation of lower-limb muscles. Muscle Nerve 2004; 29: 834-842.

154. Pap G, Machner A, Awiszus F. Strength and voluntary activation of the quadriceps
femoris muscle at different severities of osteoarthritic knee j oint damage. J Orthop Res
2004; 22: 96-103.

155. Norregaard J, Bulow PM, Vestergaard-Poulsen P, Thomsen C, Danneskiold-Samoe B.
Muscle strength, voluntary activation and cross-sectional muscle area in patients with
fibromyalgia. Br JRheumatol 1995; 34: 925-931.

156. Allen GM, Middleton J, Katrak PH, Lord SR, Gandevia SC. Prediction of voluntary
activation, strength and endurance of elbow flexors in postpolio patients. Muscle Nerve
2004; 30: 172-181.

157. Stevens JE, Mizner RL, Snyder-Mackler L. Quadriceps strength and volitional activation
before and after total knee arthroplasty for osteoarthritis. J Orthop Res 2003; 21: 775-
779.

158. Binder-Macleod SA. Variable-frequency stimulation patterns for the optimization of
force during muscle fatigue. Muscle wisdom and the catch-like property. Adv Exp M~ed
Biol 1995; 384: 227-240.









159. Neptune RR, Kautz SA, Zaj ac FE. Contributions of the individual ankle plantar flexors to
support, forward progression and swing initiation during walking. JBiomech 2001; 34:
1387-1398.

160. Aagaard P, Simonsen EB, Andersen JL, Magnusson P, Dyhre-Poulsen P. Increased rate
of force development and neural drive of human skeletal muscle following resistance
training. JApplPhysiol 2002; 93: 1318-1326.

161. Bemben MG, Tuttle TD, Bemben DA, Knehans AW. Effects of creatine supplementation
on isometric force-time curve characteristics. M~ed Sci Sports Exerc 2001; 33: 1876-1881.

162. Gerrits HL, de Haan A, Hopman MT, van der Woude LH, Sargeant AJ. Influence of
muscle temperature on the contractile properties of the quadriceps muscle in humans with
spinal cord injury. Clin Sci (Lond) 2000; 98: 31-38.

163. Shields RK. Fatigability, relaxation properties, and electromyographic responses of the
human paralyzed soleus muscle. JNeurophysiol 1995; 73: 2195-2206.

164. Ostchega Y, Dillon CF, Lindle R, Carroll M, Hurley BF. Isokinetic leg muscle strength in
older americans and its relationship to a standardized walk test: data from the national
health and nutrition examination survey 1999-2000. JAm Geriatr Soc 2004; 52: 977-982.

165. Mueller MJ, Minor SD, Schaaf JA, Strube MJ, Sahrmann SA. Relationship of plantar-
flexor peak torque and dorsiflexion range of motion to kinetic variables during walking.
Phys Ther 1995; 75: 684-693.

166. Muller R, Dietz V. Neuronal function in chronic spinal cord injury: divergence between
locomotor and flexion- and H-reflex activity. Clin Neurophysiol 2006; 117: 1499-1507.

167. Huang H, He J, Herman R, Carhart MR. Modulation effects of epidural spinal cord
stimulation on muscle activities during walking. IEEE Trans Neural Syst Rehabil Eng
2006; 14: 14-23.

168. Behrman AL, Lawless-Dixon AR, Davis SB, Bowden MG, Nair P, Phadke C, Hannold
EM, Plummer P, Harkema SJ. Locomotor training progression and outcomes after
incomplete spinal cord injury. Phys Ther 2005; 85: 1356-1371.

169. Wirz M, Zemon DH, Rupp R, Scheel A, Colombo G, Dietz V, Hornby TG. Effectiveness
of automated locomotor training in patients with chronic incomplete spinal cord injury: a
multi center trial. Arch Phys M~edRehabil 2005; 86: 672-680.

170. Hicks AL, Adams MM, Martin Ginis K, Giangregorio L, Latimer A, Phillips SM,
McCartney N. Long-term body-weight-supported treadmill training and subsequent
follow-up in persons with chronic SCI: effects on functional walking ability and
measures of subj ective well-being. Spinal Cord 2005; 43: 291-298.

171. Giangregorio LM, McCartney N. Reduced loading due to spinal-cord injury at birth
results in "slender" bones: a case study. Osteoporos Int 2007; 18: 117-120.










172. Nadeau S, Gravel D, Arsenault AB, Bourbonnais D. Plantarfiexor weakness as a limiting
factor of gait speed in stroke subjects and the compensating role of hip flexors. Clin
Biomech (Bristol, Avon) 1999; 14: 125-135.

173. Franco JC, Perell KL, Gregor RJ, Scremin AM. Knee kinetics during functional electrical
stimulation induced cycling in subj ects with spinal cord injury: a preliminary study. J
RehabilRes Dev 1999; 36: 207-216.

174. Gregory CM, Bowden MG, Jayaraman A, Shah P, Behrman A, Kautz SA, Vandenborne
K. Resistance training and locomotor recovery after incomplete spinal cord injury: a case
series. Spinal Cord 2007; 45: 522-530.

175. Stevens JE, Pathare NC, Tillman SM, Scarborough MT, Gibbs CP, Shah P, Jayaraman A,
Walter GA, Vandenborne K. Relative contributions of muscle activation and muscle size
to plantarfiexor torque during rehabilitation after immobilization. J Orthop Res 2006; 24:
1729-1736.

176. Marino RJ, Barros T, Biering-Sorensen F, Burns SP, Donovan WH, Graves DE, Haak M,
Hudson LM, Priebe MM. International standards for neurological classification of spinal
cord injury. JSpinal Cord2~ed 2003; 26 Suppl 1: S50-56.

177. Marino RJ, Ditunno JF, Jr., Donovan WH, Maynard F, Jr. Neurologic recovery after
traumatic spinal cord injury: data from the Model Spinal Cord Injury Systems. Arch Phys
M~edRehabil 1999; 80: 1391-1396.

178. Geisler FH, Coleman WP, Grieco G, Poonian D. Measurements and recovery patterns in
a multicenter study of acute spinal cord injury. Spine 2001; 26: S68-86.

179. Newton RU, Kraemer WJ, Hakkinen K. Effects of ballistic training on preseason
preparation of elite volleyball players. M~ed Sci Sports Exerc 1999; 31: 323-330.

180. Cronin J, Sleivert G. Challenges in understanding the influence of maximal power
training on improving athletic performance. Sports M~ed 2005; 35: 213-234.

181. Myer GD, Ford KR, Palumbo JP, Hewett TE. Neuromuscular training improves
performance and lower-extremity biomechanics in female athletes. J .uengthrl CondRes
2005; 19: 51-60.

182. Saunders PU, Telford RD, Pyne DB, Peltola EM, Cunningham RB, Gore CJ, Hawley JA.
Short-term plyometric training improves running economy in highly trained middle and
long distance runners. J .uengthrl CondRes 2006; 20: 947-954.

183. Herrero JA, Izquierdo M, Maffluletti NA, Garcia-Lopez J. Electromyostimulation and
plyometric training effects on jumping and sprint time. Int J Sports 2ed 2006; 27: 533-
539.

184. Behm DG, St-Pierre DM, Perez D. Muscle inactivation: assessment of interpolated twitch
technique. JAppl Physiol 1996; 81: 2267-2273.










185. Teixeira-Salmela LF, Nadeau S, McBride I, Olney SJ. Effects of muscle strengthening
and physical conditioning training on temporal, kinematic and kinetic variables during
gait in chronic stroke survivors. JRehabil2~ed 2001; 33: 53-60.

186. Ferri A, Scaglioni G, Pousson M, Capodaglio P, Van Hoecke J, Narici MV. Strength and
power changes of the human plantar flexors and knee extensors in response to resistance
training in old age. Acta Physiol Scanzd 2003; 177: 69-78.

187. Stevens JE, Walter GA, Okereke E, Scarborough MT, Esterhai JL, George SZ, Kelley
MJ, Tillman SM, Gibbs JD, Elliott MA, Frimel TN, Gibbs CP, Vandenborne K. Muscle
adaptations with immobilization and rehabilitation after ankle fracture. M~ed Sci Sports
Exerc 2004; 36: 1695-1701.

188. Perry J, Mulroy SJ, Renwick SE. The relationship of lower extremity strength and gait
parameters in patients with post-polio syndrome. Arch Phys M~edRehabil 1993; 74: 165-
169.

189. Canning CG, Ada L, O'Dwyer N. Slowness to develop force contributes to weakness
after stroke. Arch Phys M~edRehabil 1999; 80: 66-70.

190. Pohl PS, Duncan P, Perera S, Long J, Liu W, Zhou J, Kautz SA. Rate of isometric knee
extension strength development and walking speed after stroke. JRehabilRes Dev 2002;
39: 651-657.

191. Barbeau H. Locomotor training in neurorehabilitation: emerging rehabilitation concepts.
Neurorehabil Neural Repair 2003; 17: 3-11.

192. Levy CE, Nichols DS, Schmalbrock PM, Keller P, Chakeres DW. Functional MRI
evidence of cortical reorganization in upper-limb stroke hemiplegia treated with
constraint-induced movement therapy. Am JPhys 2edRehabil 2001; 80: 4-12.

193. Love FM, Son YJ, Thompson WJ. Activity alters muscle reinnervation and terminal
sprouting by reducing the number of Schwann cell pathways that grow to link synaptic
sites. JNeurobiol 2003; 54: 566-576.

194. Kim CM, Eng JJ. The relationship of lower-extremity muscle torque to locomotor
performance in people with stroke. Phys Ther 2003; 83: 49-57.

195. Neptune RR, Zaj ac FE, Kautz SA. Muscle force redistributes segmental power for body
progression during walking. Gait Posture 2004; 19: 194-205.

196. Barbeau H, Ladouceur M, Norman KE, Pepin A, Leroux A. Walking after spinal cord
injury: evaluation, treatment, and functional recovery. Arch Phys M~edRehabil 1999; 80:
225-235.

197. Edgerton VR, de Leon RD, Tillakaratne N, Recktenwald MR, Hodgson JA, Roy RR.
Use-dependent plasticity in spinal stepping and standing. AdvI Neurol 1997; 72: 233-247.










198. Edgerton VR, Kim SJ, Ichiyama RM, Gerasimenko YP, Roy RR. Rehabilitative therapies
after spinal cord injury. JNeurotrauma 2006; 23: 560-570.

199. Basso DM, Beattie MS, Bresnahan JC. Graded histological and locomotor outcomes after
spinal cord contusion using the NYU weight-drop device versus transaction. Exp Neurol
1996; 139: 244-256.

200. Rosenzweig ES, McDonald JW. Rodent models for treatment of spinal cord injury:
research trends and progress toward useful repair. Curr Opin Neurol 2004; 17: 121-131.

201. Gazula VR, Roberts M, Luzzio C, Jawad AF, Kalb RG. Effects of limb exercise after
spinal cord injury on motor neuron dendrite structure. J Comp Neurol 2004; 476: 130-
145.

202. Thomason DB, Booth FW. Atrophy of the soleus muscle by hindlimb unweighting. J
ApplPhysiol 1990; 68: 1-12.

203. Basso DM, Beattie MS, Bresnahan JC. Descending systems contributing to locomotor
recovery after mild or moderate spinal cord injury in rats: experimental evidence and a
review of literature. Restor Neurol Neurosci 2002; 20: 189-218.

204. Dietz V, Wirz M, Jensen L. Locomotion in patients with spinal cord injuries. Phys 7lher
1997; 77: 508-516.

205. Barbeau H, Rossignol S. Enhancement of locomotor recovery following spinal cord
injury. Curr Opin Neurol 1994; 7: 517-524.

206. Edgerton VR, Roy RR. Paralysis recovery in humans and model systems. Curr Opin
Neurobiol 2002; 12: 658-667.

207. de Leon RD, Roy RR, Edgerton VR. Is the recovery of stepping following spinal cord
injury mediated by modifying existing neural pathways or by generating new pathways?
A perspective. Phys Ther 2001; 81: 1904-1911.

208. Liu M, Bose P, Walter GA, Thompson FJ, Vandenborne K. A longitudinal study of
skeletal muscle following spinal cord injury and locomotor training. Spinal Cord 2008.

209. Roy RR, Zhong H, Siengthai B, Edgerton VR. Activity-dependent influences are greater
for fibers in rat medial gastrocnemius than tibialis anterior muscle. Muscle Nerve 2005;
32: 473-482.

210. Roy RR, Zhong H, Monti RJ, Vallance KA, Kim JA, Edgerton VR. Mechanical
properties and fiber type composition of chronically inactive muscles. J Gravit Physiol
2000; 7: P103-104.

211. Roy RR, Zhong H, Monti RJ, Vallance KA, Edgerton VR. Mechanical properties of the
electrically silent adult rat soleus muscle. Muscle Nerve 2002; 26: 404-412.










212. Roy RR, Zhong H, Bodine SC, Pierotti DJ, Talmadge RJ, Barkhoudarian G, Kim J,
Fanton JW, Kozlovskaya IB, Edgerton VR. Fiber size and myosin phenotypes of selected
rhesus lower limb muscles after a 14-day spaceflight. J Gravit Physiol 2000; 7: S45.

213. Talmadge RJ. Myosin heavy chain isoform expression following reduced neuromuscular
activity: potential regulatory mechanisms. Muscle Nerve 2000; 23: 661-679.

214. Lee YS, Lin CY, Caiozzo VJ, Robertson RT, Yu J, Lin VW. Repair of spinal cord
transaction and its effects on muscle mass and myosin heavy chain isoform phenotype. J
ApplPhysiol 2007; 103: 1808-1814.

215. Grossman EJ, Roy RR, Talmadge RJ, Zhong H, Edgerton VR. Effects of inactivity on
myosin heavy chain composition and size of rat soleus fibers. Muscle Nerve 1998; 21:
375-389.

216. Hyatt JP, Roy RR, Baldwin KM, Wernig A, Edgerton VR. Activity-unrelated neural
control of myogenic factors in a slow muscle. Muscle Nerve 2006; 33: 49-60.

217. Talmadge RJ, Roy RR, Edgerton VR. Myosin heavy chain profile of cat soleus following
chronic reduced activity or inactivity. Muscle Nerve 1996; 19: 980-988.

218. Talmadge RJ, Garcia ND, Roy RR, Edgerton VR. Myosin heavy chain isoform mRNA
and protein levels after long-term paralysis. Biochem Biophys Res Commun 2004; 325:
296-301.

219. Behrman AL, Harkema SJ. Physical rehabilitation as an agent for recovery after spinal
cord injury. Phys2~edRehabil Clin NAm 2007; 18: 183-202, v.

220. Harkema SJ, Hurley SL, Patel UK, Requej o PS, Dobkin BH, Edgerton VR. Human
lumbosacral spinal cord interprets loading during stepping. JNeurophysiol 1997; 77:
797-811.

221. Hopman MT, Dueck C, Monroe M, Philips WT, Skinner JS. Limits to maximal
performance in individuals with spinal cord injury. Int J Sports 2ed 1998; 19: 98-103.

222. Machida S, Booth FW. Insulin-like growth factor 1 and muscle growth: implication for
satellite cell proliferation. Proc Nutr Soc 2004; 63: 337-340.

223. Adams GR. Role of insulin-like growth factor-I in the regulation of skeletal muscle
adaptation to increased loading. Exerc Sport Sci Rev 1998; 26: 31-60.

224. Darr KC, Schultz E. Exercise-induced satellite cell activation in growing and mature
skeletal muscle. JApplPhysiol 1987; 63: 1816-1821.

225. Cabric M, James NT. Morphometric analyses on the muscles of exercise trained and
untrained dogs. Am JAnat 1983; 166: 359-368.









226. Allen DL, Monke SR, Talmadge RJ, Roy RR, Edgerton VR. Plasticity of myonuclear
number in hypertrophied and atrophied mammalian skeletal muscle fibers. JAppl Physiol
1995; 78: 1969-1976.

227. Barton ER. Viral expression of insulin-like growth factor-I isoforms promotes different
responses in skeletal muscle. JApplPhysiol 2006; 100: 1778-1784.

228. Scime A, Rudnicki MA. Anabolic potential and regulation of the skeletal muscle satellite
cell populations. Curr Opin Clin Nutr Metab Care 2006; 9: 214-219.

229. Kuang S, Charge SB, Seale P, Huh M, Rudnicki MA. Distinct roles for Pax7 and Pax3 in
adult regenerative myogenesis. JCell Biol 2006; 172: 103-113.

230. Grenier G, Rudnicki MA. The potential use of myogenic stem cells in regenerative
medicine. Handb Exp Pharmacol 2006; 299-317.

231. Grounds MD. Muscle regeneration: molecular aspects and therapeutic implications. Curr
Opin Neurol 1999; 12: 535-543.

232. Wagers AJ, Conboy IM. Cellular and molecular signatures of muscle regeneration:
current concepts and controversies in adult myogenesis. Cell 2005; 122: 659-667.

233. Caroni P. Activity-sensitive signaling by muscle-derived insulin-like growth factors in
the developing and regenerating neuromuscular system. Ann N YAcad Sci 1993; 692:
209-222.

234. Allen RE, Boxhorn LK. Regulation of skeletal muscle satellite cell proliferation and
differentiation by transforming growth factor-beta, insulin-like growth factor I, and
fibroblast growth factor. JCell Physiol 1989; 138: 311-315.

235. Adams GR, Haddad F. The relationships among IGF-1, DNA content, and protein
accumulation during skeletal muscle hypertrophy. JAppl Physiol 1996; 81: 2509-2516.

236. Adams GR, Caiozzo VJ, Haddad F, Baldwin KM. Cellular and molecular responses to
increased skeletal muscle loading after irradiation. Am JPhysiol Cell Physiol 2002; 283:
C1182-1195.

237. Resnick DK, Schmitt C, Miranpuri GS, Dhodda VK, Isaacson J, Vemuganti R. Molecular
evidence of repair and plasticity following spinal cord injury. Neuroreport 2004; 15: 837-
839.

238. Dupont-Versteegden EE, Murphy RJ, Houle JD, Gurley CM, Peterson CA. Mechanisms
leading to restoration of muscle size with exercise and transplantation after spinal cord
injury. Am JPhysiol Cell Physiol 2000; 279: C1677-1684.

239. Yablonka-Reuveni Z, Rivera AJ. Temporal expression of regulatory and structural
muscle proteins during myogenesis of satellite cells on isolated adult rat fibers. Dev Biol
1994; 164: 588-603.










240. Cornelison DD, Wold BJ. Single-cell analysis of regulatory gene expression in quiescent
and activated mouse skeletal muscle satellite cells. Dev Biol 1997; 191: 270-283.

241. Hawke TJ, Garry DJ. Myogenic satellite cells: physiology to molecular biology. JAppl
Physiol 2001; 91: 534-551.

242. Olguin HC, Yang Z, Tapscott SJ, Olwin BB. Reciprocal inhibition between Pax7 and
muscle regulatory factors modulates myogenic cell fate determination. JCellBiol2007;
177: 769-779.









BIOGRAPHICAL SKETCH

Arun Jayaraman was born in Chennai, India. He received his bachelor' s in physical

therapy from Dr. MGR Medical University in 2000 and his master' s in hospital management

from Loyola Institute of Business Administration in 2001. He also worked as an in-patient

physical therapist in cardiac rehab in the Institute of Cardio-Pulmonary Diseases in Chennai till

the year 2001. He received his advanced master' s of science in physical therapy from Georgia

State University, Atlanta, GA, in the year 2003. He j oined the doctoral program in rehabilitation

science at the University of Florida in the fall of 2003.





PAGE 1

1 SKELETAL MUSCLE ADAPTATIONS FOLLOWING INCOMPLETE SPINAL CORD INJURY AND EXERCISE TRAINING By ARUN JAYARAMAN A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2008

PAGE 2

2 2008 Arun Jayaraman

PAGE 3

3 To my mother, wife and, the almighty

PAGE 4

4 ACKNOWLEDGMENTS I am very grateful to severa l individuals for their guidance and support in my dissertation work. The greater part of this work was made possible by the guidance of my mentors and by the love and support of my family, friends and collea gues. It is with my he artfelt gratitude that I acknowledge each of them. First and foremost, I would like to sincerel y thank my advisor and mentor Dr.Krista Vandenborne, for her constant guidance, support a nd, encouragement. Under her, not only did I learn about conducting excellent research bu t also on how to be a complete academic professional. Words cannot simply summarize my gratitude towards her. Al so, I am grateful to Dr.Walter for his remarkable wisdom and thought provoking research questions. Never has a day gone by were I have not been amazed by his up to date knowledge on almost every research topic. I would like to thank Dr .Behrman for her constant encouragement and infinite support throughout my doctoral education. I would also like to express my sincere thanks and gratitude to Dr.Rosenbek for always believing in me and en couraging me to strive harder towards by goals and aspirations. A special thanks to all my lab members, wit hout whose help this di ssertation would have never been possible. I would like to thank Min, Chris, Neeti, and Jen for teaching me all the necessary skills and techniques in the lab and fo r also guiding me and encouraging me through the PhD process. I would like to thank Prithvi Fan, Gabe, Sunita, Donovan, Ravneet, and Wendy for constantly helping me and making work a lot of fun. A special thanks to Dr.Miles for helping me with my document and to the Physical Therapy department for being a very important part of my graduate student life. Finally, I would like to express my deepest gr atitude to my family. I would like to thank my grandparents for their love and support. I wo uld like to specially th ank my mother for her

PAGE 5

5 unconditional love and support thro ughout my life. I would like to say without her this PhD was never ever possible. She has encouraged me a ll throughout and always has put my education as her first priority. Last but not the least; I would like thank my beloved wife Sangeetha who has been the main pillar of support in my life. I ow e my success in life to he r. She has always had a smile on her face and her hand has held me thr ough all the ups and downs in my life. Thank you for all the sacrifices and difficulties that you ha ve patiently endured. Finally, I would like to thank the almighty and savior for trusting in me, loving me and being there for me. Thank you God! You made it all possible.

PAGE 6

6 TABLE OF CONTENTS page ACKNOWLEDGMENTS...............................................................................................................4 LIST OF TABLES................................................................................................................. ........10 LIST OF FIGURES.......................................................................................................................11 ABSTRACT...................................................................................................................................13 CHAP TER 1 BACKGROUND.................................................................................................................... 15 1.1 Introduction............................................................................................................... ....15 1.2 Demographics of Spinal Cord Injury (SCI).................................................................. 15 1.3 SCI in Humans-Pathophysio logy and Classification ....................................................17 1.3.1 Definition of SCI...............................................................................................17 1.3.2 Spinal Cord Neuro-anatomy............................................................................. 18 1.3.3 SCI Pathophysiology......................................................................................... 19 1.3.4 Classification of SCI.........................................................................................20 1.4 SCI in the Animal Model.............................................................................................. 21 1.4.1 Spinal Cord Isolation Model............................................................................. 22 1.4.2 Spinal Cord Transection Model........................................................................ 22 1.4.3 Spinal Cord Hemisection Model....................................................................... 23 1.4.4 Spinal Cord Contusion Model........................................................................... 25 1.5 Skeletal Muscle Adaptations in Human Following SCI...............................................27 1.5.1 Muscle Size....................................................................................................... 27 1.5.2 Fiber Type Composition................................................................................... 28 1.5.3 Electrically Elicited Contractile Properties .......................................................30 1.5.4 Voluntary Contractile Measurements............................................................... 31 1.6 Skeletal Muscle Adaptations in the Ani mal Models Following SCI............................ 33 1.6.1 Muscle Size....................................................................................................... 33 1.6.2 Fiber Type Composition................................................................................... 34 1.6.3 Electrically Elicited Contractile Properties .......................................................38 1.7 Rehabilitation Training St rategies F ollowing SCI........................................................ 39 1.7.1 Locomotor Training.......................................................................................... 39 1.7.1.1 Locomotor training in humans............................................................ 39 1.7.1.2 Locomotor training in the animal model............................................ 41 1.7.2 Functional Electrical Stim ulation (FES)........................................................... 42 1.7.3 Resistance Training........................................................................................... 45 1.8 Skeletal Muscle Adaptations Following SCI and Locomotor training......................... 46 1.8.1 Impact on Humans.............................................................................. 46 1.8.2 Impact on the Animal Model.............................................................. 47 1.9 Mechanisms Involved in Training Indu ced Muscle Plasticity and Recovery ............... 49

PAGE 7

7 1.9.1 Plasticity of Skeletal Muscle............................................................................. 49 1.9.2 Markers of Muscle Recovery and Regeneration............................................... 51 1.9.2.1 Adult muscle satellite cells................................................................. 51 1.9.2.2 Other stem cells.................................................................................. 54 1.8.2.3 Growth factors and muscle regeneration............................................ 55 2 OUTLINE OF EXPERIMENTS............................................................................................ 59 2.1 Experiment 1............................................................................................................... ..59 2.1.1 Specific Aim..................................................................................................... 59 2.1.2 Hypothesis......................................................................................................... 59 2.2 Experiment 2............................................................................................................... ..59 2.2.1 Specific Aim..................................................................................................... 59 2.2.2 Hypothesis......................................................................................................... 60 2.3 Experiment 3............................................................................................................... ..60 2.3.1 Specific Aim..................................................................................................... 60 2.3.2 Hypotheses........................................................................................................ 60 2.4 Experiment 4............................................................................................................... ..61 2.4.1 Specific Aim..................................................................................................... 61 2.4.2 Hypotheses........................................................................................................ 61 2.5 Experiment 5............................................................................................................... ..62 2.5.1 Specific Aim..................................................................................................... 62 2.5.2 Hypotheses........................................................................................................ 63 3 METHODOLOGY................................................................................................................. 64 3.1 Studies in People with Incomplete-SCI........................................................................ 64 3.1.1 Subjects Description......................................................................................... 64 3.1.2 Locomotor Training............................................................................64 3.1.3 Resistance and Plyometric Training.................................................................. 65 3.1.3.1 Resistance training.............................................................................. 65 3.1.3.2 Plyometric training............................................................................. 66 3.1.4 Muscle Function Assessment............................................................................66 3.1.4.1 Experimental set-up............................................................................ 66 3.1.4.2 Voluntary contractile measurements.................................................. 67 3.1.4.3 Electrically elicited cont ractile m easurements................................... 68 3.1.5 Measures of Ambulatory Function....................................................................69 3.2 Experiments in Contusion Spinal Cord Injured Animals.............................................. 70 3.2.1 Animals............................................................................................................. 70 3.2.2 Contusion Injury............................................................................................... 71 3.2.3 Locomotor Training.......................................................................................... 71 3.2.4 In-Vitro Assay of Muscle Composition and Regeneration............................... 72 3.2.4.1 Immunohistochemical analysis........................................................... 72 3.2.4.2 Western blot analysis.......................................................................... 73 4 LOWER EXTREMITY SKELETAL MUSC L E FUNCTION IN PERSONS WITH INCOMPLETE SPINAL CORD INJURY............................................................................. 77

PAGE 8

8 4.1 Introduction............................................................................................................... ....77 4.2 Methods.................................................................................................................... .....78 4.2.1 Subjects............................................................................................................. 78 4.2.2 Experimental Set-Up......................................................................................... 79 4.2.3 Voluntary Contractile Measurements............................................................... 80 4.2.4 Electrically Elicited Co ntractile Measurem ents................................................ 80 4.2.5 Voluntary Activation Deficits........................................................................... 81 4.2.6 Statistical Analyses........................................................................................... 81 4.3 Results.................................................................................................................... .......81 4.3.1 Voluntary Contractile Measurements............................................................... 81 4.3.2 Electrically Elicited Co ntractile Measurem ents................................................ 83 4.3.3 Voluntary Activation Deficits........................................................................... 83 4.4 Discussion................................................................................................................. ....83 5 LOCOMOTOR TRAINING AND MUSC LE FUNCTION AFTER INCOMPLETE SPINAL CORD INJ URY: A CASE SERIES........................................................................95 5.1 Introduction............................................................................................................... ....95 5.2 Methods.................................................................................................................... .....96 5.2.1 Subjects............................................................................................................. 96 5.2.2 Locomotor Training Protocol............................................................................ 97 5.2.3 Experimental Protocol....................................................................................... 98 5.2.3.1 Strength assessment............................................................................ 98 5.2.3.2 Voluntary contractile measurements.................................................. 98 5.2.3.3 Voluntary activation deficits............................................................... 99 5.2.4 Statistical Analyses........................................................................................... 99 5.3 Results.................................................................................................................... .......99 5.3.1 Voluntary Contractile Measurements............................................................... 99 5.3.2 Voluntary Activation Deficits......................................................................... 100 5.4 Discussion................................................................................................................. ..101 6 RESISTANCE TRAINING AND LOCOMOTOR RECOVERY AFTER INCOMPLETE SPINAL CORD IN JURY: A CASE SERIES ............................................ 111 6.1 Introduction............................................................................................................... ..111 6.2 Methods.................................................................................................................... ...113 6.2.1 Subjects........................................................................................................... 113 6.2.2 Resistance Training Program.......................................................................... 114 6.2.3 Plyometric Training........................................................................................ 114 6.2.4 Dynamometry.................................................................................................. 115 6.2.5 Voluntary Activation Deficits......................................................................... 116 6.2.6 Locomotor Data Collection............................................................................. 117 6.3 Results.................................................................................................................... .....117 6.3.1 Dynamometry.................................................................................................. 117 6.3.2 Voluntary Activation Deficits......................................................................... 118 6.3.3 Locomotor Analyses....................................................................................... 118 6.4 Discussion................................................................................................................. ..118

PAGE 9

9 7 LOWER EXTREMITY SKELETAL MUSCLE MORPHOLOGY AND FIBE R TYPE COMPOSITION FOLLOWING MODERATE CONTUSION SPINAL CORD INJURY AND LOCOMOTOR TRAINING........................................................................ 125 7.1 Introduction............................................................................................................... ..125 7.2 Methods.................................................................................................................... ...126 7.2.1 Animals........................................................................................................... 126 7.2.3 Locomotor Treadmill Training....................................................................... 128 7.2.4 Tissue Harvest................................................................................................. 128 7.2.5 Immunohistochemical Measures..................................................................... 129 7.2.6 Data Analysis.................................................................................................. 129 7.3 Results.................................................................................................................... .....130 7.3.1 Effects of IncompleteSCI and Locomotor Training on Fiber Crossectional Area (CSA) ...............................................................................130 7.3.2 Effects of IncompleteSCI and Locomotor Training on Fiber Type Com position.................................................................................................... 131 7.4 Discussion................................................................................................................. ..133 8 SKELETAL MUSCLE RECOVERY AND REGENERATI ON FOLLOWING MODERATE CONTUSION SPINAL CO RD INJURY AND LOCOMOTOR TRAINING....................................................................................................................... ....143 8.1 Introduction............................................................................................................... ..143 8.2 Methods.................................................................................................................... ...144 8.2.1 Animals........................................................................................................... 144 8.2.3 Locomotor Treadmill Training....................................................................... 146 8.2.4 Tissue Harvest................................................................................................. 146 8.2.5 Determination of IGF-I Protein Concentration............................................... 147 8.2.6 Immunohistochemistry Measurements........................................................... 147 8.2.7 Western Blot Analysis.................................................................................... 148 8.2.8 Data Analysis.................................................................................................. 148 8.3 Results.................................................................................................................... .....149 8.3.1 Effects of Incomplete Spinal co rd Injury and Locom otor Training on Insulin-Like Growth Factor-1 (IGF-1) Expression......................................... 149 8.3.2 Effects of Incomplete Spinal Cord Injury and Locom otor Training on Pax7.......................................................................................................................149 8.3.3 Effects of Incomplete Spinal Cord Injury and Locomotor Training on Myogenic R egulatory Factors (MyoD, Myf5 and Myogenin)..................... 150 8.3.4 Effects of Incomplete Spinal Cord Injury and Locomotor Training on Em bryonic Myosin.......................................................................................... 151 8.4 Discussion................................................................................................................. ..151 LIST OF REFERENCES.............................................................................................................163 BIOGRAPHICAL SKETCH.......................................................................................................182

PAGE 10

10 LIST OF TABLES Table page 4-1 Characteristics of in complete SCI subjects........................................................................88 4-2 Electrically elicited c ontractile m easurements................................................................... 88 5-1 Characteristics of inco mplete SCI subjects......................................................................105 5-2 Values of isometric peak torque an d average rate of force developm ent........................106 6-1 Preand post-RPT isometric torque data for the p lantar flexor and knee extensor muscle groups..................................................................................................................123

PAGE 11

11 LIST OF FIGURES Figure page 1-1 Etiology of SCI since 2000................................................................................................ 56 1-2 Estimated lifetime costs by age at injury........................................................................... 57 1-3 Satellite cell number in skeletal m uscle of different ages and type................................... 57 1-4 Satellite cell activity.................................................................................................... .......58 1-5 Schematic outline of a stem cell passing through the stages of m uscle regeneration........ 58 3-1 Set-up for locomotor training............................................................................................. 74 3-2 Plyometric training set-up................................................................................................. .74 3-3 Experimental set-up on a Bi odex system 3 dynamometer. ............................................... 75 3-4 Contusion injury set-up. ....................................................................................................75 3-5 anti-MHC antibodies........................................................................................................ ..75 3-6 Locomotor training in the rat model. ................................................................................ 76 4-1 Representative torque-time curve...................................................................................... 89 4-2 Peak torque (Nm) for the knee extens or and plantar flexor m uscle groups....................... 90 4-3 Torque200 (Nm) for the knee extensor and plantar flexor muscle groups.......................... 91 4-4 Average rate of torque development (ARTD)(Nm/sec) for the knee extensor and plantar flexor m uscle groups.............................................................................................. 92 4-5 Voluntary activation deficits (%) for the knee extensor and plantar flexor muscle groups. ................................................................................................................................93 4-6 Torque trace acquired during MVIC with interpolated twitch. ......................................... 94 5-1 Torque200 (Nm) measured in th e knee extensor muscle group. .................................... 107 5-1 Torque200 (Nm) measured in th e plantar flexor muscle group. ...................................... 108 5-2 Voluntary activation deficits (%) meas ured in the knee extensor m uscle group............. 109 5-2 Voluntary activation deficits (%) measur ed in the plantar flexor m uscle group............. 110 6-1 Example of plyomet ric training device ............................................................................ 124

PAGE 12

12 7-1 Average soleus muscle fiber CSA...................................................................................139 7-2 Average EDL muscle fiber CSA...................................................................................... 139 7-3 Average gastrocnemius muscle fiber CSA......................................................................140 7-4 Average TA muscle fiber CSA........................................................................................ 140 7-5 MHC based fiber type co mposition of rat soleus............................................................. 141 7-6 MHC based fiber type composition of rat TA. ............................................................... 141 7-7 MHC based fiber type composition of rat EDL............................................................... 142 7-8 MHC based fiber type composition of rat gastrocnem ius............................................... 142 8-1 Pax-7 positive staining. .................................................................................................. .157 8-2 MyoD protein levels........................................................................................................158 8-3 Myf5 protein levels..........................................................................................................159 8-4 Myogenin protein levels..................................................................................................160 8-5 Embryonic myosin positives............................................................................................ 161 8-6 IGF-1 levels.....................................................................................................................162

PAGE 13

13 Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy SKELETAL MUSCLE ADAPTATIONS FOLLOWING INCOMPLETE SPINAL CORD INJURY AND EXERCISE TRAINING By Arun Jayaraman May 2008 Chair: Krista Vandenborne Major: Rehabilitation Science Recovery of function after incomplete spinal co rd injury (incomplete-SC I) is in an exciting phase of research. Paralysis and paresis of lo wer extremity muscles following incomplete-SCI result in persistent motor dysfunction and impair ed walking. Advances in research have led to promising exercise-training stra tegies in both humans and animals following SCI. However, the mechanisms that explain the functional improvements reported following incompleteSCI and exercise training are not clea rly understood and could possibly result from musculoskeletal changes, neural adaptations, or a combination thereof. The primary purpose of this dissertation was to explore the adaptations in lower extremity skeletal muscle following incomplete-SCI and exercise training in both humans and animals. Ours findings indicate a significant loss of both peak isometric and explosive strength in lowe r extremities after incomplete-SCI in humans. Additionally, this loss in strength was attributed to a severe loss in voluntary activation of the paretic muscles. Locomotor training and resistance training were two exerci se interventions that were tested in our study, and our findings suggest that both lo comotor training and resistance training helped in sign ificantly improving both voluntary a nd explosive strength, and voluntary activation in the lower extremity muscles of pers ons with incomplete-SCI. In the rat model,

PAGE 14

14 incomplete-SCI resulted in significant atrophy in all four lower extremity muscles. In addition, SCI resulted in a shift in fi ber type composition measured us ing myosin heavy chain (MHC) composition towards faster isoforms in all four lo wer extremity muscles. Locomotor training in the rats resulted in significantly reducing the atrophy in all lower extremity muscles. In addition, there was also a significant shif t in fiber types in all hind limb muscles towards slower isoforms. In addition, our results indicate that recovery in muscle si ze following SCI and locomotor training was due to the activation of satellite cells which went to form multinucleated myotubes which repaired or replaced damaged or lost musc le fibers. The overall findings from the present work will provide essential feedback on deficits in muscle function following SCI and also effects of exercise training interventions towa rds reducing the musculos keletal deficits and promoting muscle plasticity following incomplete SCI. These findings might provide feedback for the development and integration of these exercise interventions into the community.

PAGE 15

15 CHAPTER 1 BACKGROUND 1.1 Introduction Spinal cord injury (SCI) causes a host of physical and psychosocial problem s that interferes with an individuals personal health, feeli ng of well being and so cietal interaction. The goal of rehabilitation after spinal cord injury is to enable the person to resume a life style which is physically and functionally healthy and also helps the person integrate with his family, community and society. The central role of rehabilitation requires a comprehensive understanding of all different phys iological and functiona l adaptations that occur with SCI. The main focus of this dissertation is to identify adaptations in muscle function after SCI and its response to different exercise in terventions. Background literature pertaining to all chapters is briefly discussed in chapter-1. 1.2 Demographics of Spinal Cord Injury (SCI) It is estim ated that the annual incidence of sp inal cord injury (SCI), is approximately 40 cases per million population in the U. S. or approximately 11,000 new cases each year.1 The number of people in the United States who are alive as of June 2006 who have had a SCI has been estimated to be approximately 253,000, with a range of 225,000 to 296,000 persons.1 SCI primarily affects young adults. From 1973 to 1979, the average ag e at injury was 28.7 years, and most injuries occurred between the ages of 16 and 30. Since 2000, the average age at injury is 38.0 years since the median age of the general population of the United States has increased by approximately 8 years since the mid-1970s, the average age at injury has also steadily increased over time. Moreover, the percenta ge of persons older th an 60 years of age who had a SCI has increased from 4.7% prior to 1980 to 11.5% among injuries occurring since 2000. 1Prior to 1980, 81.8% of new spinal cord inju ries occurred among males. Since 2000, 77.8% of

PAGE 16

16 SCI reported to the national data base have occurred among males.1Over the history of the database, there has been a slight trend toward a decreasing percentage of males sustaining SCI.1 Among those injured since 2000, 63.0% are Caucas ian, 22.7% are African American, 11.8% are Hispanic, and 2.4% are from other racial/ethnic groups.1, 2 Looking at the etiology of SCI, it is plausible that this injury by itself causes dramatic changes in ones lifestyle and occupational status. Howe ver, by ten years post-injury, 32.4% of persons with paraplegia become employed, while only 2 4.2% of those with te traplegia are employed during the same year.1,2 The average yearly health care and living expenses and the estimated lifetime costs that are di rectly attributable to SCI vary grea tly according to severity of injury (Fig.1-1). The last section of the facts following SCI pert ains to the causes of the death after SCI and level and extent of the lesion. The most comm on cause of death in persons with SCI is respiratory ailment, whereas, in the past it wa s renal failure. An incr easing number of people with SCI are dying of unrelated causes such as cancer or cardiovascular diseas e, similar to that of the general population. Mortality ra tes are significantly higher during the first year after injury than during subsequent years. Si nce 2000, the most frequent neurol ogical category is incomplete tetraplegia (34.1%) and incomplete paraplegia (23.1%), followed by complete paraplegia (23.0%), and complete tetraplegi a (18.3%). This makes the incide nce of incomplete injuries equals to ~57% of total SCI as of 2000. Over the past few years, the percentage of persons with incomplete tetraplegia has increased slightly while both complete paraplegia and complete tetraplegia have decreased slightly (Fig.1-2).1, 2

PAGE 17

17 In Summary, in this section the statistics a nd demographics pertaining to SCI were briefly described. A summary of the etiology, age, gender, causes of death and common types of SCI were discussed. 1.3 SCI in Humans-Pathophysiology and Classification The inf ormation in this section mainly pertains to the medical defi nition and classification of SCI. This includes the anatomy, physiology, diagnosis and classifi cation of SCI. This information will help us better unde rstand the diagnosis, extent of in jury and recovery levels of the subjects with SCI described in following chapters. 1.3.1 Definition of SCI SCI can be categorized into traum atic or non-tr aumatic injuries. The sp inal cord is often violently displaced or compressed momentarily during the injury with forceful flexion, extension, and rotation of the sp ine. The vertebral body can burst and cause pressure or scatter bone fragments into the spinal cord. SCIs are cl assified as concussion, contusion, laceration, or transection. A concussion is an injury caused by a blow or vi olent shake and results in temporary loss of function.2,3 In contusion injury, the glial tissue and spinal cord surface remain intact. There is loss of central gray matter and white matter, which creates a cavity that, is surrounded by a rim of intact white matter at the periphery of the spinal cord.3 Laceration of the cord occurs with more severe injuries in which the glia is disrupted, and the spinal cord tissue may get torn. Occasionally this can result in complete dissection of the spinal cord known as transection injuries. 3 Gun shot wounds, knife wounds, and puncture wounds fall into this category. Hemorrhages caused by contusion or laceration injuries can cause further compression of the cord.2,3 SCIs can also be classified as primary or secondary based on the modus of injury. Primary SCIs arise from mechanical disrupt ion, transection, or dist raction of neural elements. This injury

PAGE 18

18 usually occurs with fracture and/ or dislocation of the spine. Ho wever, primary SCI may occur in the absence of spinal fracture or dislocation. Pe netrating injuries due to bullets or weapons may also cause primary SCI. More commonly, disp laced bony fragments cause penetrating spinal cord and/or segmental spinal nerve injuries. Ex tradural pathology may also cause primary SCI. Spinal epidural hematomas or abscesses cause acute cord compression a nd injury. Spinal cord compression from metastatic disease is a common oncologic emer gency. Longitudinal distraction with or without fl exion and/or extension of the vertebral column may result in primary SCI without spinal fracture or dislocation.1-3 Major causes of secondary SCI are vascular in jury to the spinal cord caused by arterial disruption, arterial thrombosis, and hypoperf usion due to shock. Anoxic or hypoxic effects compound the extent of secondary SCI. In summary SCI can vary in nature hence the disability associated with it is also extremely varied ba sed on the type, level, a nd extent of injury. 1.3.2 Spinal Cord Neuro-anatomy The spinal cord is divided into 31 segm ents, eac h with a pair of anterior (motor) and dorsal (sensory) spinal nerve roots. On each side, the an terior and dorsal nerve roots combine to form the spinal nerve as it exits fr om the vertebral column through the neuro-foramina. The spinal cord extends from the base of th e skull and terminates near the lower margin of the L1 vertebral body. Thereafter, the spinal canal co ntains the lumbar, sacral, and coccygeal spinal nerves that comprise the cauda equina.4 Therefore, injuries below L1 are not considered SCIs because they involve the segmental spinal nerves and/or cauda equina. Spinal injuries proximal to L1, above the termination of the spinal cord, often invol ve a combination of spinal cord lesions and segmental root or sp inal nerve injuries.1-5 Spinal tracts : The spinal cord itself is organized into a series of tracts or neuro-pathways that carry motor (descending) and sensory (ascendi ng) information. These tracts are organized

PAGE 19

19 anatomically within the spinal cord. The corticospinal tracts are descending motor pathways located anteriorly within the spinal cord. Axons extend from th e cerebral cortex in the brain as far as the corresponding segment, where they form synapses with motor neurons in the anterior (ventral) horn. They decussate (cross over) in the me dulla prior to entering the spinal cord. The dorsal columns are ascending sens ory tracts that transmit lig ht touch, proprioception, and vibration information to the sensory cortex. They do not decussate until they reach the medulla. The lateral spinothalamic tracts transmit pain and temperature sensation. These tracts usually decussate within 3 segments of their origin as they ascend. The anterior spinothalamic tract transmits light touch. Autonomic function traverse s within the anterior interomedial tract. Sympathetic nervous system fibers exit th e spinal cord between C7 and L1, while parasympathetic system pathways exit between S2 and S4.4 1.3.3 SCI Pathophysiology Traum a to the spinal cord results in primary destruction of neurons at the level of the injury by disruption of the membrane, hemorrhage, and vascular damage. Secondary neural damage to the spinal cord extends beyond the in itial contusion. The spread of the damage is thought to be due to the activation of biochemical events leading to necrosis and excitotoxic damage and can continue for hours, days or week s. Injury to the corticospinal tract or dorsal columns, respectively, results in ipsilateral pa ralysis or loss of sens ation of light touch, proprioception, and vibration.1-3 Unlike injuries of the other tracts, injury to the lateral spinothalamic tract causes contra lateral loss of pain and temp erature sensation. Because the anterior spinothalamic tract also transmits light touch informati on, injury to the dorsal columns may result in complete loss of vibration sensa tion and proprioception but only partial losses of light touch sensation. Anterior co rd injury causes paralysis and incomplete loss of light touch sensation.1-5

PAGE 20

20 Autonomic function is transmitted in the ante rior interomedial tract. The sympathetic nervous system fibers exit from the spinal cord between C7 and L1. The parasympathetic system nerves exit between S2 and S4. Th erefore progressively higher sp inal cord lesions or injury causes increasing degrees of autonomic dysfunction. 1.3.4 Classification of SCI ASIA Impa irment Scale: Clinicians have long used a clini cal scale to grade severity of neurological loss. First devised at Stokes Manville before World War II and popularized by Frankel in the 1970's, the original scoring approach segregated patie nts into five categories, i.e. no function (A), sensory only (B), some sensor y and motor preservation (C), useful motor function (D), and normal (E). The ASIA Impair ment Scale is follows the Frankel scale but differs from the older scale in several important respects. The mechanism of the injury in fluences the type and degree of spinal cord lesion. The SCI injuries are often classified as complete and incomplete. The difference between a complete and incomplete injury depends on the survival of a small fractions of axons in the spinal cord. According to the American Spinal Injury Association (ASIA), a person is a "complete" if they do not have motor and sensory function in the anal and perineal region re presenting the lowest sacral cord (S4-S5). ASIA A is defined as a person with no motor or sensory function preserved in the sacral segments S4-S5. This defin ition is clear and unambiguous. ASIA B is essentially is the preservation of sacral S4-S5 function. It s hould be noted that ASIA A and B classification depend entirely on a single observation, i.e. the preservation of motor and sensory function of S4-5.A patient would be an ASIA C if more than half of the muscles evaluated had a grade of less than 3/5 on a manual muscle test If not, the person was assigned to ASIA D ASIA E is of interest because it imp lies that somebody can have spinal cord injury without having any

PAGE 21

21 neurological deficits at least detectable on a neurological examination of this type. Also, the ASIA motor and sensory scoring may not be sensitiv e to subtle weakness, presence of spasticity, pain, and certain forms of dyesthesi a that could be a result of spinal cord injury. Note that such a person would be categorized as an ASIA E. The ASIA committee has identified five types of incomplete spinal cord injury syndromes. A central cord syndrome is associated with grea ter loss of upper limb function compared to the lower limbs. The Brown-Sequard syndrome results from a hemisection lesion of the spinal cord. Anterior cord syndrome occurs when the injury affects the anterior spinal tracts, including the vestibulospinal tract. Conus medullaris and cauda equina syndromes occur with damage to the conus or spinal roots of the cord. Measures of ambulatory function are another commonly used method to classify people with SCI. Information can be obtained from the following references.611 In summary, in this section we briefly discussed the definiti on of the different types of SCI. This was followed by a summary of the neuroanatomy and physiology of SCI. Finally we summarized the most common classification of SCI; the ASIA scale. 1.4 SCI in the Animal Model To obtain th e necessary experimental evidence to begin clinical trials, compelling evidence for benefit must be demonstrated in reproduc ible animal models of SCI. Although no single experimental SCI animal model exactly mimics the clinical condition, an imal models allow for the rigorous study of the pathophysiology and mechanism of injury and recovery. Appropriate cat and rodent m odels that are being currently investigated include the compression, hemisection, transect ion, isolation, and contusion.12-14 With each model, injury severity and areas of damage to the spinal cord can be va ried so that a spectrum of histopathological, behavior and functional deficits can be reproduced.12-16 Rat models of SCI are

PAGE 22

22 the most commonly studied because of their low cost, size factor, ease in handling and care, and well-established SCI methods.17-20 Recently, mouse models of SCI have been developed. These models give us the ability to enhance or delete specific genes by transgenic mechanisms. Nonhuman primate models of SCI are also important in testing experimental th erapeutic strategies. In addition to various neuroanatomical considera tions, the primate spinal cord more closely resembles that of a human spinal cord and this becomes important when therapeutic and pharmaceutical interventions are focused directly towards the injured spinal cord.11-20 1.4.1 Spinal Cord Isolation Model Spinal cord isolation referred to as the classic silent preparation was attem pted in dogs by Tower in 1937.4 In this model; the lumbar region of the spinal cord is functionally isolated via complete spinal cord transections at two levels and bilateral dorsal rhizotomy between the two transection sites. This m odel eliminates supraspinal, infraspina l, and peripheral afferent input to motoneurons located in the isolated cord segments while leaving the motoneuron skeletal muscle fiber connections intact. Electromy ographic recordings (EMG) and/or reflex testing after spinal isolation have shown the hindlimb muscles to be virtually silent for prolonged periods.21-23 1.4.2 Spinal Cord Transection Model In the trans ection spinal cord injury model, the transmission of descending and ascending information between the caudal cord and the brain is mechanically eliminated. In this model, SCI is created by an incision into the spinal cord is completely transected. Following transection injury, there is an initial flacci d paraplegia stage in which the limbs of the animals are totally paralyzed.12 The animals are only able to move using their forelimbs to reach for food and water. At approximately 3 to 4 weeks following SCI, th e paralyzed hind limbs of the animals change from flaccid to spastic. After spas ticity develops, the limbs are almost always held in extension and no recovery of voluntary activity is observed. There exists a 75% decrease in the total

PAGE 23

23 integrated EMG and a 66% decrease in the total dur ation of muscle activity in the soleus muscle, 5 to 6 months after transection wh en compared to normal controls.24 Thus, in the spinal transection model hind limb muscles experien ce a significant reduction in both electrical activation and loading. The complete transection model has been used extensively to evaluate the effectiveness of interventions w ith regard to both axonal rege neration and functional recovery. The advantage of this model is a relative stabi lization of pathological changes and subsequent neurological outcomes.24-26Therefore, the effectiveness of part icular strategies can be readily assessed. Models in which the spinal cord is full y transected ensure the absolute completeness of the injury, making it somewhat easie r to evaluate the effectiveness of interventions with regard to both axonal regeneration and f unctional recovery. The implicati on in studies using transection models is that with the ensured completeness of the lesion, anterogradely labeled axons observed distal to the lesion have indeed regenerated from above and ar e responsible for the functional recovery of the animal. While this is largely accepted, and hence remains the main advantage of full transection models, there is a mounting body of literature from animal studies that describes considerable native locomotive abilities of the comp letely transected spinal cord (the so-called spinalized animal).24-26 However, the transected spinal cord model also has some disadvantages. First, due to the na tural tension present in spinal co rd, the two ends of a cut cord will separate. Such a gap is rarely present in hum an SCI. In addition, in order to cut the spinal cord, the dura has to be opened, allowing invasion by external cells leading to higher chances of infection.24-26 1.4.3 Spinal Cord Hemisection Model In hemisection models, an attempt is made to cut tracts of the spinal cord selectively. Depending on the severity of the le sion, the resulting neurologic de ficit can be relatively mild, thus making the postoperative animal care fairly easy, particularly w ith regard to bladder

PAGE 24

24 function.27 Hemisection models also may allow for comparison of the regenerative response in a particular tract with its uninjured partner on the contralateral side. The rat rubrospinal system is a useful model in this regard because the tract emerges from the red nucleus in the brain stem, crosses over nearly completely, a nd descends in the dorsolateral as pect of the spinal cord, where its lateral position makes it relatively easy to cut in a unilateral fashion while leaving the contralateral tract uninjured. In th e rat, the rubrospinal system is thought to be important for the control of skilled limb movement, particularly of the forelimbs.18,27,28 Most of the corticospinal tract in rats descends in the ve ntral aspect of the dor sal columns, just dorsally to the central canal. In dorsal hemisection models, the lesion tran sects the rubrospinal a nd corticospinal tracts bilaterally. In general, pa rtial transection models inherently ra ise the possibility that axons of the particular tract in question might have escaped injury. Retrograde tr acers are useful in identifying such spared axons.27,28,18 If a tracer is applied distally to the site of partial injury, its histologic appearance proximally in the cell body of a neuron implies that this neurons axon was not cut during the injury. Conversely, the absence of tracer confirms the injurys completeness. Partial injury models also suffer from difficulti es determining whether observed functional improvement is due to true regeneration of the injured tract or to functional compensation from other systems that are spared.27, 28, 18 Most hemisection injuries are performed on the cervical spinal cord, interrupting the descending respiratory pathways an d causing respiratory muscle pare sis or paralysis. Thus, this model has long been used to understand the mechanisms related to plasticity and recovery of the respiratory pathways after spinal cord injury. Unfortunately, a limitation of partial injury models is the difficulty in determining whether obser ved functional improveme nt is due to true regeneration of the injured tr act or to functional compensatio n from other systems that are

PAGE 25

25 spared. This is one the reasons this model is not commonly used to study adaptations of the locomotor muscles of the hind limb. 1.4.4 Spinal Cord Contusion Model In 1911, Reginald Allen described a spinal co rd injury m odel where he dropped a weight onto the spinal cords of dogs exposed by laminectomy. In 1914, he reported that midline myelotomy reduced progressive tissue damage in the contused spinal cord.29 Unfortunately, Allen died in World War I and his work was disc ontinued for nearly 50 years. In 1968, Albin and colleagues revived the contusion model when they used a primate spinal cord contusion model to assess the efficacy of hypothermic therapy following SCI.29 After that, several investigators started using the canine spinal cord contusion model again. Parker and colleagues assessed the effects of dexamethasone and chlorpromazine on edema in contused dog spinal cords. At the same time, Koozekanani and colleagues examined th e causes of variability in this model, while Collmans and others measured edema, blood flow and histopathological cha nges in the contused dog spinal cord.30 Beginning with a crude weight drop model by Reginald Allen in 1911, many models in various animals have been developed to deliver a blunt contusive force to the spinal cord, which is more representative of what occurs in most human injuries.31 Two important aspects of human injury warrant discussion because they are partic ularly relevant to inju ry models. The first is observed evolution of neuropathology over time, beginning with an early phase of spreading hemorrhagic necrosis and edema, progressing to an intermediate phase of partial repair and tissue reorganization, and reaching a chroni c phase characterized by the es tablishment of central cystic cavities within atrophic pa renchyma and glial scar.30-36 This temporal pattern of injury maturation appears to be reasonably well simulated in the spinal cords of animals after a contusion injury, thereby providi ng a setting for evaluating neuropr otective strategies in the

PAGE 26

26 acute phase of injury.30-36 The second important observation in humans is that even in the setting of complete paraplegia after blunt injury; the spinal cord rarely is completely transected, but rather leaves some residual, normal-appearing co rd parenchyma peripherally at the injury zone. Contusion injury models produce a similar lesio n, in which neuronal tissu e remains intact along a peripheral rim, the quantity of which is correlated with residual locomotor function.30-36 In general, contusion injury models app ear to induce reproducible and consistent neurologic injuries, thereby providing a good setti ng for the functional and histologic evaluation of SCI and new treatment interven tions. However, due to the incomp lete nature of injury and the complexity of the tracts, it is very difficult to verify exact changes in pathophysiology in these models. Common devices used to create contusion injuries : The Georgetown University device Wrathall, is a free falling weight down a guide tube onto a footplate resting on the cord. The NYU or MASCIS device was developed at th e NYU Neurosurgery Laboratory and first described by Gruner in 1992. In this model, a 10g rod is dropped from different heights onto the exposed dorsal surface of the spinal cord produc ing more severe neurol ogic injuries with increasing height. The ESCID device (Ohio Stat e University device displacement driven) is somewhat different rat cord contusion mode l that use a computer feedback-controlled electromechanical impactor rather than a weight drop. The Infinite Horiz on device (University of Kentucky device-force driven) is an instrument that enables th e application of standard-force injuries to the spinal cords of mice and rats. Force levels are user-selectable between 30 and 200 kDynes. A clip compression model of spinal cord injury in rats was introduced by Rivlin and Tator in 1978, in which the spinal cord was compressed for variable durations between the arms of a modified aneurysmal clip. The devices co mmonly used currently are the NYU impactor and

PAGE 27

27 the Infinite Horizon impactor device. Overall, all these devices provide consistent, reliable spinal cord injuries. However, based on the experiment al requirement or type of injury, one device might be more suitable than the other. To summarize, in this section the different types of SCI in animals was briefly described with emphasis given to the contusion SCI whic h is the model of SCI pertaining to this dissertation. In the last section we saw the different types of injury devices pertaining to the contusion injury. In the followi ng sections the NYU impactor devi ce will be used extensively to cause moderate contusion SCI in the rat model. The moderate contusion injury was used in all the animal experiments as it closely resembles th e histopathologic sequela and mechanism of an incomplete SCI in the humans, helping us to relate our animal experiments to our human studies. 1.5 Skeletal Muscle Adaptations in Human Following SCI 1.5.1 Muscle Size Num erous studies have been conducte d to study muscle atrophy after SCI.5 Of the various techniques used to measure muscle size, measur es of whole muscle cr oss-sectional area (CSA) have been identified to be the most accurate and reliable .37 Initially, muscle CSA was calculated either by measuring the limb girth by a tape meas ure or by in-vitro measurements such as fiber CSA. Gregory et al. 2003, quantified both human and rat fiber CSA after 11 weeks-SCI. Both the rat and human vastus lateralis muscle show ed significant atrophy (~50 %) with chronic SCI. 17 Adams et al. 2006 and Stewart et al. 2004 reported significant atr ophy in the vastus lateralis muscle using muscle fiber size measures following chronic SCI.38,39 Recently, muscle crossectiona l area (CSA) has been extens ively measured by means of Magnetic Resonance Imaging (MRI) and ot her non-invasive measuring tools like ultrasonography and computed tomography.14,16,18,40-47 Not only is MRI non-invasive, it is without harmful radiation, and has a unique abi lity to visualize non-muscle tissue like fat,

PAGE 28

28 connective tissue and bone. It has greater tissue sensitivity and contrast resolution with multiplanar and 3D capabilities than ultrasonography and CT. 40-47 Moreover, MRI has the advantage of visualizing the entire length of a muscle compared to a biopsy or ultrasound. Numerous studies have utilized MRI to study CSA afte r SCI in both animals and humans. Castro et al. 1999 used MRI to show that the average maximal CS A of gastrocnemius and soleus decreased by 24% and 12% within six months of SCI, while th e tibialis anterior CSA showed no change.47 The average CSA of the quadriceps femoris, the ha mstring muscle group and the adductor muscle group decreased by 16%, 14% and 16%, respectively. 47 The average CSA of atrophied skeletal muscle in the patients was 45-80% of that of ageand weight-matched able-bodied controls 24 weeks after the injury. The incomplete-SCI model in humans also showed significant skeletal muscle atrophy measured using MRI.37 Individuals with chronic incomplete-SCI showed a ~28%-33% change in their muscle size as co mpared to able bodied controls. Maximum difference was seen in the plantarflexor mu scles (32%) followed by knee extensors (31%), dorsiflexors (28%) and the knee flexors (22%).37 Skeletal muscle atrophy following SCI is a result of injury to motor neurons in the spinal cord and concurrent in activation of affected skeletal muscle along with subsequent changes in muscle length and mechanical loading conditions. Fractional presence of neural inputs to the muscle allows for variable activation of lower limb musculature after an incomplete-SCI, thus resulting in more modest atrophy in this population after injury and also better changes for positive pr ognosis compared complete-SCI group. 37-47 1.5.2 Fiber Type Composition The type of MHC expressed in human skeletal muscle also determines the characteristics of the muscle. Generally, muscle fibers in hum ans do not express more than one distinct MHC type.5 However; the atrophic response in skelet al muscle following spinal cord injury

PAGE 29

29 demonstrates a number of hybrid fibe rs co-expressing different MHC types. In general, MHC type transforms towards a faster type by the firs t year of injury with significant increases in MHC-IIx. 5 Histochemical fiber-typing studies also support the fact that there are dramatic increases in faster (type I I) fibers after SCI. Talmadge et al. ( 2002) measured the effects of SCI on the expression of sarcoplasmic reticulum, calcium-ATPase (SERCA) and MHC isoforms in the vastus lateralis (VL) muscle.48 SCI resulted in significant incr eases in fibers with MHC IIx with ~14% and ~16% increases at six weeks and 24 weeks after SCI. 48 In addition, SCI resulted in high proportions of MHC I and MHC IIa fibe rs with both SERCA isoforms (~29% and ~16% at six weeks and ~54% and ~28% at 24 weeks for MHC I and MHC-IIa fibers respectively).48 The appearance of faster isoforms of MHC after SCI suggests that the muscle will have faster contractile properties, ultimately making it highly fatigable. These changes seen in the muscle are anticipated to contribute towards the f unctional limitations observed in this patient population.48 Studies have used different muscle fiber cl assification schemes such as myofibrillar ATPase activity, and activity (or concentra tion) of specific enzymes including succinate dehydrogenase (SDH), and alpha-glycerol-phosphate dehydrogenase (GPDH), to identify and quantify skeletal muscle adaptations after SCI.5, 17 These measurement techniques visualize the activity of enzymes which are specific to each fiber type. When the enzyme reacts with an energy source a reactive product is formed. Thus th e product from the assay is used to determine if the muscle fibers are fast or slow (mATPa se), oxidative or non-oxida tive (SDH), or generate ATP aerobically or anaerobically (GPDH).5, 17 Gregory et al. (2003), quantified both human and rat VL fiber adaptations 11 weeks following SCI. The VL was sectioned and fibers were analyzed for type (I, IIa, IIb/x), SDH, GPDH, and actomyosin adenosine triphosphatase

PAGE 30

30 (qATPase) activities.17 The IIa to IIB shift was the major phenotypic adaptation that occurred in VL after SCI in both humans and rats. Rat fibers had 1.5to 2-fold greater SDH and GPDH activity compared to humans.17 The most striking differences, how ever, were the absence of slow fibers in the rat and its four-f old greater proportion of IIb/x fibers compared to humans which could be viewed as the rats abil ity to counter the grea ter decline in SDH activ ity with regard to resistance to fatigue. SCI decreased SDH activity more in rats whereas IIa to IIb/x fiber shift occurred to a greater extent in humans.17 Thus fiber type adaptations are species specific and each species has their own mechanism of counter ing an insult to its neuromuscular framework. 1.5.3 Electrically Elicited Contractile Properties Contractile properties in the hum ans have been studied in m uscles, like the quadriceps and soleus muscles after SCI. 42,49-53 Gerrits et al 1999 indicated that muscle s after SCI demonstrated faster rates of contraction and relaxation than normal control muscles and also had extremely large force oscillation amplitudes at the 10-Hz signal frequency (~65 % in SCI versus ~23% in controls).50,51 In addition, force loss and slowing of relaxation following repeated fatiguing contractions were greater in SCI muscles comp ared with controls. The faster contractile properties and greater fa tigability of the SCI muscles are in agreement with a characteristic predominance of fast glycolytic muscle fibers.50,51 Within the SCI population, the chronically paralyzed soleus on average has a 20ms shorter time to peak twitch torque and a 25% shorter twitch half-relaxation time when co mpared to individuals with acu te paralysis. This indicates that the muscle functioning gets faster with the progression of the diseased state. Fast fatigable motor units show progressive slowing during fatigue induced by repetitive activation.52 Consistent with properties of fa ster muscle and motor units, the soleus after SCI demonstrates a near doubling of the half-relaxation time during fatigue.52 This indicates that as the muscle fatigues, the calcium uptake becomes compromised or the cross-bridge cycling rate is impaired.

PAGE 31

31 Normally, as the frequency of an electrical st imulus increases, muscle contraction becomes progressively more fused and the muscle generate s greater torque. A muscle that has a slower contractile speed will fuse at a lower frequency when compared to a faster contractile speed. 52 The torque-frequency curve for a slow muscle will be shifted to the left of the torque-frequency curve of a fast muscle. Thus a torque-frequency curve for a muscle after SCI is shifted to the right of the torque-frequency cu rve for a normal muscle. Another measure that is specific to adaptations after SCI is low-fr equency fatigue, which refers to repetitive activation of a chronically paralyzed musc le at low frequencies. 52 The preferential loss of force at low frequency can be recovered at higher frequencies. Impairme nts in excitation-contraction coupling (E-C coupling) is asso ciated with low-frequency fatigue and likely represents an internal safety mechanism in skelet al muscle to prevent ATP depletion.52 Low-frequency fatigue is characterized by being delayed in onset as well as being long lasting. This type of fatigue is found to be most prominent in fast-inter mediate or fast fatigable motor units.52 1.5.4 Voluntary Contractile Measurements Paralysis of the voluntary m usculature is the most obvious effect of SCI in humans. Damage to the descending motor tracts, anterior horn cells, and/or nerv e roots leads to an impaired capacity to voluntarily contract the skeletal muscles innerv ated at or below the level of the lesion.49-52 In patients with SCI, the maximal voluntar y contractions of the affected muscles are extremely weak compared to the range of absolute forces typically produced by non-injured individuals.49-52This may relate to reduced voluntary activation of the muscle, failure of neuromuscular transmission, problems within the mu scle itself, or some combination of these possibilities. For example, if vol untary drive does not recruit all of the motoneurons that supply a muscle, the voluntary force produced will be redu ced. Failure to activate each motor unit at its maximal firing frequency will also reduce for ce production. Similarly, the force contributed by

PAGE 32

32 each motor unit will be lower if fiber size decrea ses (muscle fiber atrophy) from altered use of muscle. The sensory deficits that accompany these injuries may also exacerbate the ability of subjects to contract their muscles maximally.49-52 Few studies have measured voluntary muscle strength obj ectively afte r human SCI 54-56 or have delineated the factors th at contribute to the weakness The manual muscle test (MMT) has been used to measure strength historically in the field of physical therapy. The face and content validity of MMT in SCI is high, however manual musc le tests are subject to a ceiling effect, lack sensitivity to change and have a relatively poor inte r-rater reliability, especi ally at scores greater than 3.54-56 Studies have compared differe nt methods to assess strength after SCI (the manual muscle test (MMT), the hand-held myometry and isokinetic dynamometry (Cybex, KinCom, Biodex). These studies suggest th at the MMT method does not seem to be sufficiently sensitive to assess muscle strength, at least for grade 3 and higher and to detect small or moderate increases of strength over the course of rehabilitation. Furthe r, it has been concluded that myometry and dynamometry measurements detect increases in strength over time, which are not reflected by changes in MMT scores.55 Thus, dynamometry is curre ntly considered a more sensitive measure of voluntary strength in human SCI population. In summary, following SCI, there is signifi cant atrophy quantified using muscle and fiber crossectional, a slow to fast muscle fiber tr ansformation. Changes in contractile properties are more dramatic in fibers which ha ve a larger proportion of slow fi bers. There exists a reduction is muscle strength and voluntary muscle control. However, depending on the type of injury being incomplete or complete, the neuromuscular architecture and function are not necessarily compromised.

PAGE 33

33 1.6 Skeletal Muscle Adaptations in the Animal Models Following SCI A decrease in neurom uscular activity as a resu lt of spinal cord injury (SCI) results in significant changes in morphological, mechanical and metabolic properties of skeletal muscles below the level of injury. However, the relationship between the injury and the muscle adaptations is confounded by the variability among in juries, and the type of injury. Below is a description of various adaptations that o ccur in skeletal muscle following SCI. 1.6.1 Muscle Size This section covers atrophy m easured using muscle wet weight and fiber size. Reduced muscle activity and loading or inactivity results in a significant reduction of skeletal muscle mass and muscle fiber size following SCI.26,57 Specifically, muscle atr ophy is more pronounced in single joint muscles which are involved in weight bearing a nd postural control.26, 57 For example, the soleus muscle, a postural muscle crossing over the ankle joint, undergoes significant muscle atrophy following SCI.5,26 In contrast, the TA or EDL ar e known to show relatively less atrophy compared to the soleus. The medial gastrocn emius muscle, which crosses both the knee and ankle joints, also undergoes less atrophy than the soleus muscle even though it serves as a synergist to the soleus muscle during plantar flexion.5,26 Degree of atrophy is fiber-type specific, with the slow twitch muscles being more affected than the fast twitch muscles, and extensors atrophy more than flexors. For example, following spinal transection inju ry in adult cats, the morphological adaptations in the medial gastrocn emius (slow muscle) are higher than that seen in the tibialis anterior (fast muscle).5,26,58 Similar to fiber size, absolu te wet weight also decreases with SCI. Hutchinson et al. 2001 reported a 20-25% significant decrease in absolute wet weight in the soleus, while there was a 6% decrease in EDL wet weight when compared to matched controls.20 While it is clear that atrophied muscles pr oduce less contractile force, there appears to

PAGE 34

34 be dissociation between the percent loss of mu scle mass and percent decline in contractile tension indicating a loss in muscle specific force.20 1.6.2 Fiber Type Composition Num erous methods have been used to understa nd the differences between fiber types. In early 1800s fibers were grossl y differentiated to red and white based on their appearance.5 With sophistication of experimental t echniques different classification of fiber type have come to existence. The histochemical assay of myofib rillar ATPase activity is one of the few experimental techniques used to distinguish between fast and slow-contracting muscle fibers.5 Myosin ATPase activity is positively correlated w ith muscle contraction ve locity. Basically, fast contracting fibers hydrolyze ATP fast er than slow-contracting fibers.5 For example, crosssections of a normal soleus stained for myofibr illar ATPase show a composition with a minimal number of fast fibers, where as a transection SCI-soleus is com posed entirely of fast fibers.5 The transection SCI-soleus represents a dramatic slow to fast muscle fiber type transformation. In addition, the average area of slow fibers in the soleus decreased by about 50% following SCI. There were no changes in fiber area for the EDL after SCI.1 The soleus however generated the same absolute force in spite of its smaller musc le fibers, indicating an increase in its specific tension, and a significant conversion of its slow fi bers to the fast type.5,24,59 So, the first adaptation indicated after SCI is the reduction in fiber area and fiber type transformation from slow to fast muscle. This methodology was howev er used starting about 20 years ago and now more sensitive measures have been developed to substantiate this fibe r type conversion after SCI.24,59 Myosin, the molecular motor of the skeletal mu scle, is a protein comprised of two myosin heavy chains (MHC). The heavy chains determine the rate of cross-bridge reactions with actin filaments and hence help determine the speed of muscle contraction.59 To date, four different

PAGE 35

35 myosin heavy chain (MHC) isoforms have been id entified in varying prop ortions in the hindlimb muscles of rats. These have been identified as a slow isoform called MHC-I and three fast isoforms called MHC-IIa, MHC-IIx, and MHC-IIb.59 A number of studies have closely linked the MHC isoform composition of th e individual muscle fibers with their velocities of unloaded shortening, such that there is a gradation in th e contractile speed of fibers containing a given isoform in the order of (fastest to slowest) II b > IIx > IIa > I. Antibodies specific to these proteins identify fiber types based on these MH Cs. Animal soleus muscles stained after transection SCI for MHC compositi on analysis indicate differences in the distributions of fiber types with a greater percentage of hybrid muscle fibers which coexpress different MHCs in SCI animals and a greater shift in MHC composition towards faster isoforms.60,61 The control normal soleus primarily contains fibers reacting excl usively with type I myosin antibody (slow, 86.1 %) and a small percentage of fibers reacting excl usively with type IIa myosin antibody (fast, 13.9%).60 One-week after SCI transec tion (ST), the proportion of pure type I fibers decreased to ~75%.9 The remaining difference in the MHC com position in SCI animals was accounted for by an increase in hybrid fibers, with ~15% of fibe rs reacting to I & IIa myosin antibody and ~10% reacting to type IIa & IIx myosin antibody.60 Interestingly, the reduction in the proporti on of fibers containi ng MHC-I after spinal isolation (SI) is greater than that ob served for spinal transection. Talmadge et al. (1996) with MHC-specific antibodies demonstrat ed that the soleus from cont rol cats contained 99% type I, 1% IIa. Following ST 67% of the fibers were positive for type I, 17% IIa, 3% IIb, and 13% hybrid fibers. After SI, 48% of the fibers were positive for type I, 11% were IIa, 1% was IIb, 25% were hybrid, and 15% contained embryonic MHC.62 Roy et al. (1999) also showed that cat fast muscle (tibialis anterior) shows an ~4% in crease in the fast fiber proportion and MHC-IIx

PAGE 36

36 expression after 6 months of ST, while there is ~4% decrease in MHC-I fibers. Overall compared to control values, the percent composition of MHCs in the TA was unaffected by ST with or without training. Talmadge (1995) demonstrated th at ST results in dramatic shifts in the expression of MHC isoforms of the rat sole us (normally approx. 90% MHC-I, approx. 10% MHC-IIa), such that 1 month after ST approx. 33% of the total MHC was MHC-IIx.48,59,61,62 Rodents show a higher degree of MHC isofor m transformation after ST than cats. The proportion of MHC-I in the rat so leus is reduced from ~90% in controls to ~25% only 3 months following a complete mid-thoracic ST. The MH C-IIx, which is normally not found in the rat soleus, increased to nearly 50% and that of MHC-IIa to ~30% 6 months after ST. Immunohistochemical analyses reve aled that MHC-I was progressi vely decreased after ST, to only approx. 12% 1 year after ST. The reductions in the proportion of MHC-I were countered by increases in MHC-IIa and MHC-IIx with the increase in MHC-IIx preceding the increase in MHC-IIa. Curiously, MHC-IIb was expressed only at very low levels. Thus, a complete transformation from predominan tly MHC-I to MHC-IIb did not occur. Many fibers (up to approx. 80%) contained multiple MHCs (hybrid fibe rs) after ST. The proportion of hybrid fibers was maintained at a high leve l (approx. 50%) 1 y ear after ST. Zhong and colleagues (2005) studied the effects of short-term (4 days) and long-term (60 days) SI on the rat soleus.63 The control and SI-4d groups were 90% pure type I and 0.5 to 5% types I+IIa, I+IIa+IIx and IIa fibers in bot h groups. The SI-60d rats showed seven MHC combinations: pure type I (37%), I+IIa (32%), I+IIa+IIx (10%), I+IIx (16%), IIa (2%), IIa+IIx ( 1%), and IIx ( 2%) fibers. Thus the most dramatic adaptations in the SI-60d soleus muscles were a marked decrease in pure type I fibers, an increase in I+IIa, and appearance of fibers containing only IIx MHC. All of the hybrid fibers (fibers co expressing type I a nd II MHC isoforms) in

PAGE 37

37 control and SI-4d rats contained >50% type I MH C. In the SI-60d group, however, 21% of the hybrid fibers contained <50% t ype I MHC. Similarly in the me dial gastrocnemius (MG) and tibialis anterior (TA) muscles were also studied after short-term (4 days) and long-term (60 days) spinal isolation. Pure type I fibers were rare: 3%, 5%, and 0% in the co ntrol, SI-4d, and SI-60-d rats, respectively.63 Approximately 90% of the fibers in all groups contained only types IIx and/or IIb MHC. Fibers containing type I plus so me type II MHCs were more prevalent in the SI than control rats. There was a significant shift to wards the fastest MHC isoform with inactivity: pure IIb fibers comprised 13%, 38%, and 41% of the population of the control, 4-day, and 60day SI rats, respectively. In addi tion, there was a concomitant decr ease in fibers containing only type IIx+IIb after 4 (trend) and 60 days of SI. T hus, it appears that type IIb MHC was the default MHC isoform in the inactive MG. TA muscles from control and 4-day SI rats contained 5% pure type I fibers and 15% pure type IIa fibers. In contrast there were no pure type I or pure type IIa fibers in the 60-day SI rats. Approximately 70%, 80%, and 95% of the fibers expressed only types IIx and/or IIb MHC in the control, 4-day, and 60-day SI rats, respectively. Compared to control and SI-4d rats, there wa s a significant decrease in type IIb fibers and increases in type IIx and IIx+IIb fibers in the SI-60d rats. Thus, it appears that type IIx MHC was the default MHC isoform in the inactive TA. Thus, the magn itude of the adaptations observed following spinal isolation is more severe than after sp inal transection. This s uggests that the residual amount of electrical activation in the cat soleus after spinal transection plays a role in maintaining the levels of MHC-I expression.63 Hutchinson and colleagues (2001) measured adap tations in muscle size using muscle wet weight and MHC composition in the soleus (85% slow) and ED L (90% fast) muscles following moderate contusion SCI. They re ported a 20-25% decrease in soleus wet weight after 1-week of

PAGE 38

38 contusion, while the EDL showed a non-si gnificant 6% decrease in wet weight.20 Three weeks post contusion both the soleus and EDL wet weight s returned to normal levels. Analysis of the MHC composition showed no change in fiber type composition at 1-week after spinal contusion in either muscle, while after three-weeks ther e was an upregulation of IIx MHC in both the soleus and EDL. 20 Interestingly, the soleus muscle showed a downward trend in IIa fibers, while the EDL demonstrated an increase in IIb fibers. Preliminary results in our lab indicate ~13% decrease in Type I fibers in the soleus and EDL two-weeks following moderate contusion SCI. The soleus also shows a ~ 9% in creases in hybrid fibers of both myosin type I and IIa. The EDL shows a ~20% decrease in type IIx fibers and ~9% increase in IIb fibers following contusion SCI. 24,25,60-62 In the current study, we propose to use im munohistochemistry for MHC staining to study fiber type transformation in four important locomotor muscles with different fiber type composition and functional roles, specifically the soleus, gast rocnemius, EDL and the TA. 1.6.3 Electrically Elicited Contractile Properties The physiological m easurements such as musc le contractile speed, and force potentiation, delayed onset of fatigue, force frequency relationship, doublet potentiation, sarcolemmal membrane properties, and motoneuronal pool su ppression are useful meth odologies in assessing the mechanical adaptations in skeletal muscle after SCI. 20,48,62,64 SCI results in faster twitch properties as ev idenced by shorter time to peak tension and half-relaxation time; however the maximal isomet ric force generated is significantly reduced.1 Maximum shortening velocity is significantly increased in SCI rats whether measured by extrapolation from the force-velocity curve or by slack-test measurements.20,48,62,64 At a minimum 10Hz of electrical-stim, the soleus of th e SCI animal develops greater force and is less fused than the normal soleus, implying faster contraction and relaxation times. 5 Unfused tetani of the EDL stimulated at different frequencies did not show significant difference between the

PAGE 39

39 normal EDL and SCI-EDL.5 Time to peak tension was decreas ed by ~50% in the SCI-soleus. In the cat soleus muscle there was ~38% reduction in isometric tetanic force 10-months post-SCI, while their time to peak tension was ~41% and half-relaxation time ~50% shorter than control cats.2, 3 These changes in twitch and te tanic properties suggest a cha nge in the properties of the sarcoplasmic reticulum (SR). The change in time to peak tension suggests an increase in the calcium transportability of the SR.26,58 In the spinal transection model, reduction in maximal tetanic force is significant. Talmadge et al 2002 found ~44% reduction is maximal tetanic fo rce three months postspinal transection. In addition, the time to peak tension and ha lf-relaxation time were ~ 45% and ~55% shorter respectively. 20,48,62,64 In the contusion model, our model of interest, one week post-SCI showed a ~20% decrease in both peak tw itch and tetanic tension compared to controls and by three weeks they further decreased to ~41-51% respectively.20,65 However, no significant changes existed between the controls and injured rats for the tim e to peak tension and half-relaxation times. Overall, the decline in contractile force seen in most animal models of SCI is related to the decrease in mechanical load and neural activation associated with the injury. In summary, with SCI there is no direct dama ge to the muscle and innervation of muscles is not physically disrupted. Theref ore, the interruption of transf er of electrical activity through the motoneurons can stimulate the skeletal musc le changes that were observed in the above sessions. 1.7 Rehabilitation Training Strategies Following SCI 1.7.1 Locomotor Training 1.7.1.1 Locomotor training in humans Studies about locom otor training in people with SCI were first reported by Barbeau and colleagues (1987, 1993) where they assessed the f easibility of locomotor training on a treadmill

PAGE 40

40 using body-weight support.66,67 Currently, there is a significant increase in the use of locomotor training to retrain people to wa lk following numerous neurological conditions in the clinical setting. As described previously, this therapeu tic intervention was deri ved from the elaborate models of locomotor training in animals mode ls of SCI which showed consistent positive findings.14,68-74 Several studies in people with SCI ha ve suggested that locomotor training may increase the likelihood th at persons with upper motor neuron in juries will learn to walk over ground independently.75-78 Locomotor training guidelines compiled by Behrman and Harkema (2000)79 were derived from basic and applied science findings and include the following principles: a) maximize weight-bearing through the legs and minimize or eliminate weight-bearing through the arms, b) provide sensory input co nsistent with the motor task; spec ifically standing or walking, c) promote postural control and optimize the trunk, upper and lower extremities, and hip kinematics for walking and associated motor tasks, and d) maximize the recovery and use of normal walking patterns and minimize the use of compensatory m ovement strategies. These strategies can be applied both in the clinical setting and in community settings. Locomotor training also focuses on achievi ng independent community ambulation at normal walking speeds without assistive devices, bracing, or use of compensatory movements. Locomotor training consists of training people on a treadmill with their body-weight partially supported. Therapists manually assist in step tr aining at joint angles and timing of stance and swing phases typical of normal gait. This is followed by overground step training which consists of evaluating factors which are limiting this in dividual from walking independently in the community at normal walking speeds without an assistive device, brace, or compensatory movements. Once these are evaluated then the pe rson is trained to ambulate in the community.

PAGE 41

41 Thus locomotor training is the combination of different gait training t echniques to help get a person with incomplete-SCI to ambulate in the community independent of assistive devices. However, the largest clinical trial compari ng the efficacy of locomotor training with overground practice to defined over-ground mobility th erapy in persons with SCI reported that physical therapy strategies of body weight support on a treadmill and defined overground mobility therapy did not produce different outcomes. It was suggested that the finding was partly due to the unexpectedly high percentage of ASIA C subjects who achieved functional walking speeds, irrespective of treatment.80 Interestingly, there is still a lot of controversy surrounding the methodology and implementatio n of this clinical trial.80 1.7.1.2 Locomotor training in the animal model SCI results in the loss of moto r function due to the lack of supraspinal input. The concept that locomotor movements can be initiated even in the absence of supraspinal input was studied as early as the end of the 19th century (Freusberg 1874; Philippson 1905; Sherrington 1899, 1910; Naunyn, Dentan, and Eichorst 1874). Duri ng the 1940s and 1950s, several researchers suggested that spinally injure d animals (i.e., cats and dogs) c ould not only produce stepping as described in earlier work, but these animals c ould use all four of their limbs for walking overground (Freeman1952; Kellogg et al 1946; Shurrager 1955; S hurrager and Dykman 1951; Ten Cate 1939,1962). In 1951, Shurrager and Dykm an first reported that training could restore locomotion after spinal cord transection in cats. 81 Later, Sten Grillners laboratory in the 1980s (Forssberg 1979; Forssberg and Grillner 1973; Forssberg et al 1974, 1976, 1980a Grillner 1973) clearly showed that thoracic SC I cats could walk with their hi nd limbs on the treadmill while the forelimbs stood on a fixed platform. 81 These animals had good coordination between their hind limbs, placed their fore paws properly on th e plantar surface during the stance phase, and

PAGE 42

42 supported the weight of the hindquarters. Not only did the kinematics of the spinal cat resemble those found in the normal cat, bu t so did the muscular activity.81 However, it is only in the past 20 years that th is phenomenon of locomotor training has been vigorously explored, in concert with the growing recognition of the spinal cords considerable capaci ties for plasticity and of other new possibilities for restoring function after spinal cord injury.68-71 Today several studies have shown that recovery of motor function following spinal cord injury can be enhanced or accelerated by repetitive locomotor treadmill training. The underlying principle of locomoto r training relates to rhythmic loading and repetitive motor training that provides sufficient stimulati on of specific neural pathways to facilitate functional reorganization within the spinal cord leading to improved motor output. Furthermore, appropriate sensory input provided during training helps to achieve the optimal motor output of the spin al neuronal circuitry. 68-80 1.7.2 Functional Electrical Stimulation (FES) Functional electrical stimulation (FES) has been used as a therapeutic resistance exercise strategy to assist patients in strengthening as well as executing functional movements after SCI. 82-84 FES has been used in both the complete an d incomplete SCI population to help reverse atrophic changes, reduce muscle fatigabi lity and increase bone density after SCI. 85 FES used in combination with treadmill walk ing, cycling, external bracing holds considerable promise in assisting persons with SCI execute functional movements.82-85 In the SCI population FES and FES in combination with other exercise interventi ons have been the main resistance or strength training protocols used in pe ople with both complete and incomplete-SCI. In the following sections, we will review some the muscle adaptations following FES and FES in combination with other exercise interventions. In the current study, we will propos e to investigate the effect of resistance training on muscle function following incomplete-SCI. However, our resistance

PAGE 43

43 training protocol will be non-FES based and will include regular gym base d resistance exercise training. FES consists of a variety of stimulation para meters. It can be used at contraction times ranging from approximately 1 to 20 seconds, frequencies of 10Hz to 80Hz and voltages from 30V135V. 82-85 Gerrits et al. 2002 compared the effects of two types of FES (high-frequency and low-frequency) on neuromuscu lar activity after a motor complete SCI. Twelve weeks of FES resulted in ~20% increase in quadriceps tetanic force with no differences between the two stimulation frequencies. Neither training interven tion had a significant e ffect on the contractile properties (maximal isometric force, maximal rate of force rise, half-relaxation time, and forcefrequency amplitude) of SCI muscles.86 Crameri et al. 2000 looked at effects of 16-weeks of FES (35Hz, 70V, 60min/day) after acute SCI. Th ey found that FES helped in controlling the phenotype expression of the VL towards faster is oforms and prevented fiber atrophy after acuteSCI. 87 Dudley et al 1999 in the sub-acute SCI population, showed that 8-weeks of FES resulted in substantial increases in the quadriceps cross-sectional area. In a similar study, 24 weeks of FES resulted in a significant strength gain with increased bone density in the quadriceps muscle of persons with chronic SCI when compared to untrained controls.45 Long-term FES (two years) has also proven to yield signifi cant differences in torque, fatigue index, bone mineral density and twitch properties in persons with SCI. when compared to their untrained leg.88 FES has been the predominant means of resist ance training people with SCI. 88,89 It would be interesting to identify the effects of regular exercise based resistance training on people with SCI. FES is generally not a very favorable therap eutic intervention with the incomplete-SCI population as they have the ability to voluntarily activate their musc les to a certain extent. Bajd et al. 2000 conducted a two month FES training study on persons with incomplete-SCI. He

PAGE 44

44 concluded that long term FES re sulted in a significant improveme nt in knee extensor strength and also improved the ability to activate the dorsi and planta r flexors muscle groups in the incomplete-SCI group.90 Modlin et al. 2005 performed a FES clinical trial on 40 persons with either a conus medullaris or cauda equina lesi on. One year of FES resulted in significant increases in quadriceps muscle CSA co mpared to pre-training CSA levels. 91 Overall, FES has shown considerable promise in improving muscle function in all different models of SCI ranging from complete injuries to cauda equina inju ries. Hence, FES on its own can be used as a resistance training therapeutic modality in the SC I population. However, the point of interest is that FES in the incomplete takes longer periods of time to cause significant improvements in the incomplete-SCI population. 90-91 This can be attributed to high er levels of function and voluntary control in this population. FES stimulated cycle ergometer training (FES-CE) has been used to improve whole muscle girth and muscle mass with persons with chronic SCI.92,93 Baldi et al. 1998 examined if FES-CE was able to prevent at rophy after acute SCI. The study concluded that FES-CE prevents lower extremity muscle atroph y in acute SCI after 3 months of training, and also causes significant hypertrophy after 6 months.93 In a similar study by Crameri et al. (2002), 10weeks of FES-CE resulted in si gnificant increases in muscle fiber cross-sectional area, reduction in percentage of IIx fi bers and increase in the citrat e synthase activity, indicating a greater oxidative capacit y of muscle, in persons with chronic SCI.94 Interestingly, numerous othe r studies have reported significant improvements after FESCE on muscle morphometric and histochemical characteristics in the chronic complete SCI population.95-99 These changes include increases in whol e muscle and fiber cross-sectional area, muscle to adipose tissue ratio, fatigue resistan ce, maximal rate of force rise and speed of relaxation, and switch in MHC from fast to slower isoforms, d oubling of enzymatic activity of

PAGE 45

45 citrate synthase, and finally an increase in over-ground walking speed and endurance.95-99 Overall, FES-CE has been able to provide a resi stance exercise program w ithout the potential of over-use injury in the complete-S CI population. The actual benefit of this training intervention in improving the functional capabilities in the co mplete-SCI population remains speculative. Similarly, the functional implica tions of FES-CE on the incomple te SCI population are yet to be studied. 1.7.3 Resistance Training There is alw ays curiosity regarding the effect of exercise on the functional well being of people with SCI. 100-102 In the above section we saw the e ffect of electrically stimulated resistance training either using weights or usi ng CE. However, various impediments exist in the SCI population to complete successful regula r resistance exercise training protocols. Specifically, only the incomplete-SCI populati on with limited voluntary muscle control can perform regular non-electrically induced resist ance training. Although they have certain degree of voluntary control, they are st ill structurally and functionally ill-suited for strong propulsive and weight-bearing exercises. One has to be awar e of inducing overuse bone and muscle injury, nociceptive and neuropathic pain, reflex sympathetic dystrophy, and some cases, cardiovascular complications.103,104 Few studies have looked at the effect s of resistance or strength training protocols on the SCI population. Nilsson et al. 1975 was the first to report significant improvement in the triceps muscles in persons with incomplete-SCI following resistance training. Cooney et al. 1986 used a hydraulic device in a nine-week training program which improved upper extremity power output in the chronic SCI population.105,106 Persons with SCI as we know exhibit deficits in voluntary control a nd sensation that limit not only the performance of daily tasks but also the overall functional and social activity. This leads to extremely sedentary lifestyle with an increased incide nce of secondary complications

PAGE 46

46 including diabetes mellitus, hypertension and lipid profiles. As the daily lif estyle of the average person with SCI is without adequate activity, struct ured exercise activities must be added if the individual is to reduce the like lihood of secondary complications a nd/or to enhance their physical capacity. The acute exercise responses and the ca pacity for exercise conditioning are related to the level and completeness of the SCI. Appropriate exercise testing and training of persons with SCI should be based on the individual's exercise capacity as determined by accurate assessment of the spinal lesion. Other issues that need to be taken into considera tion before resistance training can be incorporated as a therapeutic act ivity. For example, the scientific basis for the exercises needs to be identified, training parameters; like dosage refinement, safety instructions, and inclusion-exclusion criteria need to be postulated. Overal l, clinicians involved in SCI rehabilitation need to consider resistance trai ning as a therapeutic intervention rather than concentrate on compensation as their modus ope randi. Wheelchair strength training has shown considerable promise in improving muscle power and strength in the SC I population. However, these studies are either limited to the upper ex tremity or are for wheelchair athletes only.107,108 To conclude, important strides need to be taken in the research field on studying the effects of resistance training on improving skeletal muscle function after SCI. In current study, we will examine the effect of gym based resistance exercise training on muscle function on people with chronic incomplete-SCI. The current study will be one of the first stud ies which will look at strength training lower extremity locomotor muscles in persons with incomplete-SCI. 1.8 Skeletal Muscle Adaptations Following SCI and Locomotor training 1.8.1 Impact on Humans Current rehabilitation research has described loss of skeletal muscle function as one of the significant problem s impacting the health care and quality of life of persons after SCI.1,2 A significant portion of the SCI related costs can be attributed to degradation of the

PAGE 47

47 musculoskeletal system resulting in decreased skeletal muscle function.1,2 Even though locomotor training is not consid ered a therapeutic intervention designed to induce muscle hypertrophy; previous studies have shown that in the incomple te-SCI population the training stimulus and loading can be of sufficient magn itude to induce muscle plasticity. Giangregorio et al. 2006 reported increases in whole-body lean ma ss, from ~45.kg to ~47kg and increases in muscle CSAs by an average of 4.9% and 8.2% at the thigh and lower leg after 144 sessions of locomotor training in persons with chronic incomplete-SCI.109 In a similar study performed in the acute-SCI population, 48 sessions of locomotor trai ning resulted in increases in muscle CSAs ranging between ~4% to ~58%.110 The study concluded that twic e-weekly locomotor training appeared to partially reverse muscle atr ophy after SCI, but failed to prevent bone loss.109,110. These findings are supported by research examini ng changes at the muscle fiber level. Stewart et al. 2004 reported a 25% increase in the mean muscle fiber area of type I and IIa fibers in the vastus lateralis following 6 months of body weight supported treadmill training in chronic incomplete-SCI subjects.39 Adams et al. 2006 in a single case study (c hronic ASIA B) reported that the vastus lateralis mean fiber area in creased by 27.1% and type I fiber % distribution increased to 24.6%, whereas type IIa and type IIx fibe r % distributions both decreased following 48 sessions of locomotor training.38 1.8.2 Impact on the Animal Model The effects of locom otor training on SCI-induced muscle adaptations ha ve been studied to a limited extent over the past two decades. Roy et al. as early as 198626 identified that spinalized adult cats who exercised on a treadmill for a week showed less atrophy and fiber type adaptations, especially in the postural muscles (slow extensors).26 In a similar study, the same group identified that only 30 min of daily step training em phasizing weight support on a treadmill ameliorated, and in some cases prevented, the contractile and morphological

PAGE 48

48 adaptations in the soleus muscle associated with a complete low thoracic spinal cord transection in adult cats.111 Similarly, a few studies have also been conducted in the rode nt model, identifying muscle adaptations after SCI and lo comotor training. Versteegden et al (1999, 2000) reported that locomotor training resulted in an increase in muscle fiber size, m yonuclear number, satellite cell count and a decrease in the apoptot ic nuclei in the soleus muscle af ter spinal transection in the rat model.112,113 A unique finding in these studies was that satellite cell fusion and restoration of myofiber nuclear number contributed to increased muscle size in the soleus after locomotor training.113 Stevens et al. 2006 reported that locomotor traini ng following contusion SCI resulted in a significant improvement in overall locomo tor function (32% improvement in BBB scores) when compared to no training group. Also, the in jured animals that trained for one week had 38% greater peak soleus tetanic forces, a 9% decrease in muscle fatigue, 23% larger muscle fiber CSA, and decreased expression of fast myosin heavy chain fiber types compared to rats receiving no training.65,114 Overall, locomotor training has shown significant promise in attenuating the adaptations in skeletal muscle seen after SCI. This includes prevention of atrophy following SCI, reduced fatigability, improved mu scle force production and transformation of fiber type towards slower isofor ms. However, further investigati on is required to identify the training effects on specific models of SCI. In conclusion, locomotor training has shown to induce positive alterations in skeletal muscle function in both humans and animals. Ho wever, most of the current data still revolve around the complete SCI model. Further investig ation in the incomplete SCI model in both humans and animals is warranted. In this dissert ation we will to answer some of the questions

PAGE 49

49 regarding skeletal muscle adaptations followi ng locomotor training in both the animal and human model. 1.9 Mechanisms Involved in Training Indu ced Muscle Plasticity and Recovery The prim ary functions of skeletal muscle are production of movement, posture control, and respiration. Interestingly, skeletal muscle is susceptible to injury from direct trauma (e.g., intensive activity, stab wounds, gun shots et c.) or resulting from indirect causes such as neurological disease or genetic complications.115 Direct or indirect injuries may lead to loss of muscle mass and strength leading to a functiona l limitation. The maintenance of a working skeletal muscle is conferred by its remarkable ability to regenerate.115 Indeed, upon muscle injury a finely orchestrated set of cellular and molecular respons es is activated, resulting in the regeneration of a well-innervated, fu lly vascularized muscle apparatus.115 Muscle fibers are the sing le cells that form skeletal muscles. They are individually surrounded by a connective tissue layer (endomysium) and grouped into bundles surrounded by the perimysium, and these bundles are surrounde d by the epimysium to form a skeletal muscle.115 As the muscle fiber or myofiber matures, it is contacted by a single motor neuron and expresses molecules for contractile function, principally different MHC isoforms and metabolic enzymes. Both the origin of the myoblast a nd the motor neuron play an important role in specifying the contractile propertie s of their myofiber. Nevertheless, adult skeletal muscles are composed of a mixture of myofibers with different physiological properties, ranging from a slow/fatigue-resistant type to a fast-/non-fatigue-resistant type. The proportion of each fiber type within a muscle determines its overall contractile property.115 1.9.1 Plasticity of Skeletal Muscle Adult skeletal muscle is a ve ry stable tissue with little turnover of nuclei. Minimal damage inflicted by daily wear and tear elicits only a slow turnover of its multinucleated muscle fibers. It

PAGE 50

50 is estimated that in an adult rat muscle no more than 1% of myonuclei are replaced every week.116 Nonetheless, mammalian skeletal muscle has the ability to complete rapid and extensive regeneration in response to severe injury or damage. The ma jority of this rege neration is carried out by the activation, proliferation and differentia tion of a resident popula tion of myogenic cells called satellite cells. Under normal conditions, sate llite cells are quiescent but become activated in response to injury giving rise to prolifer ating myogenic precursor cells that eventually differentiate and fuse to form multinucleated myotubes. Quiescent satellite cells and their descendant myogenic precursors are the key effectors of muscle regeneration.116-118 The early phase of muscle injury is usually accompanied by the activation of mononucleated cells, principally inflammatory cells and myogenic cells. The factors released by the injured muscle activate inflammatory cells within the muscle. Neutrophils are the first inflammatory cells to invade the injured muscle. After neutrophil infiltration, macrophages infiltrate the injured site to phagocytose cellular debris and initiate muscle regeneration by activating myogenic cells. Thus muscle fiber necrosis and/or increase d number of non-muscle mononucleated cells within the damaged site are the main histopathological characteristics of the initial activity following muscle injury.117-119 Muscle degeneration after injury is followed by the activ ation of a muscle repair process. The myogenic cells provide an ample source of new myonuclei for muscle repair. On crosssection, classic characteristics of muscle regeneration are small newly formed myofibers with centrally located myonuclei. 120,121 Newly formed myofibers are often basophilic and express embryonic/developmental forms of MHC which reflect new fiber formation. 120,121 Fiber splitting or branching is also a characteristic feature of muscle regeneration and is likely due to the incomplete fusion of fibers regenerating within the same basal lamina. Once fusion of myogenic

PAGE 51

51 cells is completed, newly formed myofibers increase in size, and myonuclei move to the periphery of the muscle fiber. Under normal conditions, the regenerated muscle is morphologically and functi onally indistinguishable from undamaged muscle.120,121 1.9.2 Markers of Muscle Recovery and Regeneration 1.9.2.1 Adult muscle satellite cells Muscle satellite cells are a population of undifferentiated, mononuclear myogenic cells found in skeletal muscles including muscle spindles. Even though the temporal appearance of satellite cells follows the appearance of both embryonic a nd fetal myoblasts, satellite cells display specific characteristics in culture allowing their distinction from embryonic and fetal myoblasts. 116,122,123 Satellite cells are situated between the plasma membrane and the basal lamina of the muscle fiber. These cells are furt her identifiable by their relatively minute amount of cytoplasm, sparse organelles and high ratio of heterochromatin to euchromatin, indicative of the inactive state of these cells. Sa tellite cells are presen t in different types of skeletal muscles and are associated with all fiber type s, although the distri bution might be unequal. For instance, the percentage of satellite cells in adult slow soleus muscle is twoto thre efold higher than in the adult fast tibialis anterior or extensor digitorum longus muscle.124 Similarly, high numbers of satellite cells are found associated with slow muscle fibers compared with fast fibers within the same muscle.116-122-124 Increased density of satellite cells have been observed at the motor neuron junctions and adjacent to capillaries, suggesting that some factors associated with these structures may play a role in homing satellite cells to specific locations or in regulating the satellite cell pool by other means. The regulation of satellite cell density at the single fiber level is also suggestive of a role for the muscle fiber in regulating the satellite cell pool (Fig.1-3).116,124 Satellite cells are activated upon muscle injur y, resulting from mechanical stress, direct injury to the muscle or in course of a disease to help in muscle regenera tion. In the initiation of

PAGE 52

52 muscle regeneration, satellite cells first change from their quiescent state to a highly proliferating stage. After proliferating several times, the majori ty of satellite cells fuse to form new myofibers or join and repair the damaged one. During proliferation, a certain percentage of satellite cells are restored underneath the basal lamina for subsequent rounds of regeneration (Fig.1-4).122 The gene or the marker responsible for sp ecification of muscle pr ogenitor cells to the satellite cell lineage is pax-7.125 The Pax7 gene is a member of th e paired box containing gene family of transcription factors implicated in de velopment of the skeletal muscle of the trunk and limbs, as well as elements of the central nervous system. 125,126 The number of Pax7 expressing cells corresponds well with the expected number of satellite cells. Pax7 ex pression is upregulated in proliferating satellite cell-der ived myoblasts and a rapid down regulation of Pax7 transcripts is seen upon myogenic differentiation. Pa x7 is not expressed at detectab le levels in a variety of non-muscle cell lines. In addition, analysis of R NA from selected mouse tissues revealed only a low level expression of Pax7 in adult skeletal muscles. 125 Normally Pax7 mRNA and protein are found in less than 5% of satelli te cells in undamaged skeletal mu scle. However, the number of Pax7-positive cells increases in muscles undergoing regeneration such as in MyoD/, mdx, and mdx: MyoD/ skeletal muscles. 125,126 Centrally located nuclei within newly regenerated muscle fibers are also associated with Pax7 expression, suggesting that recently activated and fusing satellite cells express Pax7. Together, these data demonstr ate the specific expression of Pax7 in quiescent and activated muscle satellite cells.124,125,126 The analysis of Pax7/ skeletal muscles demonstrates the important role this gene has in satellite cell development.125-128 Pax7/ mice appear normal at birth but fail to grow post-natally, leading to a 50% decrease in body weight by 7 days of age compared w ith wild-type littermates. Pax7 mutant animals fail to thrive and usually die within 2 weeks after birth. These animals are

PAGE 53

53 also characterized by a decreased skeletal muscle mass resulting from a fiber size decrease rather than a decrease in fiber number.125-128Pax7/ skeletal muscles have a striking absence of satellite cells. Overall, the data suggest a key role for Pax7 in lineage determination, especially in the specification of myogenic proge nitors to the satellite cell lineage. Pax7 is unequivocally required for satellite cell development (Fig.1-5).125-128 In the next stage, proliferating satellite ce lls are referred to as myogenic precursor cells (mpc). At the molecular level, activation of mpcs are characte rized by the upregulation of two muscle regulatory factors (MRF), Myf5 or MyoD MRFs are part of a super family of basic helix-loop-helix (bHLH) transcrip tion factors. The MRF subfamily consists of MyoD (Myf-3), Myf-5, myogenin (Myf-1), and MRF4 (Myf-6/Herculin).123,129 In general, quiescent satellite cells do not have any detectable levels of MRFs. Upon satellite cell activation, MyoD upregulation appears the earliest within 12 hrs of activation. Activation of MyoD and Myf5 expression following muscle injury has also been observed in various in vivo models for muscle regeneration and in varying muscle types.129,123 A study by Megeney et al indicated that MyoD/ mice show increase in mpc population compared to normals, however they have a decrease in the number of regenerated m yotubes. Furthermore, MyoD/ muscles display an increased occurrence of branched myofibers suggestive of chronic or inefficient muscle regeneration.129-135 In vitro cultures of MyoD/ satellite cells demonstrate a myogenic cell population with abnormal morphology characterized by a stellate, flattened appearance in contrast to the compact rounded appearance displayed by normal myoblasts. Overall, these data suggest an important role for MyoD in the process of satellite cell differen tiation during muscle regeneration.129-135 Myf5-deficient mice display a delayed epaxial (back muscle) embryonic myogenesis and a normal hypaxial (trunk and limb muscles) embryonic myogenesis.129-131 These data combined

PAGE 54

54 with the reciprocal delay in hypaxial m yogenesis in MyoD-deficient mice and the mutually exclusive expression of Myf5 and MyoD in early stages of embryonic muscle precursor cells have led to the hypothesis that Myf5 and MyoD support di stinct myogenic lineages during embryonic muscle development. 129-131 Myf5 promotes satellite cell self-renewal, whereas MyoD promotes satellite cell progression to terminal differentia tion. There is new compelling evidence that the satellite cell population is composed of hierarch al subpopulations of stem cells: the Pax7+/ Myf5+ satellite cells preferentially differe ntiate and become committed myogenic progenitors, while the Pax7+/ Myf5satellite cells extensively c ontribute to the satellite cell compartment.129-131 After the mpc proliferation pha se, expression of Myogenin and MRF4 is upregulated in cells, beginning their terminal differentiation program. This is followed by cell cycle arrest and permanent exit from the cell cycle. The differentiation program is then completed with the activation of muscle-specific proteins, such as MHC, and the fusion of mpc to repair damaged muscle or form their own fibers. Overall, Myf5, MyoD, and Myogenin possibly play distinct roles in myofiber maturation.122 Gross defects in embryonic muscle development of mutant mice for Myogenin and MRF4 have impeded further study of these genes in muscle regeneration. Mice lacking myogenin display a normal number of myoblasts but die at birth because of an absence of myofibers. It has also been suggest ed that Myogenin help s in the conversion of myoblasts to myotubes and helps in the maturation of myotubes.122 1.9.2.2 Other stem cells Mammalian skeletal muscle regeneration invo lves the activation of the quiescent muscle satellite cell population to prolif erate, differentiate, and fuse to provide new myonuclei for muscle repair. Pax7 is required for muscle satell ite cell specification/survival, whereas MRFs are essential in satellite cell pro liferation and differentiation. Mult ipotential stem cells in adult

PAGE 55

55 muscles (adult muscle-derived stem cells) are also capable of myogenic commitment. 122,123 Adult muscle-derived stem cells contribute to both muscle satellite cell pool and myonuclei. Similarly, stem cells capable of myogenic commitment can be isolated from other adult tissues (bone marrow stem cells, neuronal stem cells, and various mesenchymal stem cells)can be used for repair following muscle damage or towards new fiber formation.122,123 1.8.2.3 Growth factors and muscle regeneration Muscle regeneration is a complex process in wh ich growth factors play an important role. Mechanisms that are controlled or altered by growth factors include satellite cell activation, migration to the injury site, proliferation of satellite cell-derived mpcs and differentiation to myotubes and myofibers. Insulin-like growth fact ors (IGFs) I and II are involved in almost all stages of muscle regeneration; they promote satellite cell activation and proliferation, are upregulated in regenerating mu scle and may protect cells fr om apoptosis. Both IGF-I and hepatocyte growth factor/scatte r factor (HGF) are upregulated during muscle regeneration, HGF being crucial during the initial st ages and IGF during the initial to mid stages of regeneration. A significant increase in muscle regeneration was obs erved when human mpcs were cultured with IGF-I prior to their implan tation into damaged muscle.122 IGF-I has a significant effect on proliferate arrest and hypertr ophy of myotubes derived from human fetal mpcs in culture and causes an increase in myosin heavy chain content. HGF is known to increase the chemotaxis of mpcs.122,132-134 C2C12 myoblasts treated with HGF reorganized their actin cytoskeleton and developed a polarized cell shape.136 HGF appears to increase the mpc population by means of mitogenic and chemotactic activities, possibly resulting in an optimal myoblast density. IGFs most certainly promote muscle repair by signaling to both the satellite cells and the myofibers. Whether distinct

PAGE 56

56 roles are played by different IGFs is possible, since IGF-II ap pears to be upregulated later during the process of mu scle regeneration.122,132-134 Fibroblast growth factors (F GF) are also involved with satellite cell activation, proliferation and differentiation. FGF-2 acts as a regulator of satellite cell activity and FGF-6 expression is upregulated during muscle regeneration. Expression of FGF-6 in C2C12 cells induces morphological changes; redu ces cell adhesion and differentiation.137 A greater proportion of the cells which expresses FGF-6 were side population cells, suggesting that FGF-6 may be involved in the maintenance of the rese rve pool of progenitor cells in skeletal muscle. Also the role of FGF in muscle regenera tion may reside in the revascularization process during regeneration through thei r recognized angiogenic properties. 122,132-134 In summary, mammalian skeletal muscle has li ttle turn over of sate llite cells under normal conditions. However, upon injury, skeletal muscle activates satellite cells to both repair and regenerate muscle fibers to prevent atrophy and damage. There are certain key phases in the regeneration process and these processes are supported by different growth factors. Figure 1-1. Etiology of SCI since 2000 modified from www.spinalcord.uab.edu

PAGE 57

57 Severity of Injury 25 years old 50 years old High Tetraplegia (C1-C4) $2,924,513 $1,721,677 Low Tetraplegia (C5-C8) $1,653,607 $1,047,189 Paraplegia $977,142 $666,473 Incomplete Motor Functional at any Level$651,827 $472,392 Figure 1-2. Estimated lifetime costs by age at injury modified from www.spinalcord.uab.edu. Figure 1-3. Satellite cell num ber in skeletal muscle of different ages and type modified from Charge SB, Rudnicki MA. Cellular and molecu lar regulation of mu scle regeneration. Physiol Rev 2004; 84: 209.

PAGE 58

58 Figure 1-4. Satellite cell activity modified from Shi X and Garry DJ. Muscle stem cells in development, regeneration, and disease. Genes Dev. 2006; 20: 1692. A) Schematic outline of satellite cell activity in muscle regeneration, B) gene expression of satellite cells and myogenic precursor cells. Figure 1-5. Schematic outline of a stem cell pass ing through the stages of muscle regeneration modified from Pault B, Rudnicki M, Torrent e Y, Cossu G, Tremblay JP, Partridge T, Gussoni E, Kunkel LM, Huard J Stem and progenitor cells in skeletal muscle development, maintenance, and therapy. Mol Ther 2007; 15: 867.

PAGE 59

59 CHAPTER 2 OUTLINE OF EXPERIMENTS 2.1 Experiment 1 2.1.1 Specific Aim To charac terize and specifically quantify impairments in lower extremity skeletal muscle function after chronic incomplete spinal cord injury (SCI). Peak isometric torque, torque developed w ithin the initial 200 ms of contraction (torque200), average rate of torque development (ART D), and voluntary activation deficits will be calculated as indices of muscle function. Measures of muscle f unction and electrically elicited contractile measurements will be quantitatively performed in the knee extensor (KE) and ankle plantar flexor (PF) muscle groups in ten i ndividuals with chronic incomplete SCI. 2.1.2 Hypothesis Reduced peak and instantaneous torque produc tion, as well as greater voluntary activation deficits of th e lower extremity muscles will be found to be characteristic of individuals with chronic incomplete SCI compared to healthy controls. In addition, significant bilateral asymmetries will exist between limbs, with one limb being more affected than the other. 2.2 Experiment 2 2.2.1 Specific Aim To determ ine the impact of nine weeks of lo comotor training on lower extremity skeletal muscle function in persons with chronic incomplete SCI. Five individuals with chroni c incomplete SCI will undergo ni ne weeks (five sessions per week) of locomotor training (LMT). Indices of muscle function will be determined for the KE and PF muscle groups before and after LMT.

PAGE 60

60 2.2.2 Hypothesis Nine weeks of locomotor training will result in positive alterations in the lower extremity muscles that include improved vol untary activation as well as an improved ability to generate both peak and explosive torque about the knee and ankle joints in persons with chronic incomplete SCI. 2.3 Experiment 3 2.3.1 Specific Aim a) To determ ine the impact of a 12-week resistance and plyometric training on lower extremity skeletal muscle function in pe rsons with chronic incomplete SCI. b) To determine the effect of 12-weeks of re sistance and plyometric training on gait speed in persons with chronic incomplete SCI. Three ambulatory individuals with chroni c motor incomplete SCI (18.7.2 months post injury) will complete 12-weeks of lower extremity resistance training combined with plyometric training (RPT). Indices of muscle function will be determined for the KE and PF muscle groups before and after RPT. Maximal, as well as self-sel ected gait speeds will also be determined preand post-RPT. 2.3.2 Hypotheses a) Twelve weeks of RPT will result in improved ability to generate peak and instantaneous torque as well as improved voluntary activation and re duced time to peak torque in the lower extremities of persons with chronic in complete SCI. In addition, the magnitude of improvements in these outcomes will be most pronounced in the PF versus the KE muscle group. b) Twelve weeks of RPT will result in si gnificant improvements in both self-selected and maximal gait speed in persons w ith chronic incomplete SCI.

PAGE 61

61 2.4 Experiment 4 2.4.1 Specific Aim a) To determ ine the effect of moderate T8 contusion spinal cord injury (rat model of incomplete SCI) on muscle fiber cross-sectional area (CSA) and fiber type composition in four lower extremity muscles (soleus, gastrocnemius, tibialis anterior and extensor digitorum longus) with different fiber type com positions and functional roles. b) To compare the effect of 1-week of locomotor training on fiber CSA and fiber type composition in lower extremity muscles with di fferent fiber type composition and functional roles in rats following moderate T8 contusion spinal cord injury. c) To determine the effect of one week of locomotor training on fibe r crossectional area and fiber type composition in four lower extremity muscles (soleus, gastrocnemius, tibialis anterior, and extensor digitorum longus) in healthy controls. Rats (n=6 per group) will be assigned to four groups; a SCI-treadmill training group, a SCI-no training group, control-treadmill traini ng group, control-no training group. Moderate spinal cord contusion injuries will be produced using a standard NYU (New York University) impactor. Animals assigned to the training groups w ill be trained continuously for 1week (5 days/week, 2 trials/day, 20minutes /trial), starting on post-operative day eight for the SCI training group. Fiber CSA will be assessed at two weeks post-injury for the slow-twitch, plantarflexor muscle, soleus, fast-twitch plantarflexor, gast rocnemius and fast-twitch dorsiflexor muscles (tibialis anterior [TA] and extensor digitorum longus [EDL]). 2.4.2 Hypotheses a) Two weeks following m oderate T8 spinal contusion injury, the injured rats will experience the maximum decrease in fiber CSA in the slow-twitch pl antarflexor (extensor) soleus when compared to the non-injured control rats. The next largest decline in muscle fiber

PAGE 62

62 CSA will be seen in the gastrocnemius, followe d by the fast-twitch dorsiflexors, TA and EDL. Similarly, following moderate T8 spinal contusio n injury, all muscles in the injured rats will show a shift in fiber type towards faster myos in isoforms. Specifically, the soleus from the injured rats will show a fiber type shift from a slower isoform to a faster isoform (MHC-I to MHC-IIa) compared to the soleus of controls. Similarly, the gastrocnemius, TA and EDL of the injured rats will show a fiber type shift towa rds faster isoforms (MHC-IIa to MHC-IIx and MHC-IIxMHC-IIb) when co mpared to controls. b) One week of treadmill training will attenuate the decrease in fiber CSA of the injured rats in all the muscles. Specifically, the soleus will experience maximum gains in fiber CSA, followed by the gastrocnemius, and then the dor siflexors. In addition, treadmill training will attenuate the fiber type shift observed following a moderate T8 contusion spinal cord injury. Specifically, the soleus will show fiber type tr ansformation towards slower isoforms (MHCIIa to MHC-I), while the gastrocnemius, TA and EDL will show a transformation from MHCIIb towards IIx or IIa. c) Healthy control rats trained for one week will show increases in fiber CSA compared to untrained control rats. However, we anticipate that there will be no difference in the CSA values between the trained and the untrained group. 2.5 Experiment 5 2.5.1 Specific Aim a) To dete rmine the impact of moderate T8 contusion SCI on satellite cell activity on the slow-twitch (soleus) and fast -twitch (TA) rat muscles. b) To determine the influence of one week of locomotor training on satellite cell activity on the slow (soleus) and fa st twitch (TA) muscles on spinal cord-injured rats.

PAGE 63

63 c) To determine the impact of one week of locomotor training on satellite cell activity on the slow (soleus) and fast twitc h (TA) muscles on control rats. Rats (n=6 per group) will be assigned to either a SCI-treadmill training group, a SCI-no training group, control-treadmill training group or control-no training group. Expression of markers of muscle regeneration will be assess ed for all four traini ng groups. Specifically, immunofluorescence techniques will be used on th e soleus and TA to quantify for Pax-7 and EM-MHC expression and Western blot analysis will be used to quantify for MyoD, Myf5, and Myogenin expression. 2.5.2 Hypotheses a) Two weeks following m oderate T8 spinal c ontusion injury, both the slow twitch (soleus) and the fast twitch (TA) muscles will show incr ease in the regulation of muscle regeneration markers compared to controls. Specifically, the le vels of the markers will be higher in the slow twitch muscles when compared to the fast twitch muscles. b) One week of locomotor training, will re sult in increased regulation of muscle regeneration markers in both the muscles types, compared to untrained SCI rats. Specifically, the slow twitch (soleus) will show si gnificant elevations in regenerati on markers after the training in comparison to the fast twitch (TA) following mo derate T8 contusion sp inal cord injury. c) SCI rats trained for one week will show increased regulation of regeneration markers compared to trained control rats.

PAGE 64

64 CHAPTER 3 METHODOLOGY 3.1 Studies in People with Incomplete-SCI 3.1.1 Subjects Description Subjects who participate in the first three experim ents are persons with chronic upper motor neuron lesions and motor incomplete-SCI. Criteria for inclusion in clude: 1) age 18-70; 2) first time SCI (C5-T10); 3) medically stable a nd asymptomatic for bladder infection, decubitis, cardiopulmonary disease or other significant me dical complications prohibiting testing and/or training; 4) if using antispasticity medication, agreement to maintain current levels throughout study; Exclusion criteria will be: 1) participation in a rehabilitation or research protocol that could influence outcomes of this study; 2) histor y of congenital SCI or other disorders that may confound treatment, study, and/or evaluation procedur es; Prior to participation, written informed consent will be obtained from all subjects, as a pproved by the Institutional Review Board at the University of Florida. 3.1.2 Locomotor Training The locom otor training intervention consists of 45 training sessions (5x/ week) spread over nine weeks, with each session consisting of 30 mi nutes of step training on the treadmill with body weight support (BWS) immediately followed by 20 minutes of level overground walking and community ambulation training. Includi ng pre-training stretc hing, donning/doffing the harness, and additional time spent on the treadmill for stand training and standing rest breaks, the total session duration will be approximately 75 to 90 minutes per day. Each subject is expected to complete all of the training sessions. With the aid of the body weight support, treadmill and manual trainers, the treadmill training environment will facilitate delivery of locomotor specific practice. Trunk, lower limb, and upper limb kinematics w ill be consistently assisted and/ or

PAGE 65

65 monitored by trainers to assure appropriateness in relation to normal walking. Speed of treadmill stepping will be kept in a range consistent with normal walki ng (2.2-2.8 miles/hr). Progression of training will be achieved by decreasing BWS, altering speed, increasing trunk control, decreasing manual assistance for limb control an d increasing the time spent walking on the treadmill per bout. A more detailed description of the training principles, parameters and progression has been provided by Behrman & Harkema et al. 2000. Overground training will consist of an immediate assessment of the pa rticipants ability to stand and/or walk independently overground and an evaluation of th e deficits limiting achievement of this goal. These deficits became the focus for goal setting in the next days training session. Additionally, overground training addressed translation of the skills from the treadmill to the home and community identifying practical ways for the partic ipants to incorporate new skills into everyday activities (Figure 3-1). 3.1.3 Resistance and Plyometric Training 3.1.3.1 Resistance train ing Lower extremity progressive resistance training will be 12 weeks in duration and subjects will complete 2 to 3 sessions/week for a total of 30 sessions. Resistance exercises will include unilateral leg press, knee extens ion/flexion, hip extension/flex ion and ankle plantar flexion exercises performed on adjustable load weight machines. During the initial training session a predicted one-repetition maximum (1-RM) will be calculated for each subject and for each exercise. 1-RM will be determined using a pred iction table based on a single set to volitional failure with load that allowed between 6 and 12 repetitions. During subs equent training sessions, subjects will perform 2-3 sets of 6-12 repetitions at a relative intensity of ~70-85% of predicted 1-RM. Maximal strength will be evaluated weekly to assess for training-related improvements and exercise loads will be adjusted accordingly. Specifically, if the subject achieved the target

PAGE 66

66 number of repetitions for all prescribed sets of a given exercise, a new predicted 1-RM will be prescribed and resistance will be increased for subsequent training sessions. 3.1.3.2 Plyometric training Unilateral plyometric jump-training exercises will be performed in both limbs in a supine position on a ballistic jump-t raining device (ShuttlePro MVP Contemporary Design Group, Figure3-2). Session intensity for this exercise will be modified by changing either the resistance or the number of ground contacts and progresse d over the training period, accordingly. Briefly, after familiarization with the training device, subjects will complete a total of 20 unilateral ground contacts (e.g. jumps) with each limb at a resistance of ~25% of body mass. Thereafter, upon successful completion of at least 20 ground c ontacts per limb (e.g. complete clearance from the foot plate), resistance will be increased in increments of 10 lbs. When a new resistance is set, repetition goal will be set at 10 ground contacts per limb for the initial session. Subsequent sessions allowed for up to 20 contacts per lim b. Thus, a minimum of tw o sessions at a given resistance will be required before load is increased. Resistance will be held constant between limbs throughout the training program. 3.1.4 Muscle Function Assessment 3.1.4.1 Experimental set-up Voluntary and electrically elicited contractile measurem ents ar e performed in the self-reported more-involved and less-involved limbs for the kn ee extensor and plantarflexor muscle groups, using a Biodex System 3 Dynamometer. Knee exte nsor testing will be performed with subjects seated in an upright positi on with hips flexed to ~85 and knees flexed to ~90. The axis of rotation of the dynamometer will be aligned with the axis of the knee joint and the lever arm secured against the anterior aspe ct of the leg, proximal to the lateral malleolus. Testing of the

PAGE 67

67 plantar flexor muscle group will be perf ormed with the hips flexed at 90100, the knee flexed at ~10 and the ankle at ~0 plantar flexion. The anatomical axis of the ankle will be aligned with the axis of the dynamometer, while the foot was secu red to the footplate with straps placed at the forefoot and ankle. Proximal stabilization was ach ieved with straps across the chest, hips and thigh (Figure3-3). 3.1.4.2 Voluntary contractile measurements Prior to testing, subjects perf orm three warm-up contractions to get familiarized with the testing procedures. This was followed by three maximal voluntary isometric contractions (~5 second each with 1 minute rest intervals) while being given verbal encouragement. Peak torque will be defined as the highest value obtained duri ng the 3 maximal isometric contractions. In the event that the peak torque values differed by more than 10%, additional contractions will be performed. In addition to peak torque we also will dete rmine the average rate of torque development (ARTD) and the torque200, as indices of explosive muscle strength. The ARTD will be defined as the average increase in torque generated in unit time, and will be calculated in the time interval corresponding to 20% to 80% of peak am plitude, starting from muscle perturbation. This time interval was selected to reduce the effect of errors in calculating peak amplitude. Hence ARTD was calculated through numerical differentiation as: N i it f N ARTD11 Where, N is the total number of time slots for numerical differentiation, f iis the change in torque in the time slot i and tis the unit time duration for a slot. Torque200 will be defined as the absolute torque reached at 200ms duri ng a maximal voluntary contraction (Nm).

PAGE 68

683.1.4.3 Electrically elicited contractile measurements Peak twitch torque, time to peak twitch and twitch half-relaxation time will be determined by delivering a supra-maximal electrical stimulus ( 600s pulse duration) to the muscles at rest. Supramaximal intensity will be determined by incr easing the current voltage until twitch torque production plateaued. Time to peak twitch and twitch half-relaxation time will be calculated from the peak twitch contractions. Voluntary activation deficits. are performed using self-adhesive electrodes covering the width of the muscles with sizes ranging from 3. 8 6.35 cm to 7.6.7 cm. For the KE, electrodes will be placed across the width of the distal portion of the thigh (quadriceps muscles), just above the knee joint and across the prox imal portion of the thigh, near the origin of the muscle group. For the PF, electrodes will be placed across the width of the proximal portion of the calf (triceps surae muscles) just below the knee joint line and across the distal portion of the soleus superior to the Achillies tendon (Figure3-3). Voluntary activation deficits will be dete rmined using the twitch interpolation method. A Grass S8800 stimulator with a Grass Model SIU8T stimulus isolation unit (Grass Instruments, West Warwick, RI) will be used to briefly de liver a single biphasic, and supra-maximal pulse was delivered at rest and during maximal voluntary isometric contra ction. The stimulator and the dynamometer will be interfaced with a personal computer through a commercially available hardware system (MP150 system). The data will be sampled at 400H z and analyzed with commercially available software (AcqKnowledge 3.7.1). The voluntary activation deficit will be calculated based on the ratio between the to rques produced by the superimposition of a supramaximal twitch on a peak isometric contra ction (a) and the torque produced by the same stimulus in the potentiated, resting muscle (b). Voluntary activation deficit (%) = (a/b)*100

PAGE 69

693.1.5 Measures of Ambulatory Function Lower extremity motor scores (LEMS). The voluntary muscle strength of 5 key muscles (hip flexors, knee extensors, ankle dorsiflexors, toe extensors, ankle plantar flexors) of both lower extremities is tested in accordance with the standard neurologic assessment developed by ASIA. Each muscle will be given a value between 0 and 5 according to the strength of voluntary muscle contraction. Maximum and mini mum LEMS are 50 and 0, respectively. Walking index for spinal cord injury (WISCI II). Physical limitation for walking secondary to impairment is defined at the pers on level and indicates the ability of a person to walk after spinal cord injury. The development of this assessment index required a rank ordering along a dimension of impairment, from the level of most severe impairment (0) to least severe impairment (20) based on the use of devices, braces and physical assistance of one or more persons. The order of the levels suggests each successive level is a less impaired level than the former. The ranking of severity is based on the se verity of the impairme nt and not on functional independence in the environment. Level description. 0. Unable to stand and/ or participate in assisted walking. 1. Ambulates in parallel bars, with braces and physical assistance of two persons, less than 10meters 2. Ambulates in parallel bars, with braces and physical assistance of two persons, 10 meters. 3. Ambulates in parallel bars, with braces and physical assistance of one person, 10 meters. 4. Ambulates in parallel bars, no braces and physical assistance of one person, 10 meters. 5. Ambulates in parallel bars, with braces and no physical assistance, 10 meters. 6. Ambulates with walker, with braces and phys ical assistance of one person, 10 meters. 7. Ambulates with two crutches, with braces and physical assistance of one person, 10

PAGE 70

70 meters. 8. Ambulates with walker, no braces and physic al assistance of one person, 10 meters. 9. Ambulates with walker, with braces and no physical assistance, 10 meters. 10. Ambulates with one cane/crutch, with braces and physical assistance of one person, 10 meters. 11. Ambulates with two crutches, no braces and physical assistance of one person, 10 meters. 12. Ambulates with two crutches, with braces and no physical assistance, 10 meters. 13. Ambulates with walker, no braces a nd no physical assistance, 10 meters. 14. Ambulates with one cane/crutch, no braces and physical assistance of one person, 10 meters. 15. Ambulates with one cane/crutch, with braces and no physical assistance, 10 meters. 16. Ambulates with two crutches, no braces and no physical assistance, 10 meters. 17. Ambulates with no devices, no braces and physi cal assistance of one person, 10 meters. 18. Ambulates with no devices, with braces and no physical assistance, 10 meters. 19. Ambulates with one cane/crutch, no braces and no physical assistance, 10 meters. 20. Ambulates with no devices, no braces a nd no physical assistance, 10 meters. 3.2 Experiments in Contusion Spinal Cord Injured Animals 3.2.1 Animals The animal model will consist of young adult, female Sprague Dawley rats (16-20 weeks, weighing 250-290gms). The animals will be housed in an AALAC accredited animal facility in a temperature (22C), humidity (50%) and light controlled room (12:12 hours light: dark cycle), and will be provided rodent chow and water ad libitum. The rats will be acclimatized for a week prior to the start of experiments. All pr ocedures will be performed in accordance with the

PAGE 71

71 US Government Principle for the Utilization and Care of Vertebrate Animals and will be approved by the Institutional Animal Care & Use Committee at the University of Florida. 3.2.2 Contusion Injury Spinal cord contusion injuries will be produced using a NYU (New York University) impactor device (Figure3-4). A 10g weight will be dropped from a 2.5-cm height onto the T8 segment of the spinal cord exposed by laminect omy under sterile conditions. Procedures will be performed under ketamine (100mg/kg)-xylazine (6 .7mg/kg) anesthesia. Subcutaneous lactated Ringers solution (5 ml) and antibiotic spray wi ll be administered after completion of the surgery. Animals will receive two doses of Ampici llin per day for 5 days, starting at the day of surgery. Animals will also be given Buprenorphi ne (0.05mg/Kg IM) and Ketoprofen (5.0 mg/Kg SC) for pain and inflammation over the first 36h rs after SCI. The animals will be kept under vigilant postoperative care, including daily ex amination for signs of distress, weight loss, dehydration, and bladder dysfunction. Manual expre ssion of bladders will be performed 2 to 3 times daily, as required, and animals will be m onitored for the possibility of urinary tract infection. Animals will be housed in pairs with the exception of the first few hours following surgery. At post-operative day 7, open field loco motion will be assessed using the Basso-BeattieBresnahan (BBB) locomotor scale and animals that do not fall within a preset range (1-4) will be excluded from the locomotor training study. 3.2.3 Locomotor Training Animals that will receive locomotor treadmill training will be trained for five consecutive days (1 week of training), 2 trials/day, 20 minutes/trial, starting on post-operative day eight. Training will consist of a quadrapedal treadmill ste pping (Figure3-5). On the first day of training, animals will be given five minutes to explore the treadmill and then encouraged to walk on the moving treadmill at a speed of 11 meters per minut e, for a series of four, five-minute bouts. A

PAGE 72

72 minimum of five minutes rest will be provide d between bouts. Body weight support will be provided manually by the trainer. Th e level of body weight support will be adjusted to make sure that the rats can bear their weight and there will be no collap se of their hind limbs. Typically, the rats will start stepping when they have experi enced some small load on their hind limbs. In addition, during the first week of training, wh en all rats have profound hind limb paralysis, assistance will be provided to place rat hind lim bs appropriately for plantar stepping during training. On the second day of training, animal s will complete two 10 mi nute bouts, twice a day. Starting on day three, animals will be traine d continuously for 20 minutes with a minimum interval between trials. Bodywei ght support through the trunk and th e base of the tail will be provided as necessary and gradually remove d as locomotor capability improved. 3.2.4 In-Vitro Assay of Muscle Composition and Regeneration 3.2.4.1 Immunohistochemical analysis The muscles required for analysis will removed from one of the hind limbs of the animal. The muscles were subsequently rapidly frozen in isopentane pre-cooled in liquid nitrogen (storage at -80C) for the following immunohistological measurements. Fiber CSA measures: Cryostat sections (10 m) in a transverse plane will be prepared from the central portion of each muscle taken from the both legs and mounted serially on gelatincoated glass slides. Immunocytochemical reactions will be performed on each cryostat section with anti-laminin to outline the muscle fibers for cross-sectional area (CSA) quantification. The fiber CSAs will be analyzed using the SCION image program. The pixels setting used for conversion of pixels to micr ometer is 1.50 pixels1 m2 for a 10 X objective. The average maximal CSA for the soleus, gastrocnem ius, TA and EDL will be quantified. MHC Measures: Immunocytochemical reactions for qua ntifying fiber type transformation will be performed on serial cryostat sections with anti-laminin and anti-MHC antibody at various

PAGE 73

73 dilutions. Rabbit anti-laminin (Neomarker, Labvisi on, Fremont, CA) will be used to outline the muscle fibers for cross-sectional area quantific ation. Four anti-MHC antibodies (BA-D5, SC-71, BF-F3, and BF-35) will be selected on the basi s of their reactivity to ward adult MHC (figure36). Sections will be incubated with rabbit anti-laminin and one of the anti-MHC antibodies (40C over night), followed by incubation with rhoda mine-conjugated anti-rabbit IgG and Fitcconjugated anti-mouse IgG (Nordic Immunological Laboratories). Stained sections will be mounted in mounting medium for fluorescence (Vector Laboratories, Burlingame, CA) and kept at 40C to diminish fading. Stained cross sections will be photographed (10X magnification) by using a Leica fluorescence microscope with a di gital camera. A region of the stained serial sections from each muscle will be randomly selected for MHC composition analysis. The proportions of each fiber type wi ll be determined from a sample of 150-250 fiber across the entire section of each muscle. Immunohistochemical measures for Pax7 and embryonic myosin will be performed using similar methodologies. 3.2.4.2 Western blot analysis Quantification and expression of MyoD, My f5 and Myogenin will be measured using Western blot analysis. Muscles will be homogenized in a lysis buffer with Fast-Prep homogenizer machine at 13,000 RPM at 40C for five minutes. The supernatant will be preserved for protein assay. Protein will be denatured by heating samples to 95-100 0C for 5 minutes. Protein will be measured using BCA protein assay kit from Pi erce. Electrophoresis will be performed by mixing 40-50 g protein with 5X loading buffer and loading it to 4-15% SDS page gel from Bio-Rad. Protein will then be transferred from gel to nitrocellulose membrane. Blocking will be conducting using 5% non fat dry milk in TBS/T (Tris Buffer Saline, Tween-

PAGE 74

74 20). Blot with be incubated with primary antibody overnight at 40C according to manufacturers instruction. Blot will then be incubated with HRP-conjugate secondary antibody for 40 minutes to one hour at room temperature. Finally pr otein will be detected using Western Blotting Luminal Reagent from Santa Cruz. Figure 3-1. Set-up for locomotor training. Figure 3-2. Plyometri c training set-up.

PAGE 75

75 Figure 3-3. Experimental set-up on a Biodex System 3 Dynamometer. Figure 3-4. Contusion injury set-up modified from Meyer et al.2003. Figure 3-5. anti-MHC antibodies.

PAGE 76

76 Figure 3-6. Locomotor training in the rat model.

PAGE 77

77 CHAPTER 4 LOWER EXTREMITY SKELETAL MUSC L E FUNCTION IN PERSONS WITH INCOMPLETE SPINAL CORD INJURY 4.1 Introduction Approximately 200,000 persons with spinal cord injury (SCI) live in the United States alone; with roughly 11,000 new injuries occurring each year.1 In addition, the relative number of new injuries is rising and expected to have increased by approximately 20% by 2010, compared to the 1994 prevalence. The reported costs associated with the care and treatment of persons after SCI is estimated to range between $15,000$125,000 annually, with an approximate lifetime total of $435,000-$1,590,000.1 Interestingly, a significant propor tion of these costs can be related to the loss of skeletal muscle mass and asso ciated secondary health -related complications 52, (i.e. non-insulin dependent diabetes mellitus, cardio-vascular disease, osteoporosis). A decrease in skeletal muscle function has been described as one of the most signi ficant problems impacting the health care and quality of life of persons after SCI.52, 39 Consequently, the potential to decrease costs and improve quality of life by main taining or partially rest oring skeletal muscle size and function seems high. Due in part to advancements in the quality of emergency care, the relative number of injuries classified as incomplete has risen dramatically over the past 20 years.2 In fact, the majority of new injuries occurring annua lly are now classified as incomplete.1 Despite the rise in the proportion of persons with incomplete-SCI ; the preponderance of scientific literature describing the effects of SCI on skeletal musc le involves persons with complete injuries.49,138-140 To date very little data exits describing muscle function in persons with chronic incomplete-SCI. Interestingly, the innate plasticity associated with incompleteSCI furnishes these persons with the potential to progress functionally to a greater extent than the complete SCI population.141,142 In addition, novel intervention therapies have shown promise in promoting spinal plasticity and

PAGE 78

78 motor function after spinal cord injury. However, improvements in functional capacity in persons with I-SCI with rehabilitation vary greatly and the incidence of disability still remains high.143,144 In order to provide a foundation for the develo pment of rehabilitation strategies targeting neuromuscular deficits in persons with incomp lete-SCI, a need exists to characterize and objectively quantify existing impairments. Therefor e, the purpose of this study was to quantify lower extremity muscle function in persons with chronic incomplete-SCI compared to age-, gender-, heightand body weight matched healthy controls. Specifically, we measured isometric peak torque and performed measures of explosiv e or instantaneous muscle strength in the knee extensor and ankle plantar flexor muscle groups and quantified voluntary activations deficits using a combination of voluntary contractile measurements and superimposed electrical stimulation. 4.2 Methods 4.2.1 Subjects Ten persons with chronic, upper motor neuron lesions and motor incomplete-SCI participated. Characteristics of the persons with incomplete-SCI are provided in Table 4-1. Average age, height and body mass standard devi ation (SD) at the time of the study enrollment were 45.4 14.8yrs, 155.9 9.4cm, and 79.9 12 .2kg. Eight of the subjects were classified ASIA D and two as ASIA C. Four subjects were able to ambulate over ground, while six used a wheelchair as their primary mode of locomotion. The incomplete-SCI subjects were matched on the basis of age, gender, hei ght and weight with ten recreationally active controls (45.1 14.9yrs, 159.1 9.0cm, and 78.0 11.7kg). Prior to participating in the study, written informed consent was obtained from all subjects, as approved by the Institutional Review Board at the University of Florida, Gainesville.

PAGE 79

794.2.2 Experimental Set-Up Voluntary and electrically elic ited contractile measurements were performed in the selfreported more-involved and less-i nvolved limbs for the knee extens or and plantar flexor muscle groups, using a Biodex System 3 Dynamometer. Knee extensor testing was performed with subjects seated in an upright pos ition with hips flexed to ~85 and knees flexed to ~90. The axis of rotation of the dynamometer was aligned with the axis of the knee jo int and the lever arm secured against the anterior aspect of the leg, proximal to the lateral malleolus. Testing of the plantar flexor muscle group was performed with the hips flexed at 90100, the knee flexed at ~10 and the ankle at ~0 plantar flexion, as previously described.13, 14 The anatomical axis of the ankle was aligned with the axis of the dynamomete r, while the foot was secured to the footplate with straps placed at the forefoot and ankle. Proximal stabilization was achieved with straps across the chest, hips and thigh. Electrical stimulation was performed using a Grass S8800 1stimulator with a Grass Model SIU8T stimulus isolation unit*. Electrically induced contractions were delivered through two 3.0 by 5.0 self-adhesive neuromuscular stimulation electrodes placed over the proximal and distal portions of the muscle group being tested. The stimulator and the dynamometer were interfaced with a personal computer through a commercially available hardware system (MP150 system) The data were sampled at 400Hz and analyzed with commercially available software (AcqKnowledge 3.7.1). 1 Grass Instruments, West Warwick, Rhode Island, USA. Biodex Medical Systems, Inc., 20 Ramsay Road, Shirley, New York 11967 BIOPAC systems Inc., Goleta, CA

PAGE 80

804.2.3 Voluntary Contractile Measurements Prior to testing, subjects performed three warm -up contractions to get familiarized with the testing procedures. Subjects then performed thre e maximal voluntary isometric contractions (~5 second each with 1 minute rest intervals) while being given verbal encouragement. Peak torque was defined as the highest value obtained during the 3 maximal isometric contractions. In the event that the peak torque values differed by more than 10%, additional contractions were performed. In addition to peak torque we also determin ed the average rate of torque development (ARTD) and the torque200, as indices of explosive muscle st rength. The ARTD was defined as the average increase in torque generated in unit time, and was calculated in the time interval corresponding to 20% to 80% of peak amplitude, starting from muscle perturbation. This time interval was selected to reduce the effect of e rrors in calculating peak amplitude. Hence ARTD was calculated through numerical differentiation as N i it f N ARTD11 where N is the total number of time slots for numerical differentiation, f iis the change in torque in the time slot i and tis the unit time duration for a slot. To rque200 was defined as the absolute torque reached at 200ms during a maximal voluntary contraction (Nm). 4.2.4 Electrically Elicited Contractile Measurements Peak twitch torque, time to peak twitch and tw itch half-relaxation time were determined by delivering a supra-maximal electrical stimulus ( 600s pulse duration) to the muscles at rest. Supra-maximal intensity was determined by increas ing the current voltage until twitch torque production plateaued. Time to peak twitch and twitch half-relaxation time were calculated from the peak twitch contractions.

PAGE 81

814.2.5Voluntary Activation Deficits Voluntary activation deficits were determ ined using the twitch interpolation method.15 Briefly, a single biphasic, and supra-maximal pul se was delivered at rest and during maximal voluntary isometric contraction. The voluntary activation defic it was calculated based on the ratio between the torques produced by the supe rimposition of a supra-maximal twitch on a peak isometric contraction (a) and the torque produced by the same stimul us in the potentiated, resting muscle (b). Voluntary activation deficit (%) = (a/b)*100. 4.2.6 Statistical Analyses Independent sample T-tests were used to determine if differences existed between the groups. Comparisons were made between the self -reported dominant side of the controls and both the self-reported more involv ed side and less involved side of the incomplete-SCI group For all analyses, significance was established when P< 0.05. Data are presented as means standard error of mean. All statistical anal yses were performed using SPSS for Windows, Version 11.0.1. 4.3 Results 4.3.1 Voluntary Contractile Measurements Individuals after incomplete-S CI demonstrated significant deficits in their ability to generate peak isometric torque relative to noninjured controls in bot h the knee extensor and plantar flexor muscle groups (p<0.05). A repr esentative ankle plantar flexor torque trace acquired during a peak isometric contraction of both incomplete-SCI and control subject is provided in Figure 4-1. The peak torque deficit measured in both muscle groups was of similar magnitude (Figure. 4-2). Specifically, persons after incomplete-SCI were able to produce 36%

PAGE 82

82 and 24% of the knee extensor tor que and 38% and 26% of the plan tar flexor torque generated by non-injured controls in the le ss-involved and more-involved limbs, respectively (p<0.01). Significant bilateral asymmetries were noted in peak torque pr oduction between the self-reported more-involved versus the less-i nvolved limb in both the knee extensor (57 vs. 85 Nm) and plantar flexor muscle groups (26 vs. 39 Nm; p<0.01). Both indices of explosive muscle strength, ARTD and torque200, were significantly lower in persons with incomplete-SCI relative to c ontrols in both muscle groups tested (P<0.01; Figure4-3, 4-4). Bilateral asymmetries in torque200 and ARTD were specific to the ankle plantar flexor muscles. Of interest to note is that both indices of explosive muscle strength, showed more pronounced deficits in the ankle pl antar flexor muscles compared to the knee extensor muscles. In particular large deficits were noted in the torque200 of the ankle plantar flexor muscles with an 11.7 fold difference between the torque200 measured in the self-reported more involved limb and a 5 fold difference in the less-involved limb comp ared to control muscles (Figure4-1&4-3). The torque200 was 4.2.6 Nm in the plantar flexor musc les of the more-involved limb, 9.2.6 Nm in the less-involved limb and 47. 2.2 Nm in the non-injured contro ls, respectively. In contrast, a 5.5 fold difference and 3.7 fold difference was noted in the torque200 measured in the knee extensor muscles of the self -reported more involved (27.013.2 Nm) and less involved limb (39.9.3 Nm) of incomplete-SCI persons compar ed to non-injured controls (148.6.3 Nm). Torque200 and ARTD data are summari zed in Figures 4-3 & 4-4.

PAGE 83

834.3.2 Electrically Elicited Contractile Measurements No significant differences were found either within or betwee n subject groups for measures of peak twitch torque, time to peak twitch or ha lf-relaxation times in either the knee extensor or plantar flexor muscle groups (Table 4-2). 4.3.3Voluntary Activation Deficits A significant injury related effect on the ability to voluntarily activate the plantar flexor and knee extensor muscle groups was noted. Activa tion deficits in the knee extensors were 42 % and 66% in the less involved and more invol ved side, respectively, compared to only a 5% deficit in non-injured controls. The incomp lete-SCI group also demonstrated a 53 % voluntary activation deficit in th e less involved side and a 64 % deficit in the more involved side for the plantar flexor muscle group, compar ed to a 5% deficit in non-injured controls (Figure 4-5). Significant bilate ral asymmetries existed for bo th muscle groups for voluntary activation deficits (p<0.05, Fi gure 4-5). A representative torq ue trace acquired during a peak isometric voluntary contraction with interpolated twitch is provided in Figure 4-6. 4.4 Discussion The development of novel intervention therap ies to promote the recovery of skeletal muscle function after incomplete-SCI is one of the exciting paths of current rehabilitation research.87,145-149 However, the translation of these experimental ther apies to the SCI population is enormously challenging given the extreme heterogeneity in presentation and response to treatment of this population. As such, a comprehensive examination of skeletal muscle function in this patient population might aid in the deve lopment of targeted th erapies aimed at the recovery of muscle function af ter incomplete-SCI. Accordingly, the present study demonstrates that after chronic upper motor lesi ons and incomplete-SCI, both kn ee extensor and plantar flexor skeletal muscles 1) generate ~70% less peak torque, 2) demonstrate significant bilateral

PAGE 84

84 asymmetry in peak torque, which matches the hi erarchy for self-reported functional deficits, 3) experience voluntary activation deficits ranging between 42% and 66%, and 4) demonstrate large deficits in the rate of torque development and instantaneous muscle strength. While in this study both muscle groups demonstrated signif icant impairments in ARTD and torque200, more pronounced deficits were noted in the ankle plantar flexor muscles and bilateral asymmetries in ARTD and torque200 were specific to the ankle plantar fl exor muscles. Given the role of the ankle plantar flexor muscles in propulsion during gait we put forward that the latter impairments should be targeted in rehabilita tive interventions aiming to restore or promote locomotion in this population. The deficits noted between persons after incomp lete SCI and controls in their ability to generate peak torque in the plantar flexor and knee extensor muscle groups may appear somewhat intuitive. In additi on, the bilateral asymmetries obs erved may be considered obvious by many after this type of injury. However, no quantitative measuremen ts of muscle function have previously been reported in this population. Moreover, we contend that the methodologies described here are more suitable than traditional evaluative tests in assessing impairments of muscle function in persons with incomplete-SCI. Muscle strength assessments in persons with incomplete-SCI are typically performed using ma nual muscle tests during ASIA evaluations. The ASIA is used routinely to describe the level of injury and impairment and imply severity of injury.150,151 However; this evaluative t ool may not be adequate to di rect targeted rehabilitation interventions in persons with incomplete-SCI. Manua l muscle tests are subject to a ceiling effect, lack sensitivity to change and have a relatively p oor inter-rater reliability, especially at scores greater than 3.54,143

PAGE 85

85 A myriad of physiological changes occur in pers ons after spinal cord injury. Many of these changes are due to the direct effects of the injury (i.e. neural circ uitry disruption) while others are secondary in nature and attribut able to a resultant decrease in neuromuscular activity. An inability to voluntarily activate skeletal muscles may be a product of both primary and secondary mechanisms. Twitch interpolation is a commonly used method to estimate the extent to which a person can voluntarily activate a given muscle or muscle group.152-155 Our findings of small activation deficits (~5%) in the quadriceps and ankle plantar flexor muscles of non-injured controls are consistent with those from other laboratories.155 The activation deficits measured in persons with incomplete-SCI (42-66%) are larger in magnitude compared to those measured in patients early after surgery or long-term immobilization.156,157 Accordingly, persons with incomplete-SCI may benefit from rehabilitation stra tegies that target vol untary activation deficits to maximize skeletal muscle function, i.e. f unctional electrical stimul ation or bio-feedback.158 While these interventions may not directly imp act the primary injury, they may be able to ameliorate the loss of muscle function secondary to disuse or lack of neuromuscular activity. Perhaps the most functionally relevant characteristics of muscle torque production for persons with incomplete-SCI are the indices of explosive strength. ARTD is reflective of the average rate of contractile torque developm ent during maximum voluntary contraction while torque200 is the absolute torque generated within the initial 200ms of contr action and is indicative of the magnitude of instantaneous torque. Both ARTD and torque200 were significantly reduced in the ankle plantar flexor and quadriceps mu scle groups of persons with incomplete-SCI. However the deficit in instantaneous strength was more pronounced in the ankle plantar flexor muscles. It is our contention that the initial ra te of torque development and the instantaneous strength may be most critical fo r performance of functional tasks (i.e. walking). For example,

PAGE 86

86 steady state walking is characteri zed by repetitive, reciprocal contractions of the plantar flexor muscles (i.e. propulsion at push off) that must be accomplished in finite periods of time. A speed commonly deemed necessary for persons to safely ambulate in the community is 1.2 m/s.145 At this speed, the time it takes to complete one gait cycl e (i.e. right heel strike to right heel strike) is ~1.0 seconds. Given that the plantar flexor muscle s are reported to be acti ve for ~40% of the gait cycle and approximately of that time is spent generating con centric torque, roughly 200ms is available for torque generation by this muscle group.159 Given this available time, plantar flexor muscles must generate torque of sufficient magnit ude and at precise rates so as to propel the mass of the body forward, translat ing to movement or walking.160 We speculate that the large deficits in instantaneous torque in the ankle plantar flexors obs erved in this study (11.7 and 5 fold difference in torque200) may potentially limit locomotor function in persons with incomplete-SCI. Thus, rehabilitative strategies must be employed that result in improved rates of torque production and enhanced instantaneou s torque to meet the imposed demands of walking at community ambulating speeds.161 Although we chose to examine torque generation at 200ms based on our calculations of muscular demands at a functionally minimal gait speed (1.0 m/s), consideration should also be given to the fact that as f unctional improvements are realized, the contractile demands (i.e. magnitude and rate of for ce production) will continue to increase and the available time to generate torque will decrease. An interesting finding in the present study was the lack of difference in the electrically elicited contractile properties be tween persons after incomplete-S CI and non-injured controls. Previous studies have used these properties as a means to explain molecular and histochemical changes that occur in skeletal muscle.52 Studies using both animal and human models have provided evidence for faster cont ractile properties following SCI.50,52,162,163 However, we

PAGE 87

87 observed no differences in rate of rise or relaxa tion of electrically elicited contractions in muscles after incomplete-SCI re lative to non-injured controls. Th is is somewhat surprising in that both the knee extensor and plantar flexor muscle groups have been characterized by faster contractile speeds following SCI 50,162. These findings have been us ed to support the idea of a fiber type transformation following SCI (slow fast). However, whether a fiber type transition occurs after incomplete-SCI and the timeline for any potential shift are yet unclear. Thus, further research and tissue sampling is warranted befo re we can make any suggestions towards the muscle fiber type transformation based on c ontractile properties in this population. In conclusion, this study characterizes the impa irments in lower extremity skeletal muscle function in persons after incomplete spinal co rd injury relative to non-injured controls. The examination of knee extensor and plantar flexor muscle groups in this study is clinically meaningful given the anti-gravity responsibili ties of each of these muscle groups and their purported roles in standing and locomotor function.164,165 Reduced peak torque production, ARTD and torque200, as well as increased voluntary act ivation deficits were found to be characteristic of affected muscles below the level of incomplete spinal cord injury. In addition, a hierarchy of these impairments existed between limbs with significant bilateral asymmetries in the plantar flexor muscle group for all variables tested. This characteri stic asymmetry suggests that recovery and response to rehabilitation ma y be specific to each side, with rate limiting factors to functional performance potentially being limb rather than subject specific. We speculate that the large deficit in the rate of to rque development and instantaneous torque in the ankle plantar flexors of persons with incomplete-SCI limits locomotor function.

PAGE 88

88 Table 4-1. Characteristics of incomplete SCI subjects Level of injury ASIA Classification Duration of injury (mos) LEMS WISCI-II Mobility Status S1 C6 D 20 35 19 Ambulator S2 T4 D 7 44 19 Ambulator S3 C4 D 16 45 13 WheelchairS4 C6 C 14 15 8 WheelchairS5 C6 D 37 40 16 WheelchairS6 C4 D 18 48 20 Ambulator S7 C8 D 28 37 16 WheelchairS8 C4 C 22 26 9 WheelchairS9 C5 D 16 34 13 WheelchairS10 C6 D 39 38 19 Ambulator Table 4-2. Electrically elicited contractile measurements Incomplete-SCI Controls more-involved less-involved Knee Extensors Peak twitch force (Nm) 29.1 2.3 27.3 2.8 31.6 3.9 Time to peak twitch (ms) 123.3 5.8 135.6 5.3 129.3 5.8 Twitch-half relaxation time (ms) 73.8 5.7 107.1 20.1 95.1 11.0 Plantar Flexors Peak twitch force (Nm) 13.8 1.5 14.8 0.8 14.5 0.8 Time to peak twitch (ms) 143.8 7.2 143.9 7.3 144.3 5.2 Twitch-half relaxation time (ms) 116.2 7.2 125.7 9.2 127.3 11.2

PAGE 89

89 Figure 4-1. Representative torque-time curve. Drop down arrows indicate time points at which peak torque is reached in a representative incomplete-SCI and control subject. Shaded areas indicate torque200 in both subjects.

PAGE 90

90 Figure 4-2. Peak torque (Nm) for the knee extens or and plantar flexor mu scle groups, comparing the dominant side of the c ontrol with the more involved (more-involved) and less involved limb (less-involved) of the inco mplete-SCI group. Significant difference between control group and incomplete-SCI group. Significant difference between the less-involved versus the more-involved (p<0.05).

PAGE 91

91 A B Figure 4-3. Torque200 (Nm) (A) knee extensor and (B) plantar fl exor muscle groups, comparing the dominant side of the c ontrol with the more involved (more-involved) and less involved limb (less-involved) of the inco mplete-SCI group. Significant difference between control group and incomplete-SCI group. Significant difference between the less-involved versus the more-involved (p<0.05).

PAGE 92

92 A B Figure 4-4. Average rate of torque developmen t (ARTD)(Nm/sec) for the (A) knee extensor and (B) plantar flexor muscle groups, comparing th e dominant side of the control with the more involved (more-involved) and less involved limb (less-involved) of the incomplete-SCI group. Significant difference between control group and incomplete-SCI group. Significant differe nce between the lessinvolved versus the more-involved (p<0.05).

PAGE 93

93 Figure 4-5. Voluntary Ac tivation Deficits (%) for the knee extensor and plantar flexor muscle groups, comparing the dominant side of th e control with the more involved (moreinvolved) and less involved limb (less-involved) of the incomplete-SCI group. Significant difference between contro l group and incomplete-SCI group. Significant difference between the less-involved versus the more-involved (p<0.05).

PAGE 94

94 Figure 4-6. Torque trace acquired during MVIC with interpolated twitch to quantify muscle activation deficit. A single supramaximal intensity electrical stimulus was superimposed on a maximal voluntary isomet ric contraction (a), as well as on a resting, potentiated plantar flexor muscle (b).

PAGE 95

95 CHAPTER 5 LOCOMOTOR TRAINING AND MUSCLE FUNCTION AFTER INCOMPLETE SPINAL CORD INJURY: A CASE SERIES 5.1 Introduction Traumatic spinal cord injury (SCI) is one of the most disabling health problems facing adults today. Despite advances in treatment in terventions individuals wi th SCI often lose the ability to walk and are at risk to develop s econdary health complicat ions. Muscle atrophy and reduced ability to generate force play essential ro les in the development of disability after SCI. Individuals with chronic complete spinal cord in jury show 42-68% atrophy in the calf and thigh muscles one year after injury, while subject s with incomplete-SCI demonstrate a 25-30% reduction in average lower extremity muscle cross-sectional area (CSA).46 Few studies have performed a quantitative analys is of skeletal muscle strength after incomplete-SCI.37 However, we recently demonstrated in persons with chro nic upper motor lesions and incomplete-SCI, that both knee extensor and plantar flexor skeletal mu scles generate ~70% less peak torque, with even larger reductions in measures of instantaneous or expl osive peak torque.53 Repetitive locomotor training with body weight support has emerged as a potential promising therapeutic intervention to promot e motor recovery and ambulation following incomplete-SCI. 166-168 Locomotor training has been suggested to have a positive impact on walking ability, 168,169 functional independence and subjective well being.170 Giangregorio et al.109, 171 and Stewart et al.39 have also shown that locomotor training involves sufficient mechanical loading to induce muscle plasticity increasing muscle size and altering the muscle phenotype both after acute and chronic incomplete -SCI. Interestingly, studies involving animal models of incomplete-SCI have also shown th at LMT has the potential to augment the force generating capabilities of aff ected lower hind limb muscles. 111,114 To our knowledge, no study has systematically investigated the effect of locomotor training on lower extremity muscle force

PAGE 96

96 production and instantaneous power in persons wi th incomplete-SCI. Mo stly; studies rely on manual muscle tests and ASIA motor scores to assess voluntary strength in persons with incomplete-SCI. However, ASIA scores have been criticized to lack sensitivity and to have a limited ability as indicators of neur omuscular recovery in chronic SCI.54,78,143,168,169 Therefore, the purpose of this study was to dete rmine the effect of nine weeks of locomotor training on lower extremity muscle function in persons with chronic incomplete-SCI using isokinetic dynamometry. Specifically, we measured peak isometric torque, torque developed within the initial 200 ms of contraction (Torque200) and the average rate of torque development (ARTD) in the knee extensor and ankle plantar fl exor muscle groups. In addition, we quantified voluntary activations deficits usi ng superimposed electrical stimulation. The knee extensor and plantar flexors muscles groups were selected fo r study because of their purported role during human locomotion. 5.2 Methods 5.2.1 Subjects Five persons (one woman, four men) with chronic motor incomplete-SCI underwent nine weeks (45 sessions, 5-times /week) of locomo tor training. A summary of the subjects demographics is provided in Table 5-1. Criteria for inclusion included: 1) age 18-70; 2) history of SCI as defined by the American Spinal In jury Association (ASI A) Impairment Scale categories C or D; 3) first time traumatic SCI at cervical or thoracic leve ls (C4-T12) resulting in upper motor neuron lesions in the lower extremity ; 4) medically stable and asymptomatic for bladder infection, decubitis, cardiopulmonary dis ease or other significant medical complications prohibiting testing and/or traini ng; and 5) if using anti-spasticity medication, agreement to maintain current levels throughout the study. Exclus ion Criteria were as fo llows: 1) participation in a rehabilitation or research pr otocol that could infl uence the outcome of this study. Prior to

PAGE 97

97 participating in the study, written informed consent was obtained from all subjects, as approved by the Institutional Review Board at the University of Florida, Gainesville. 5.2.2 Locomotor Training Protocol The locomotor training intervention consisted of 45 training sessions (5x/ week) spread over nine weeks, with each session consisting of 30 minutes of step training on the treadmill with body weight support (BWS) immediately followed by 20 minutes of level overground walking and community ambulation training. Includi ng pre-training stretching, donning/doffing the harness, and additional time spent on the treadmill for stand training and standing rest breaks, the total session duration was approximately 75 to 90 minutes per day. Each subject completed all of the training sessions. With the aid of the body weight support, treadmill speed and manual trainers, the treadmill training environment fac ilitated delivery of locomotor specific practice. 79,168 Trunk, lower limb, and upper limb kinematics were consistently assisted and/ or monitored by trainers to assure appropriateness in relation to normal walking. Speed of treadmill stepping was kept in a range consistent with normal wa lking (2.2-2.8 miles/hr). Progression of training was achieved by decreasing BWS, altering speed increasing trunk contro l, decreasing manual assistance for limb control and increasing the time spent walking on the treadmill per bout. A more detailed description of the training pr inciples, parameters and progression has been provided by Behrman & Harkema et al. 2000. 79 Overground training consis ted of an immediate assessment of the participants ability to stand and/or walk independently overground and an evaluation of the deficits limiting achievement of this goal. These deficits became the focus for goal setting in the next days training sessi on. Additionally, overground training addressed translation of the skills from the treadmill to the home and community identifying practical ways for the participants to incorporate new skills into everyday activities.

PAGE 98

985.2.3 Experimental Protocol 5.2.3.1 Strength assessment Voluntary contractile measurements were dete rmined in the self-reported more-involved and less-involved limbs for the kn ee extensor and plantar flexor muscle groups before and after locomotor training, using a Biodex System 3 Dynamometer. Testing was performed with subjects seated with hips flexed to ~85, as previously described. 53 For knee extensor testing, the knees were flexed to ~90 and the axis of rotation of the dynamometer was aligned with the axis of the knee joint and the lever arm secured against th e anterior aspect of the leg, proximal to the lateral malleolus. Plantar flexor testing was performed with the knee flexed at ~30 and the ankle at ~0 plantar flexion. The anatomical axis of the ankle was aligned with the axis of the dynamometer, while the foot was secured to the foot plate with straps placed at the forefoot and ankle. Proximal stabilization for all testing was ac hieved with straps across the chest, hips and thigh. 5.2.3.2 Voluntary contractile measurements Prior to testing, subjects performed three wa rm-up contractions to become familiar with the testing procedures. Subjects then performed three maximal vol untary isometric contractions (~5 seconds each with 1 minute rest intervals) wh ile being given verbal encouragement. Peak torque was defined as the highest value obtained during the 3 maxima l contractions. In the event that the peak torque values differed by more than 5%, additional contractio ns were performed. In addition to peak torque we also determined th e absolute torque generated during the initial 200ms of contraction (Torque200) as well as the average rate of torque development (ARTD) during the contractile effort, as previously described. 53

PAGE 99

995.2.3.3 Voluntary activation deficits Voluntary activation deficits were determ ined using the twitch interpolation method. 152 Briefly a single biphasic, supra-maximal elect rical pulse was delivered at rest and during maximal voluntary isometric contraction. Volunt ary activation deficit was calculated using the ratio between the torques produced by the supe rimposition of a supra-maximal twitch on a peak isometric contraction (a) and the torque produced by the same stimul us in the potentiated, resting muscle (b). Voluntary activation deficit (%) = (a/b)*100. Electrical stimulation was elicited using a Gr ass S8800 stimulator with a Grass Model SIU8T stimulus isolation unit. Electrically induced cont ractions were delivered through two 3.0 by 5.0 self-adhesive neuromuscular stimulation electrodes placed over the proximal and distal portions of the muscle group being tested. The stimulat or and the dynamometer were interfaced with a personal computer through a commercially available hardware system (Biopac MP150 system) sampling at 400Hz and data were analyzed with commercially available software (AcqKnowledge 3.7.1). 5.2.4 Statistical Analyses A longitudinal, prospective case series was used in which participants completed nine-weeks of locomotor training. Individual data have b een summarized in tables and as plots. 5.3 Results 5.3.1 Voluntary Contractile Measurements All individuals with chronic incomplete SCI demonstrated a significant improvement in their ability to generate peak isometric torque following locomotor training. The most robust increase in isometric peak tor que production was observed in th e ankle plantar flexor muscles (average increase 43.9.0%) of the self-repo rted more involved limb, followed by the knee

PAGE 100

100 extensor muscles of both the more involved ( 21.1.3%) and less involved (19.8.3%) limb. Individual gains in peak torque ranged from 8% to 45% in knee extensor and 14% to 98% in the plantar flexor muscle groups. Note that four out of five subjects showed an increase in isometric peak torque in at least three of the tested muscle groups. Individual torque data prior to and after nine weeks of locomotor training are summarized in Table 5-2. Both indices of explosive muscle torque generation, ARTD and Torque200, showed large improvements in the ankle plantar flexor and knee extensor muscles with locomotor training. In particular, large bilateral improve ments in plantar flexor Torque200 measures were realized, with average relative improvements of 587% and 219% in the more-involved and lessinvolved limbs, respectively. Individual increases in a nkle plantar flexor Torque200 ranged from 8% to 835%. A more variable response was not ed in the knee extensor s with some subjects showing an enhancement in the more involved limb (subjects 2, 3 and 5) and others in the less involved limb (subjec t 1 and 4). Torque200 data for both the knee extensors and ankle plantar flexors are presented in Figures 5-1A-D. ARTD values showed a similar pattern with relatively larger improvements in the ankle plantar flexor muscles compared to the knee extensors (Table 5-2). The mean ARTD in the ankle pl antar flexor muscles improved from 36.3 16.5Nm/s to 46.9 13.3 Nm/s in the more involved limb and from 68.2 23.2 Nm/s to 102.8 32.7 Nm/s in the less involved limb. The mean ARTD in the knee extensor muscles increased from 207.9.9 Nm/s to 252.1.7Nm/s and from 325.5.6Nm/s to 392.7.0Nm/s. 5.3.2 Voluntary Activation Deficits All subjects showed voluntary activation deficits in both the knee extensor and ankle plantar flexors muscles prior to LMT. Interestingly, a si gnificant training effect was noted in the ability to voluntarily activate th e bilateral knee extensor muscle gr oups as well as the more-involved

PAGE 101

101 plantar flexor muscles. Mean activation deficits in the knee exte nsors improved from 63 15% to 43 10% and from 41 16% to 31 16% in the more involved and less involved sides, respectively. Only one subject (subject 4), the subject with th e highest pre-LMT knee extensors strength, did not show any improvement in knee ex tensor activation deficit after LMT. Similar to the knee extensors, activation deficits in th e more involved plantar flexors improved from 61 10% to 41 11% after nine weeks of locomotor trai ning. Individual data are summarized in Figure 5-2A & B. 5.4 Discussion The results of this case series suggest that ni ne weeks of locomotor training in persons with chronic motor incomplete SCI results in positive alterations in lower extremity skeletal muscles that include an improved ability to generate both peak and instantaneous torque about the knee and ankle joints. Intere stingly, increases in force producti on were more pronounced in the ankle plantar flexor muscles versus the knee extensor muscles, consistent with previous literature suggesting that the ankle plantar flexors are criti cal for propulsive force generation during locomotion and experience high loads.159,172 Superimposed electrical stimulation further showed that improvements in muscle strength with loco motor training are accompanied with a decrease in voluntary activation deficit. A myriad of physiological changes occur in pers ons as a results of tr aumatic spinal cord injury. Many of these changes are due to direct effects of the injury (i.e. neural circuitry disruptions), while others are linked to pharmaco logical side effects or due to the lack of neuromuscular activity and loading. Among the phys iological changes is a dramatic loss in the ability to voluntarily produce muscle force, leading to impaired motor function and disability. We previously demonstrated that isometric p eak torque generation in the knee extensor and

PAGE 102

102 plantar flexor muscle groups is reduced by about 70% in person with chronic incomplete SCI (>1 year), compared to agegenderand body weightmatched control subjects.53 Individuals in the present study demonstrated similar reduced planta r flexor and knee extensor peak torque values prior to locomotor training. Forty-five sessions of locomotor training resulted in a robust increase in isometric peak torque production in the ankl e plantar flexor muscles (average increase 43.9.0%) of the self-reported more involved limb and the knee extensor muscles of both the more involved (21.1.3%) and less involved (19.8.3%) limb. The ability to improve peripheral muscle strength in persons with in complete-SCI seemingly adds to the positive attributes previously contributed to this experimental therapeutic intervention. In addition to peak torque generation, we suggest that the functionally more relevant characteristics of muscle torque production in person with incomplete SCI are re presented by the indices of explosive or instantaneous streng th, ARTD and Torque200. ARTD represents the average rate of contractile torque development during maximum voluntary contraction, while Torque200 measures the absolute torque generated within the initial 200ms of contraction. We previously showed that both ARTD and Torque200 are significantly reduced in person s with incomplete SCI, with more pronounced deficits in the ankle plantar flexor muscles compared to the knee extensor muscles.53 In particular large deficits were noted in the Torque200 of the ankle plantar flexor muscles with an 11.7 fold difference between the Torque200 measured in the self-reported more involved limb and a 5 fold difference in the less-involved limb compared to control muscles. With nine weeks of locomotor training large improvements in both measures of instantaneous muscle strength were noted. In par ticular, large bilateral improvements in plantar flexor Torque200 measures were realized, with average relative improvements of ~600% and 200% in the more-involved and less-involved limbs, respectively. Smaller and less consistent

PAGE 103

103 relative gains were realized in the knee extens or muscle group. The large increase in the Torque200 of both ankle plantar flexor muscle groups with locomotor training deserves special attention, given these muscles importance dur ing bipedal walking. At a speed commonly deemed necessary for persons to safely ambulate in the community (1.2 m/s),173 a time window of only about 200ms is available to generate the n ecessary concentric torque in the plantar flexor muscle group to produce forward propulsion.159 Data from our previous and current study combined indicate that the torque produced by the ankle plantar flexors in this time window is significantly reduced in persons with incomplete SCI and can be considerably improved with intense locomotor training.53 An improved ability to generate instantaneous torque may be critical to facilitating functional recovery and ambulation in patients with incomplete SCI. The suggested importance of plantar flexor muscle torque generation for improving ambulation in persons with central nervous system injuries is not new and has been reported in persons poststroke.172,159 The ability to drive -motoneurons to elicit maximal muscle recruitment is often referred to as maximal voluntary activ ation and can be estimated using superimposed electrical stimulation, a method commonly implem ented in a variety of populations.154,155,174 In a previous study, we measured voluntary activation deficits ranging betw een 42% and 66% in the lower extremity muscles of incomplete-SCI subjects, whereas control subjects showed a ~5% voluntary activation deficit.53 Similar voluntary activation deficits were found in this study prior to locomotor training. Interestingly, voluntary activation deficits were partially attenuated following 45 sessions of locomotor training (3040% post-training), even though they did not return to normal values. In particular in the knee extensor muscles bilateral improvements in voluntary muscle activation contribu ted significantly to gains in muscle force production, while

PAGE 104

104 muscle cross-sectional area was relatively unchanged. Improvements in muscle activation in persons with incomplete SCI with locomotor tr aining have also been reported using iEMG.165 Of interest to note is that voluntary activation deficits can also be observed following disuse or immobilization. However, in these models the phenomena is transient and normalization in muscle activation is typically observed after 3 to 4 weeks of rehabilitation.175 Despite the measured increases in inst antaneous and peak force production, and improvements in voluntary activation only one of th e five participants in this study showed any change in their lower extremity motor scores (LEMS) after locomotor training. In specific, subject 3 improved his LEMS score from 35 to 38 In all other subjects no change in LEMS score could be detected. These data are consiste nt with other locomotor training studies, which often fail to demonstrate a change in ASIA sc ores with training in persons with chronic injuries.79,168,169 We believe that the lack of change in ASIA motor scores in the present study reflects a limitation in the measurement tool. Compared to isokinetic dynamometry, manual muscle tests have a limited inter-rater reliability and have been criticized to lack sensitivity, especially at scores above 3 (out of 5).54,143 Others have argued that while ASIA scores are valuable in predicting motor recovery in acute pa tients, they may be less powerful as measures of neuromuscular recovery in chronic SCI.110,176-178 In conclusion, nine weeks of locomotor tr aining resulted in improved lower extremity skeletal muscle function in persons after incomp lete spinal cord injury. Specifically, extensor muscles about the ankle and knee joint demonstrat ed an improved ability to generate both peak and instantaneous torque. Relative gains in musc le function were greatest in the ankle plantar flexor muscles, consistent with their critical role for propulsive force generation and high loading during locomotion. Ankle plantar flexor muscles also showed a significant increase in maximal

PAGE 105

105 CSA, while increases in knee extensor force production were mainly linked to improvements in voluntary muscle activation. Finally, we suggest that skel etal muscle alterati ons contribute to the functional improvements reported with locomotor training in person with incomplete-SCI. Table 5-1. Characteristics of in complete SCI subjects Age (yrs) Height (cm) Body Mass (kg) ASIAR Impairment Classification Level of Injury Duration of injury (months) Mobility status LEMS (preLMT) LEMS (postLMT) S1 44 154.9 74.8 C C6 20 Powerwheelchair 33/50 34/50 S2 21 185.4 68.0 D T4 8 Bilateralcanes 44/50 44/50 S3 48 198.6 77.0 D C6 39 Bilateralcrutches 35/50 38/50 S4 58 183.0 90.7 D C4 14 Wheelchair 45/50 45/50 S5 36 176.9 83.9 C C6 16 Wheelchair 17/50 17/50

PAGE 106

106 Table 5-2. Values of isometric peak tor que and average rate of force development Isometric Peak Torque ________________________________________________________________________ _______________ S1 S2 S3 S4 S5 Knee Extensors More Involved Pre-LMT 35.8 95.8 26.8 176.5 10.8 Post-LMT 42.6 84.6 39.0 181.8 15.7 Less Involved Pre-LMT 65.0 136.5 62.6 179.0 15.7 Post-LMT 70.3 190.5 78.0 199.9 18.0 Plantar Flexors More Involved Pre-LMT 11.7 45.5 19.4 65.2 12.5 Post-LMT 23.3 51.9 29.1 63.3 20.0 Less Involved Pre-LMT 24.8 52.3 42.7 90.2 21.9 Post-LMT 35.5 60.7 51.7 82.5 21.0 Average Rate of Torque Development ________________________________________________________________________ _______________ S1 S2 S3 S4 S5 Knee Extensors More Involved Pre-LMT 92.6 281.5 62.3 572.1 30.8 Post-LMT 182.5 337.1 81.8 613.3 46.0 Less Involved Pre-LMT 270.4 338.0 233.1 727.1 58.1 Post-LMT 325.9 588.1 239.2 746.8 63.3 Plantar Flexors More Involved Pre-LMT 17.8 27.4 20.0 94.7 21.2 Post-LMT 30.8 44.4 45.9 91.2 22.2 Less Involved Pre-LMT 36.5 47.0 103.8 130.3 22.9 Post-LMT 101.0 82.7 105.0 203.5 21.9

PAGE 107

107 A S1S2S3S4S5 0 20 40 60 80 100 120 140 160Torque200 (Nm/s) Pre -LTM Post-LTM B Figure 5-1. Torque200 (Nm) measured in the kn ee extensor muscle group of the (A) more involved and (B) less involved li mb of individuals with in complete-SCI before (preLMT) and after locomotor training (post-LMT).

PAGE 108

108 S1S2S3S4S5 0 5 10 15 20 25 30 35Torque200 (Nm/s) Pre -LTM Post-LTM A S1S2S3S4S5 0 5 10 15 20 25 30 35 40 45 50Torque200 (Nm/s) Pre -LTM Post-LTM B Figure 5-2. Torque200 (Nm) measured in the pl antar flexor muscle group of the (A) more involved and (B) less involved li mb of individuals with in complete-SCI before (preLMT) and after locomotor training (post-LMT).

PAGE 109

109 0% 20% 40% 60% 80% 100% 120%PrePost PrePost More involved Less involved Voluntary activation deficits S1 S2 S3 S4 S5 Figure 5-3. Voluntary activation deficits (%) meas ured in the knee extensor muscle group, of the more involved and less involved limb of i ndividuals with incomplete-SCI before (Pre) and after locomotor training (Post).

PAGE 110

110 0% 20% 40% 60% 80% 100% 120%PrePost PrePost More involved Less involved Voluntary activation deficits S1 S2 S3 S4 S5 Figure 5-4. Voluntary activation deficits (%) meas ured in the plantar flexor muscle group of the more involved and less involved limb of i ndividuals with incomplete-SCI before (Pre) and after locomotor training (Post).

PAGE 111

111 CHAPTER 6 RESISTANCE TRAINING AND LOCOMOTO R RECOVE RY AFTER INCOMPLETE SPINAL CORD INJURY: A CASE SERIES 6.1 Introduction The proportion of persons that suffer a spinal co rd injury (SCI) resulting in an incomplete lesion has risen dramatically over the past 20 ye ars. As a result ~55% of the new injuries sustained in the United States are now classified as incomplete. In addition, the life expectancy for persons with an incomplete injury is higher th an after a complete SCI and is approaching that of non-injured persons, regardless of age at injury.1 As such, the increased incidence and prevalence of persons with this type of injury necessitates a co mprehensive understanding of the adaptations that occur and the potential for reha bilitative interventions to impact persons with incomplete-SCI. Unfortunately, despite the prop ortion of persons sustaining and subsequently living with incomplete SCI, the preponderan ce of scientific lite rature describing the physiological and functional adap tations to SCI involves persons with complete injuries. Accordingly, limited data are available that describe motor function and its impact on functional ability in this large subject cohort. Th e ability to independently ambulate is a primary goal of many persons after SCI. However, ev en though a large number of individuals with incomplete SCI regain some ability to walk, lim itations in gait speed may make this method of mobility impractical for activities of daily livi ng. Slow speed combined with other mobility deficits (e.g. difficulty climbing stairs, curbs, etc...), coul d negate the ability to safely ambulate in the community, resulting in a perceived disability Interestingly, rehabi litation practice focusing on compensatory approaches to locomotion has largely been based on the prevailing assumption that neural as well as functional recovery is lim ited in persons with chronic SCI. However, recent evidence from both animal and human studies indicat es that with the appropriate training stimuli,

PAGE 112

112 neural as well as muscular plasticity can be induced even years after injury 47,140 Improvements in functional ability, however, vary greatly and the inciden ce of disability remains high.52,141 Previous data suggest that persons after incomplete SCI produce less voluntary torque about the knee and ankle than non-injured contro ls. Perhaps more importantly, impairments in the ability to produce torque in a timely manner as well as a reduced walking velocity is also common to these persons.53 It is our belief that reduced mu scle power generation significantly impacts locomotor function and that functional re covery can be facilitated with rehabilitation interventions that attenuate this impairment. Specifically, the ankle plantar flexor and knee extensor muscle groups are of interest primarily because of their purported roles during bipedal locomotion, with torque demands at these joints during walking represen ting the two highest in the lower extremity. As such, the potential for impaired torque pr oduction about these joints to be a limiting factor in locomotor performance seems high. The common goal of resistance training programs is to increase maximal strength in the trained musculature. In additi on, the focus of plyometric tr aining, which incorporates highvelocity stretch-shortening type contractions, has been to improve performance in activities requiring fast contractions (e .g. jumping or sprinting).179,180 The combination of these two types of training has been shown to be effective in improving both maximal strength as well as muscle power production and 179,181 result in improved jump height and sprint speed in neurologically healthy individuals Interestingl y, the potential for rehabilitativ e-training induced changes in muscle strength and power to affect functional ab ility after incomplete SCI is largely unstudied. In addition, whether potential in creases in muscle function in these persons identified during strength testing are reflectiv e of improved muscle power output during functional tasks is unknown and of obvious value. Acco rdingly, the challenge is to now develop, evaluate and

PAGE 113

113 implement strategies that maximize neuromuscular plasticity in individuals after incomplete SCI with the hopes of resultant improvements in functional capacity and a subsequent decreased disability. As such, the purpose of this study was to determine if improvements in muscle function accompanied by improvements in loco motor ability can be realized following a combined resistance and plyometric jump training program in persons with chronic incomplete SCI. 6.2 Methods 6.2.1 Subjects Three independently ambulatory males with chronic motor-incomplete SCI participated in this study. Criteria for inclusion included 1) ag e 18-70; 2) first time SCI (C5-T10); 3) medically stable and asymptomatic for bladder infection, decubitis, cardiopulmonary disease or other significant medical complications prohibiting testing and/or traini ng; 4) if using antispasticity medication, agreement to maintain current leve ls throughout study; Exclus ion criteria were 1) participation in a rehabilitation or research protocol that could in fluence outcomes of this study. 2) history of congenital SCI or other disorders that may c onfound treatment, study, and/or evaluation procedures; Prior to participation, written informed consent was obtained from all subjects, as approved by the Institutional Re view Board at the University of Florida. Subject 1, a 22 year-old male (69 kg, 185 cm ), suffered a traumatic SCI (T4, 17 months post-injury) and was classified as American Spinal Injury Association (ASIA) impairment level D, with a lower extremity motor score (LEMS) of 44/50. Prior to RPT this subject had a selfselected gait speed of 0.71 m/s and a maximal gait speed of 1.01 m/s. This subject completed 29 sessions of RPT over the 12-week study period. Subject 2, a 61 year-old male (93 kg, 189 cm ), suffered a traumatic SCI (C5, 27 months post-injury) and was classified as ASIA D with a LEMS of 48/50 prior to RPT. Subject 2 had a

PAGE 114

114 self-selected gait speed of 0.82 m/s and a maximal gait speed of 1.18 m/s. Subject 2 completed 30 sessions of RPT over the 12-week study period. Subject 3, a 58 year-old male (88 kg, 178 cm ), suffered a traumatic SCI (C5, 24 months post-injury) and was classified as ASIA D with a LEMS of 35/50. Prior to RPT this subject had a self-selected gait speed of 0.78 m/s and a maxi mal gait speed of 1.06 m/s. This subject completed 30 sessions of RPT over the 12-week study period. 6.2.2 Resistance Training Program Lower extremity progressive resistance trai ning was 12 weeks in dur ation and subjects completed 2-3 sessions/week for a total of 30 sessions. Resistance exercises included unilateral leg press, knee extension/flexion, hip extensi on/flexion and ankle plan tar-flexion exercises performed on adjustable load weight machines During the initial training session a predicted 1repetition maximum (1-RM) was calculated for e ach subject and for each exercise. 1-RM was determined using a prediction table based on a si ngle set to volitional failure with load that allowed between 6 and 12 repetitions. During subs equent training sessions, subjects performed 2-3 sets of 6-12 repetitions at a relative intensity of ~7085% of predicted 1-RM. Maximal strength was evaluated weekly to assess for tr aining-related improvements and exercise loads were adjusted accordingly. Specifically, if the su bject achieved the target number of repetitions for all prescribed sets of a given exercise, a ne w predicted 1-RM was prescribed and resistance was increased for subsequent training sessions. 6.2.3 Plyometric Training Unilateral plyometric jump-training exercises were performed in both limbs in a supine position on a ballistic jump-t raining device (ShuttlePro MVP Contemporary Design Group, Figure 1). Session intensity for this exercise was modified by changing either the resistance or the number of ground contacts and progressed over the training period, accordingly. Briefly,

PAGE 115

115 after familiarization with the training device, su bjects completed a total of 20 unilateral =ground contacts (e.g. jumps) with each limb at a resistance of ~25% of body mass. Thereafter, upon successful completion of at leas t 20 ground contacts per limb (e.g. complete clearance from foot plate), resistance was increased in increments of 10 lbs. When a new resistance was set, repetition goal was set at 10 ground contacts per limb for the initial session. Subsequent sessions allowed for up to 20 contacts per limb. Thus, a minimum of two sessions at a given resistance was required before load was increased. Resistance was held consistent between limbs throughout the training program.182,183 6.2.4 Dynamometry Strength measurements were performed in th e PF and KE muscle groups using a Biodex isokinetic dynamometer (Biodex Corp., Shirley, NY). PF strength was assessed with subjects seated in a semi-reclined (~70 hip flexion) posi tion, with the knee flexed ~15 and the ankle in an anatomical neutral position (0 of plantar flexion). The axis of the dynamometer was aligned with the lateral malleolus, and the foot was secured with straps placed at the forefoot and ankle. Proximal stabilization was achieved with straps ac ross the chest, hips, and knee. KE strength assessments were performed with subjects seated in the same position used for PF testing, with the exception that the knee was fl exed to 90. The axis of the dynamometer was aligned with the knee joint line, and the leg was secured to the lever arm. Peak torque (Nm) was defined as the highest isometric torque achieved during 3 maximal contractions (~3 sec contractions separated by a minimum of 60 seconds rest ). In the event that the peak torque values during the three trials differed by more than 5%, additional contractions were performed. In addition to peak torque, values for T20-80, torque200 and ARTD were also determined both preand post-RPT. These measures were used as indices of a subjects ability to produce torque in an explosive manner and accoun t for potential differences in both the timing

PAGE 116

116 and magnitude of torque production. T20-80, used to represent the time to peak tension, was defined as the amount of time to generate from 20% to 80% of peak isometric torque. This time interval was chosen to minimize potential errors in the determin ation of the precise onset and nadir of torque development while still representing a majority of the time interval for achieving maximal torque production. Average rate of to rque development (ARTD) was defined as the average increase in torque gene rated in unit time (Nm/s), and was calculated over the same interval as T20-80. Hence ARTD was calcu lated through numerical differentiation as N i it f N ARTD11 where N is the total number of time slots for numerical differentiation, f iis the change in torque in the time slot i and tis the unit time duration for a slot. Torque200 was defined as the absolute torque reached at 200ms during a maximal voluntary contraction (Nm). Torque220 was defined as the absolute amount of to rque generated during the initial 220ms during a maximal voluntary contraction and is based on the calculated time that is available for concentric torque generation du ring a typical gait cycle at a speed designated necessary for community ambulation159. For example, the speed commonly deemed necessary for persons to safely ambulate in the community is 1.2 m/s 159. At this speed, the time it takes to complete one gait cycle (i.e. right heel strike to right heel strike) is ~1.1 seco nds. Given that the plantar flexor muscles are reported to be active for ~40% of th e gait cycle and approximately 1/2 of this active time is spent generating concentric torque, roughly 200 milliseconds is available for force generation (e.g. propulsion) by this muscle group. 6.2.5 Voluntary Activation Deficits Voluntary activation deficits were determ ined using the twitch interpolation method.152,184 Briefly, a single biphasic, supra-maximal pulse (600 sec pulse duration) was delivered at rest

PAGE 117

117 and during maximal voluntary isometric contracti on. Voluntary activation deficit was calculated using the ratio between the torques produced by the superimposition of a supra-maximal twitch on a peak isometric contraction (a) and the to rque produced by the same stimulus in the potentiated resting muscle (b). Voluntary activ ation deficits were e xpressed as: voluntary activation deficit (%) = (a/b)*100. 6.2.6 Locomotor Data Collection Subjects performed repeated 10 meter walk s over a 14 ft. long mat (Gait Rite) that measures the geometry and the applied pressure of each footfall as a function of time in order to determine both self-selected and maximal overg round walking speed (3 trials each). Gait analyses were performed 3 months prior to trai ning as well as at both preand post-RPT time points. Multiple baseline tests were conducted to control for im provements resulting from natural recovery. 6.3 Results 6.3.1 Dynamometry All subjects demonstrated improveme nts in peak torque production, T20-80, torque200 and ARTD during postversus pre-RPT dynamometric te sting. On average, RPT resulted in a 35.0 9.1% and 28.9 4.4% improvements in peak isometri c torque production in the PF and KE muscle groups, respectively. Indi vidual gains ranged from 17% to 76% in the plantar flexors and from 22% to 45% in the knee extensors. Time to peak tension, represented by T20-80, decreased from 470.8 82.2 ms to 312.0 65.7 ms in the PF and from 324.5 35.4 ms to 254.2 34.5 ms in the KE muscle groups following training. In addition, both indi ces of muscle power generation, ARTD and torque220, were noticeably improved following training. Of interest to note is that both torque220 and ARTD showed more pronounced improvements in the PF

PAGE 118

118 compared to the KE muscles with training. Sp ecifically, a 62.1% and 122.2 % improvement in torque220 and ARTD were seen in the PF muscles, with only a 33.4% improvement in torque220 and a 66.4 % improvement in ARTD in the KE mu scle group. In addition, the largest relative gains in indices of explosive muscle strength (T20-80, torque200 and ARTD) occurred in the PF muscle group of the more-involv ed limb. Peak torque, torque200, T20-80 and ARTD data are summarized in Table 6-1. 6.3.2 Voluntary Activation Deficits Significant voluntary activation de ficits were noted in both th e PF and KE muscle groups prior to training. RPT resulted in reductions in activation deficits in both the PF and KE muscle groups in each subject. Individua l data for activation deficits ar e presented in Table6-1. Although significant bilateral asymmetries existed prior to and following the intervention, these differences were seemingly attenuated in both muscle groups following RPT. 6.3.3 Locomotor Analyses Values for maximum and self-selected gait speeds did not differ by more than 0.04 m/s and 0.02 m/s, respectively, for any of the subjec ts in this study when comparing tests done 3 months prior to the onset of tr aining and immediately prior to tr aining. Following RPT, a 36.1 % average increase in maximum gait speed and a 34 .7% average improvement in self-selected gait speed were realized. 6.4 Discussion The results of this study suggest that a combination of resistance and plyometric training in persons with motor incomplete SCI results in bilateral improvements in 1) peak torque production, 2) time to peak torque and 3) rate of torque producti on in the plantar flexor and knee extensor muscle groups. These improvements in muscle function can be attributed to both an increase in muscle cross-sectional area as well as an increased ability to voluntarily activate

PAGE 119

119 affected skeletal muscles. Interestingly, the magnitude of improvement in these outcomes was most pronounced in the moreversus the less-involve d limb and in the PF versus the KE muscle group. In addition, improvements in both self-s elected and maximum gait speeds were realized and were explained by increased propulsion in the more-involved li mb as well as increased lower extremity joint powers, suggestive of improved task specific muscle function (i.e. during walking). Injury to descending spinal pa thways as well as decreased activation history both has the physiological consequence of reducing the ability to voluntarily activate affected skeletal muscles. Although restoration or repair of the injured spinal cord is not a reasonable expectation with training, the potential to improve deficits resulting from disuse seems likely and has been demonstrated after periods of inactivity in other populations.160,185,186 In this study, significant activation deficits existed prior to RPT that are comparable to other models of disuse (i.e. cast immobilization, limb-suspension).187 Interestingly, these deficits were partially attenuated with training and this enhancement of neural function could serve to explain a portion of the strength gains realized post-RPT. In addition to enhan ced neural transmission, muscle hypertrophy postRPT cannot be ignored as a mechanism for im proved muscle torque production during both dynamometric testing as well as during walking. However, though significant skeletal muscle hypertrophy (e.g. larger effector) might suggest improve d torque generation independent of the activation pattern, the magnitude of strength gains would suggest th at the majority of these gains were accounted for by means other than muscle hypertrophy. In this study we chose to examine the morphol ogical and contractile ch aracteristics of the ankle plantar flexor and knee extensor muscle gr oups primarily because of their purported roles during bipedal locomotion. Torque demands at thes e joints during walking are the two highest in

PAGE 120

120 the lower extremity. In addition, we have previo usly shown that torque generation about these joints is limited in persons after incomplete SCI.53 Similarly, subjects in the present study presented with reduced PF and KE peak torque va lues prior to RPT, as well as a reduced gait speeds. Interestingly, marked improvements in PF and KE isometric torq ue generation and gait speed were realized following RPT. However, pos t-RPT measures of peak torque about these joints as well as maximum gait speeds are still reduced relative to control values 53, thereby suggesting the potential for further functional im provements if additional increases in torque production by these muscle groups can be realized. In addition to absolute torque production, a likely mechanism explaining impaired muscle function during locomotor tasks may be an in ability to produce properly graded and timed muscle output. This impairment has been identif ied in this and other populations with central nervous system dysfunction 53,188-190 and shown to relate to reduced gait speed.190 The combination of a prolonged time to peak torque and a decreased ability to generate maximal torque in these persons suggests th at at least some of limitations in gait speed in persons with incomplete SCI might result from impaired muscle function. However, the dramatic improvements in muscle function demonstrated in the present study highli ght the potential for this type of training to attenuate existing deficits in neuromuscular function and facilitate functional improvements. Recent therapeutic interventions examining gait in persons after CNS injury have largely focused on the task specificity of training with little focus on impairment level deficits 151,168,191 Although the rationale for task-specific training in terventions to result in improvements in motor function is quite strong and shown to be e ffective in producing co rtical reorganization 192,193 we feel that in-vivo muscle function is also limiti ng in these persons and appropriate training can

PAGE 121

121 also induce neuroplastic changes in these tissu es that facilitate locomotor improvements by improving the element of muscle function dictated by locomotor task performance. Accordingly, given that few studies have attempted to ex amine the relationship between lower extremity strength and gait in persons after incomplete SCI, comparisons to other populations with CNS involvement yield valuable information. For exam ple, data examining the relative importance of lower extremity strength in persons after stroke demonstrate significant correlations between the strength of the paretic hip flexors (r = .57), knee extensors (r = .41) and primarily the ankle plantar flexors (r = .85), with maximal gait speed.172,194 In addition, previous simulation work suggests that force production by the soleus an d gastrocnemius is critical to trunk forward progression, swing initiation and power generation during gait.159,195 Thus, one might predict slower gait speeds if force production by these muscles is abnormal during locomotion. Indeed, the negative impact of reduced plantar flexor function is suppo rted by experimental data. For example, Lamontagne et al. suggested that more than 50% of the variance in gait speed in persons post-stroke was explained by the peak activation of the medial gastrocnemius. In addition, Mulroy et al. demonstrated that ankle moments were substantially reduced in two groups of hemiparetic persons compared to sl ow walking controls, with household walkers having reduced moments relative to limited community walkers.172 These same investigators also found that at two different time points, walking speed was strongly associated with plantar flexor voluntary strength. Specifically, deficits in plantar flexor strength were pronounced, with the slow subject group (~10% of normal age-matched speed) demonstrating stre ngth equal to ~18% of normal age-matched strength upon admission to re habilitation. Interestingly, at six months post stroke, plantar flexor strengt h increased to 22% of control va lue, an increase of ~20%, and was associated with increased walking speed (~ 20%). Thus, these data provide support to

PAGE 122

122 suggest a relationship may exist between changes in plantar flexor streng th and gait speed, at least at slow velocities. Interestingly, the relati ve gains in plantar flexor strength in the present study (35.0%) are almost identical to the increases in fastest (36.1%) and self-selected (34.7%) gait speeds postRPT. In conclusion, the importance of the proposed work revolves around the fact that little is known about the extent to which skeletal muscle plasticity may impact functional outcomes after incomplete SCI. The desire to be more normal with respect to locomoto r ability is one that many persons after this type of injury possess. Accordingly, the development of appropriate rehabilitation strategies that target improvement s in locomotor ability with the goal of increasing functional independence could have a tremendous impact on this population. The data in the present study provide support for the use of phys ical rehabilitation in terventions aimed at attenuating neuromuscular impairments as a means for improving not only gait speed but also the strategies utilized by these pers ons to ambulate. As such, we s uggest that the benefits reported following a combination of resistan ce and plyometric training represent a first step in the use of these modalities to facilitate the recovery of motor function and functional ability in this population. Although, we report si gnificant gains in strength a nd gait speed following 12 weeks of RPT, at this point we do not know if the subj ects in thus study reached a plateau in any of the outcomes measured. Therefore, future studies ex amining the impact of physical rehabilitation training programs after incomplete SCI should focus on the optimal volume (e.g. duration and frequency) and intensity of training, as well as the potential of this type of training to serve as an adjunctive therapy in the overall treatment of thes e persons. In addition, these studies need not only focus only on gait, but other functional outcomes (e.g. stair climbing, sit to stand) as well as

PAGE 123

123 the potential psychosocial benefits (i.e. community integration) that likely parallel increased functional capacity Table 6-1. Preand post-RPT isometric torque data for the plantar flexor and knee extensor muscle groups. KNEE EXTENSORS Peak Torque T20-80Activation Deficit (%) Peak Torque T 20-80 Activation Deficit (%) M ore-involve d S1 99.8 283.039.0125.7 241.6 31.0 S2 100.3 440.834.0123.8 210.6 25.0 S3 65.1 370.450.081.6 280.4 35.0 L ess-involve d S1 136.4 254.132.0177.6 250.3 29.0 S2 143.9 240.720.0176.1 300.9 14.0 S3 112.5 360.219.0162.7 215.5 18.0 PLANTAR FLEXORS Peak Torque T20-80Activation Deficit (%) Peak Torque T 20-80 Activation Deficit (%) M ore-involve d S1 45.4 807.336.056.1 587.7 28.0 S2 27.3 430.142.036.1 240.1 31.0 S3 17.0 490.941.026.8 280.6 28.0 L ess-involve d S1 56.7 403.218.066.4 252.2 16.0 S2 32.7 380.634.042.8 300.4 15.0 S3 33.2 315.947.058.6 215.4 41.0 P re-RP T P os t -RP T ARTD Torque 200 ARTD Torque 220 282.4 67.5 478.688.9 204.6 44.1 497.471.0 196.1 28.1 244.530.5 482.1 78.2 706.2108.5 501.8 69.7 827.3102.4 330.9 53.8 570.554.2 ARTD Torque 220 ARTD Torque 220 P reR P T P ostR P T 59.1 13.7 95.722.8 50.4 12.1 119.527.5 28.6 5.6 102.99.5 105.2 14.4 259.826.4 95.0 26.7 164.934.4 84.5 12.5 256.116.9

PAGE 124

124 Figure 6-1. Example of pl yometric training device.

PAGE 125

125 CHAPTER 7 LOWER EXTREMITY SKELETAL MUSCLE MORPHOLOGY AND FIBE R TYPE COMPOSITION FOLLOWING MODERATE C ONTUSION SPINAL CORD INJURY AND LOCOMOTOR TRAINING 7.1 Introduction Spinal cord injury (SCI) is a devastati ng condition which causes severe long lasting neurological dysfunction and morbidity in humans.12,52,196 In addition to effects directly related to CNS dysfunction, common problems experienced with SCI are skeletal muscle atrophy and impaired muscle function leading to walking disabilities.45,52,69,197,198 Animal models of SCI are commonly used to evaluate the pathology of SCI a nd to ensure the feasibility and efficacy of new therapeutic interventions. Commonly used an imal models of SCI include transection, isolation, and contusion injuries.199,200 While the transection and isolation models successfully reproduce complete SCI, the contusion model is a clinically more releva nt model as it is known to closely mimic the mechanism and histopathologi c sequela of the majority of current human SCI (>55%, incomplete-SCI), thus making it a relevant model to study.20,200 In contrast to the complete SCIs in which animals experience signif icant atrophy and a complete loss of locomotor capabilities, animals with contusion injury199,201 show some spontaneous r ecovery of muscle size and regain some locomotor function wit hout any specific therapeutic intervention.199,202,203 Locomotor treadmill training has recently gain ed momentum as a therapeutic intervention to improve lower extremity function and walking after SCI. Locomotor training is based on the principle that stepping can be ge nerated by virtue of the neurom uscular systems responsiveness to phasic, peripheral sensory inform ation associated with locomotion.72,79,204,205 Although locomotor treadmill training programs promote changes in spinal cord properties, motor unit morphology, and functional recovery40,198,206,207, the impact of this training intervention towards ameliorating atrophy and improving muscle function after SCI are not cl ear. Currently, a few

PAGE 126

126 studies have looked at skeletal muscle adaptations after contusion SCI and locomotor training. Min et al.2008208 characterized the longitudinal changes in rat lower hindlimb muscle morphology following contusion SCI and locomo tor training by using magnetic resonance imaging over a three month period. The greatest amount of atrophy was obs erved at 2-week postinjury and locomotor training as early one-week post injury significantly reduced atrophy and improved function. In a follow up study, Stevens et al. 200665 evaluated therapeutic potential of early locomotor training in the soleus muscle. Lo comotor training appeared to ameliorate soleus muscle atrophy and attenuate the shift in m yosin heavy composition (MHC) towards faster isoforms. However, this study was limited to the slow postural muscle soleus only and information on other lower extremity muscles is still warranted. Since it is known that different lower extremity muscles adapt differently to unloading conditions ba sed on their specific function role and phenotype, it is important to in vestigate the influence of locomotor training on muscle size and fiber type distribution in muscle s with different functional roles and fiber type composition. The objectives of this study were 1) to qua ntify changes in fiber size and fiber type composition following incomplete SCI in lower extr emity muscles with different functional roles and fiber type composition in the rat 2) to study the therapeutic infl uence of one-week of locomotor training on lower extremity muscle wi th different functional roles and fiber type composition in spinal cord-injured animals. 7.2 Methods 7.2.1 Animals Twenty-four SpragueDawley rats (femal e, 228 g, weighing 250-290gms; Charles River, NJ, USA) were used in this study. Six rats per group were assigned to either a SCItraining group, a SCI-no training group, a contro l group, or a control training group. Six of the

PAGE 127

127 injured rats received treadmill locomotor training (TM) starting 1 week after SCI, when the surgical staples were removed and soft tissue had healed sufficiently to to lerate training without increasing the risk of trauma at the incision si te. Training in the TM group was implemented for 5 consecutive days, 20 min/trial, 2 trials/day. Th e additional 8 injured rats received no exercise intervention (no TM). The rats were housed in a temperature-controlled room at 21 C and were provided unrestricted access to f ood and water. All procedures we re approved by the Institutional Animal Care and Use Committee at the University of Florida. 7.2.2 Contusion Spinal Cord Injury Spinal cord contusion injuries were produced using a prot ocol described previously. A NYU (New York University) impactor was used to produce the injuries. Br iefly, a 10g weight was dropped from a 2.5-cm height onto the T8 se gment of the spinal cord which was exposed by laminectomy. The entire procedure was carried out under sterile conditions. All injuries were performed under ketamine (100mg/kg)-xylazine (6 .7mg/kg) anesthesia. Animals received two doses of Ampicillin (100mg/kg) per day for 5 days starting on the day of surgery. To prevent dehydration, subcutaneous lactated Ringers solution (5 ml) was administered after completion of the surgery. Animals were given Buprenophine (0.05 mg/kg) and Ketoprofen (5.0 mg/kg s.c.) for pain and inflammation over the first 36 hours after SCI. The anim als were kept under vigilant postoperative care, including daily examination for signs of distress, weight loss, dehydration, and bladder dysfunction. Manual expression of bladders was performed 2-3 times daily until spontaneous voiding returned (~2 weeks), and an imals were monitored for the possibility of urinary tract infection. Animals were housed in pairs with the excepti on of the first few hours following surgery.

PAGE 128

1287.2.3 Locomotor Treadmill Training Animals with spinal cord injury were exposed to treadmill locomotor training. Training was started on post-operative day 7. There were two reasons for this First, on day 8 the surgical staples were removed and soft ti ssue had healed sufficiently so th at trauma could be avoided at the incision site. Second, red por phyrin expression around the eyes, a symptom associated with stress, disappeared within a week post SCI. Therefore, animals could be trained without apparent discomfort and stress at this time. Animals assigned to the treadmill training group were given five minutes to explore the treadmill on the first training day and then encouraged to walk on the moving treadmill (11 meter/minute) for a series of four, five-minut e bouts. A minimum of five minutes rest was provided between bouts. On the second day of training, animals completed two bouts of ten minutes each, twice a day. Starting on day 3, an imals trained continuously for 20 minutes with a minimum interval between training sessions of 2 hours. Training consisted of quadrapedal treadmill stepping. Body weight support was prov ided manually by the trainer as necessary. The level of body weight support was adjusted to ma ke sure that the animals hind limbs did not collapse and was gradually removed as locomotor capability improved. Typically, when all rats had profound paraplegia, assistance was provided to place the rat hind paws in plantar stepping position during training. 7.2.4 Tissue Harvest Muscle samples will be harv ested from the normal control ra ts, two weeks post injury on the SCI-no training rats, and one week posttraining on the traine d SCI and trained control rats. The muscles will then be dissected and snap-frozen at resting length in isopentane, pre-cooled in liquid nitrogen and stored at 0C.

PAGE 129

1297.2.5 Immunohistochemical Measures Cryostat sections (10m) in a transverse plan e were prepared from the central portion of the soleus, TA, EDL and Gastroc muscles taken fro m both legs and mounted serially on gelatincoated glass slides. Immunocytochemical reactions were performed on serial cryostat sections with anti laminin and anti-MHC antibody at vari ous dilutions. Rabbit anti-laminin (Neomarker, Labvision, Fremont, CA) was used to outline the muscle fibers for cross-sectional area quantification. Four anti-MHC abs (BA-D5, SC-7 1, BF-F3, and BF-35) were selected on the basis of their reactivity toward adult MHC. Secti ons were incubated with rabbit anti-laminin and one of the anti-MHC antibodies (4C over night), followed by incubation with rhodamineconjugated anti-rabbit IgG and Fitc-conjugated anti-mouse IgG (Nordic Immunological Laboratories, Tilburg, The Nether lands). Stained sections were mounted in mounting medium for fluorescence (Vector Laboratories, Burlingame, CA) and kept at 4C to diminish fading. Stained cross-sections were photographe d (10x magnification) by using a Leica fluorescence microscope (Leica Microsystems, Bannockburn, IL) with a digi tal camera. A region of the stained serial sections from each muscle was randomly selected for MHC composition analysis. The proportions of each fiber type were determined fr om a sample of 150 fibers across the entire section of each muscle. The pixels setting used for conversion of pixels to micrometers were 1.5 pixels to 1 m2 for a 10x objective. The average fiber CSA of all the circle fibers was determined. However, in order to minimize the risk of including nonmuscle tissue, areas consisting of less than 100 pixels were excluded from the analysis. 7.2.6 Data Analysis All statistical analyses were performed with SPSS, Version 13.0.1. Tests for normality will be performed on all of the measured variables before proceeding with tests of statistical inference. Results are expressed as mean standard error of mean One-way ANOVA was used

PAGE 130

130 to test for differences among the four experiment al groups. In an effort to control for multiple comparisons, post-hoc analysis was implemented. For all analyses, significance was established when p< 0.05. 7.3 Results 7.3.1 Effects of IncompleteSCI and Locomoto r Training on Fiber Crossectional Area (CSA) The effect on SCI on fiber size was determined in four different hindlimb muscles-Soleus, Tibialis Anterior (TA), Extensor Digitorum Longus (EDL), and Gastrocnemius (Gastroc). Immunohistochemistry was used to quantify the ch anges in these lower extremity muscless size after contusion SCI. Measurements were made in animals two weeks after SCI (SCI group) and in animals with one week of locomotor traini ng one week after SCI (SCI +locomotor training group). The degree of atrophy in this study seemed not to be muscle phenotype or functional role specific. The slow extensor soleus showed the maximum atrophy followed by the predominantly fast flexor EDL, then the mixed extensor gastrocnemius, and finally the fast flexor TA. Two weeks following SCI, the soleus showed signi ficant reduction in fiber CSA (~29%) in comparison to un-trained controls (p <0.05, Fig. 7-1). Locomotor training lead to a significant increase in soleus CSA in comparison to untra ined SCI group. This change in CSA observed after locomotor training was not si gnificantly different from the c ontrol group. Interestingly, one week of locomotor training in the control rats did not result in any change in fiber CSA. As shown in Figure 7-2, SCI also produced a significant loss in muscle fiber CSA in the EDL in comparison to the control group (~28%, p<0.05). Lo comotor training resulted in significant increase in fiber CSA compared to th e untrained SCI group. Even though the training intervention was only partially effective in re storing fiber CSA toward s control levels, the

PAGE 131

131 difference between the control and SCI + loco motor training groups were not significantly different (p<0.05). Gastrocnemius being an extremely large muscle, the fiber CSA was determined from a sample of 150 fibers located at areas which mostly stained positive for MHC type I. This method was chosen also to help us study fibe r type transformation in the Gastroc muscle following moderate contusion SCI. Two weeks of SCI resulted in si gnificant reduction in average fiber CSA in the predominantly slow Gastroc (~22%, p<0.05). Interestingly, locomotor training resulted in no change in average fibe r CSA in the Gastroc in comparison to the untrained-SCI group (p<0.05). However, we feel our results did not subs tantially justify the influence of locomotor training in restoring fi ber CSA in the Gastroc because our study was specific to areas containing only t ype I MHC fibers and we strongly feel that the training might have significantly influenced the other MHC fiber types (results not reported, Fig.7-3). Finally in the TA muscle, two weeks of contusion SCI resulted in a non-significant ~12.6% decrease in average CSA in comparison to controls,( p<0.05, Fig.7-4). Locomotor training once again resulted in restoring muscle CSA in the TA towards pre-injury levels. No changes in CSA were observed in the control trai ned group compared to the contro l group. Overall, these results indicate atrophy following incomplete-SCI is fibe r type and functional ro le specific with slowextensor showing maximum atr ophy while the fast-flexor showi ng the least amount of atrophy. In addition, locomotor training significantly contri buted in reducing the exte nt of atrophy in all lower extremity muscles except the gastrocn emius in spinally contused-animals. 7.3.2 Effects of IncompleteSCI and Locomoto r Training on Fiber Type Composition The myosin heavy chain (MHC) molecule is an actin-based protein which plays an important role in specifying skeletal muscle c ontractile properties. Therefore, we used MHC staining to identify fiber type composition in animals following SCI and locomotor training.

PAGE 132

132 The soleus muscle from the control untrained animals primarily contained fibers reacting exclusively with type I monoclonal antibody (m AB) (~85%), indicating slow MHC isoforms, and a small percentage of fibers reacting with type IIa mAb, exclusively. Two weeks following moderate T8 contusion SCI, the proportion of ty pe I fibers was reduced by ~10% compared to controls and subsequently the reduction in type I fibers was replaced by fibers that co-expressed both MHC-I and MHC-IIa and IIa and IIx (mixed fibers). Locomotor training prevented the appearance of fibers that co expressed both t ype IIa and IIx in the soleus. In addition the proportion of fibers that were st ained positively with both types I and IIa were lower in the SCItrained animals than the SCI-untrained animals (Fig.7-5). The TA muscle from untrained controls prim arily consisted of fast MHC isoforms (i.e. fibers reacting exclusively with type IIb mAb [~50%], followed by the type IIx [~25%] and IIa [~20%]), and only a small percentage of pure type I fibers (Fig.7-6). In the SCI no training group, the proportion of type IIb fibers were high er by ~15% compared to the controls, while the proportion of IIx and IIa fibers were lower. In addition, the TA from SCI animals also showed mixed fibers that co-expressed both MHC-I a nd MHC-IIa and IIx and IIb, which were not present in the controls. Locomotor training one week post-SCI resulted in the MHC fiber type distribution recovering towards phe notypes represented by control TA muscles. In addition, there were also a higher proportion of mixed fibers which co-expressed type IIx and IIa instead of the faster IIb and IIx as seen in the SCI-untrained group. There wa s no difference in fibers that expressed only type I among the SCI trai ned and untrained a nd control groups. The EDL muscle is a mixed fast muscle containing primarily of fast MHC isoforms. The percentage composition of types I, IIa, IIx and IIb MHC isoforms in the EDL of control rats was ~4, 16, 34, and 45% re spectively (Fig.7-7). Two-weeks of contusion

PAGE 133

133 SCI shifted the MHC profile toward faster isoforms i.e. the type IIb from 45 to 48% and the type IIx from 34-28%. The other changes include the a ppearance of IIa + IIx and IIx + IIb fibers. One-week of locomotor training resulted in a significant decrease of the t ype IIb fibers from 4532% and increase in the type IIa from 1621% in comparison to SCI untrained group. Furthermore, in comparison to the SCI untrain ed group, locomotor training resulted in the reduction of IIx + IIb fibers which seemed to be replaced by an increase in the I + IIa fibers. Even though, there seems to be a slight shift in MHC isoforms af ter training towards the slower isoforms, these data suggest that type IIb is the default MHC isoform in the EDL both after SCI and training, while training seems to have a positive influence in causing some shift in the MHC isoform from fast to slow just one-week following contusion SCI. The gastrocnemius is a muscle which is significantly compartmentalized relative to fiber type composition. In order to study changes in fiber type composition with contusion SCI, we choose to study only areas with the Gastroc which st ained mainly for type I fibers. In the control rats, our regions of choice compromised 54, 28, 15, and 3% pure type I, IIa, IIx and IIb fibers respectively. Following of two weeks of moderate contusion SCI, the MHC isoform distribution was nearly even acr oss groups. There was a ~28% type I, ~20% IIa, ~31%IIx, and ~22% IIb fibers. Interestingl y, there was no appearance of fibers co-expressing two MHC isoforms. However, the trend was different fo llowing one-week of locomotor training one week post-SCI. The type I, IIa, IIx and IIB fibers were approximately 51%, 23%, 17%, and 9%. In summary, the early training intervention just one-week post-SCI seemed to change the expression of MHC in the Gastroc compar able to control levels (Fig.7-8). 7.4 Discussion One of the major problems associated with spinal cord injury (SCI) irrespective of the type of injury is loss of muscle mass as manifested by a reduction in cross-sec tional area (CSA). This

PAGE 134

134 reduction in CSA has been historically accompan ied by fastening of the muscle contractile properties manifested by an increased expression of faster myosin heavy chain (MHC) isoforms.20,58,209-212 However, these adaptations seen vary based on the functional role or fiber type composition of the observed skeletal muscle.20,65,213 To better understand the impact of contusion-SCI on skeletal muscle mass and pheno type, we studied changes in fiber CSA and MHC composition in four lower extremity muscles w ith different functional roles and fiber type compositions. In addition, the therapeutic influen ce of locomotor training was also examined in restoring muscle mass and attenuating the cha nge in MHC composition. The findings of the current study demonstrate that contusion SCI results in significant atrophy in all lower extremity muscles (soleus, extensor digitorum longus, tibia lis anterior and gastrocnemius), and this was accompanied by a shift in MHC composition in all the muscles towards faster isoforms. Interestingly, locomotor training was effectiv e in restoring muscle mass and MHC composition to pre-injury levels. Numerous studies have been conducted in looking at the loss of muscle mass following SCI.63,208,213,214 The majority of these studies have b een performed following spinal transection or spinal isolation were minimal loading or mi nimal muscle activity was recorded following the injury. In a few studies simila r to our study, were changes in muscle mass were quantified twoweeks following spinal transecti on and isolation they observed an atrophy of ~41-50% in the soleus, ~36-49% in the medial Gastroc, ~45% in the TA, and ~40% in the EDL.23,63,215,216 These studies have indicated that muscle adaptive resp onses following transection and isolation SCI are similar in the early stages of atrophy (14-15 days), however fo llowing chronic in activity musclespecific atrophic response is more in the slow-t witch muscles compared to the fast-twitch, and more in the extensors compared to the flexors. 23,63,215,216 In comparison, onl y a few studies have

PAGE 135

135 looked at skeletal muscle mor phology following contusion SCI and results from these studies are conflicting. In the first study by Hutchinson et al. 200120 reported a decrease of 20-25% and 1621% in all lower extremity muscles, at 1 and 3 weeks following moderate contusion SCI. In this study they reported that muscle at rophy occurred in flexor as well as the extensor muscle and that the extent of atrophy was similar in the fast and the slow musc les. In contrast Min et al. 2008208, using MRI observed at 2 weeks post contus ion SCI showed a hier archal pattern of atrophy, with the extensor tricep s surae having more atrophy than flexors muscles. In the current study, at 2-weeks post contusion injury, atr ophy quantified through fiber CSA showed the following hierarchy of atrophy: soleus>EDL>Gas troc>TA. The overall atrophy of ~12-29% was observed in all the lower extremity muscles. Sign ificant difference in fiber CSA after contusion SCI was observed only between the slow-extensor so leus and fast-flexor TA. In summary, in this study we demonstrated that there was signi ficant atrophy in all lower extremity muscles 2 weeks following contusion SCI and the extent of atrophy measured through fiber CSA was maximal in the soleus but similar between a slow -twitch and fast-twitch muscle and also similar between a extensor and flexor. We feel this muscle response may be attributed to the spontaneous recovery and muscle activity observed following the contusion injury. The appearance of the different MHC isoforms in a muscle plays a defining role in regulating the contr actile and histochemical characteristics of the muscle.5,24,209 The maximal velocity of shortening of muscle at least in part is dependant on the MHC composition of the muscle. Research over the years has identified at least four different MHC isoforms being highly expressed in rat muscles. 5,24,209 They have been identified as MHC-I, IIa, IIx and IIb isoforms. Hybrid fibers which coexpress multiple MHC is oforms also exist. Reduction in loading and neuromuscular activity following SCI leads to fa stening of the muscle contractile properties

PAGE 136

136 resulting from an increased expression of faster MHC isoforms.62,213,217,218 There is also an increased expression of hybrid fibers which co-e xpress different MHC isoforms. Findings of our current study are consistent with other studies performed following contusion injury, like studies by Hutchinson et al. 200120 and Stevens et al. 200465 who reported increases in faster MHC isoforms and appearance of hybrid fibers co -expressing different MH C isoforms two weeks following contusion SCI. Specifi cally in our current study, th e soleus had an increased expression of IIx fibers and also the appearance of hybrid fibers expressing faster isoforms, while both the TA and EDL had increases in IIb MHC expression. In the Gastroc the fibers which were predominantly type I shifted to ex pressing equal levels of all MHC isoforms. To summarize, the findings of our study are consistent with those of other studies indicating that 2 weeks post-SCI there is a signifi cant shift in MHC composition in all lower extremity muscles irrespective of functional role or fiber type to switch to faster is oforms. We feel the influence of injury in modulating MHC composi tion is similar across all muscle groups at earlier time points (2-weeks) and this might turn muscle specific at more chronic time points. Motor recovery following spinal cord injury can be enhanced or accelerated by locomotor treadmill training.72,79,204,205 Locomotor training uses the principles showing that rhythmic loading of the limbs and force feedback from the hindlimb muscles induces task appropriate activity-dependent plasticity. Following moderate contusion in ra ts, locomotor training has been shown to induce substantial hind limb muscle and motor recovery.65,114,208 Locomotor training using treadmill has also produced significant impr ovement in locomotor recovery (limb axis, base of support, BBB locomotor scale) compared with those of untrained injured controls. 65,114,208In the current study, we monitored the impact of one-week of locomotor treadmill training on the lower extremity muscles one weekpost mid-thoracic spinal cord contusion injury

PAGE 137

137 by studying changes in fiber CSA and MHC compositi on. In this context our findings are unique and suggest that early locomotor training can be effective in halting the atrophic process and improving the rate of recovery by restoring fibe r CSA and phenotype of lower extremity muscles following contusion SCI. At the end of one week of locomotor training, no significant differences in fiber CSA were noted between the locomotor trained group and the control group in all the lower extremity muscles, except for the gastrocnemius which showed slightly lower CSA values. One possible explanation for the appa rent smaller changes in the Gastroc is that measures for CSA were restricted to only the type I fibers and we feel that the training could have impacted fibers of other phenotypes which are predominant in the Gastroc and if we had averaged CSA across fibers of all the phenotype s we would have had a significant training impact. An interesting finding in this study is ea rly training intervention re sulted in similar rates of recovery in fiber CSA in all lower extremity muscles irrespective of their functional role or fiber type composition. In addition, locomotor training was effective in attenuating the shift MHC composition towards faster is oforms. There was a significant recovery in the proportion of fibers expressing slower isoforms in all the four lower extremity muscles. The restoration of the slow MHC phenotypes following locomotor traini ng may also reflect a potential modulatory decrease in the velocity of shortening in the muscle. In summary, the findings of this study are encouraging because they demonstrate that skeletal muscle atrophy and changes in muscle phenotype following two-weeks contusion SCI are similar across muscles with different functional roles and fiber types and early locomotor training starting one week post-SCI is effec tive in restoring fiber CSA and MHC isoform phenotype irrespective of the muscle functional ro le or fiber type. Alt hough there are limitations in using animal models to unders tand human SCI recovery with locomotor training, the present

PAGE 138

138 study demonstrates that early training interv entions will be effective in ameliorate the debilitating effects of SCI in all the lower extremity muscles.

PAGE 139

139 0 500 1000 1500 2000 2500 3000 SoleusFiber CSA (um2) Control Control+TM SCI SCI+TM Figure 7-1. Average soleus muscle fiber CSA for control, control+TM, SCI no TM, and SCI + TM groups at 2 weeks post SCI. *Significantly smaller average muscle fiber CSA in SCI no TM compared to control, control+TM, and SCI + TM groups, p<0.05. 0 500 1000 1500 2000 2500 Extensor Digitorum LongusFiber CSA (m2) control control+TM SCI SCI+TM Figure 7-2. Average EDL muscle fiber CSA for control, control+TM, SCI no TM, and SCI + TM groups at 2 weeks post SCI. *Significantly smaller average muscle fiber CSA in SCI no TM compared to control, contro l+TM, and SCI + TM groups, p<0.05.

PAGE 140

140 0 500 1000 1500 2000 2500 GastrocnemiusFiber CSA (m2) control control+TM SCI SCI+TM *# Figure 7-3. Average gastrocnemius muscle fibe r CSA for control, control+TM, SCI no TM, and SCI + TM groups at 2 weeks post SCI. *Si gnificantly smaller average muscle fiber CSA in SCI no TM compared to control and control+TM groups, p<0.05. #Significantly smaller average muscle fiber CSA in SCI + TM compared to control and control+TM groups, p<0.05. 0 500 1000 1500 2000 2500 Tibialis AnteriorFiber CSA (m2) control control+TM SCI SCI+TM Figure 7-4. Average TA muscle fiber CSA for control, control+TM, SCI no TM, and SCI + TM groups at 2 weeks post SCI.

PAGE 141

141 0 10 20 30 40 50 60 70 80 90 100 I I +IIaIIaIIa+IIxSoleus Control SCI SCI + TM Figure 7-5. MHC based fiber type composition of rat soleus from control, control+TM, SCI, and SCI+TM groups. 0 10 20 30 40 50 60 70 80 type II+IIaIIaIIa+IIxIIxIIb+IIxIIb Tibialis Anterior% Fiber type distribution control SCI SCI+TM Figure 7-6. MHC based fiber type composition of rat TA from c ontrol, control+TM, SCI, and SCI+TM groups.

PAGE 142

142 0 10 20 30 40 50 60 type II+IIaIIaIIa+IIxIIxIIb+IIxIIb Extensor Digitorum Longus% Fiber type distribution control SCI SCI+TM Figure 7-7. MHC based fiber type composition of rat EDL from c ontrol, control+TM, SCI, and SCI+TM groups. 0 10 20 30 40 50 60 70 type I IIa IIx IIb Gastrocnemius% Fiber type distribution control SCI SCI+TM Figure 7-8. MHC based fiber type composition of rat ga strocnemius from control, control+TM, SCI, and SCI+TM groups.

PAGE 143

143 CHAPTER 8 SKELETAL MUSCLE RECOVERY AND REGENERATI ON FOLLOWING MODERATE CONTUSION SPINAL CORD INJU RY AND LOCOMOTOR TRAINING 8.1 Introduction Incomplete spinal cord injury (SCI) is a debilitating human conditi on resulting in severe motor and sensory impairments below the level of injury.52,196,219 In addition to effects directly related to CNS dysfunction, atrophy of skeletal muscle is a common problem associated with incomplete SCI.45,52,163,206 Animal models of SCI have been used to characterize lesions, study mechanisms of recovery, and to devel op and test therapeutic interventions.14,73,220 Although the majority of current SCIs are incomplete (>55% ), most animal studies of skeletal muscle adaptations after SCI have been done following complete spinal cord injuries.45,93,221 Therefore, it may be relevant to use an animal model of in complete SCI to study skel etal muscle adaptations and the effects of therapeutic in terventions. One such model is the contusion injury model which mimics the mechanism and histopathologic sequela associated with human incomplete SCI. Skeletal muscle possesses a remarkable ability to recover after damage or disuse atrophy. One way skeletal muscle can recover involves the activation, prolif eration, and differentiation of a resident population of myogenic cells called satellite cells.122,133 These satellite cells induce muscle plasticity by differentiating and fusing to form multinucleated myotubes which repair or replace damaged or lost muscle fibers. Activatio n of satellite cells seems to require growth factors, such as insulin-like growth factor 1 (IGF-1), which ha ve also been shown to increase muscle protein and DNA content.122,222,223 Once activated, the upregulat ion of these cells can be identified by using various molecular markers.123,128 Activated satellite cells committed to myogenic lineage express the tran scription factor Pax-7. The m yogenic regulatory factors, MyoD and Myf5 are involved in satellite cell proliferation, and the expr ession of transcription factor myogenin signals satellite cell terminal differentia tion into myotubes. Fina lly, the appearance of

PAGE 144

144 embryonic myosin signals new fiber formation. Desp ite the fact that SCI results in significant muscle atrophy, only a few studies have been done to assess satellite cell activity after SCI, and they have been done only after complete SCI. Fu rthermore, the results of these studies have suggested greater satellite cell activity in slow-twitch extensor muscles than in fast-twitch flexor muscles. Therefore, it may be relevant to study satellite cell activity after incomplete SCI and to do this study in both slow-twitch extensor and fast-twitch flexor muscles. Locomotor treadmill training has been used as a therapeutic intervention to improve lower extremity function and/or walking after SCI. Although locomotor treadmill training programs promote changes in spinal cord proper ties, motor unit morphology, and functional recovery,40,198,206,207 the particular contributions of this therapy towards skeletal muscle plasticity and function are still unclear. Numerous studies have shown that therapeutic interventions like treadmill training, resistance exercise, and cycling training result in the activation of satellite cells.224-226 However, to our knowledge, no studies have been done to assess the effects of short term locomotor training after incomplete SC I on satellite cell activation and regulation. The objectives of this study were 1) to invest igate the effects of incomplete SCI (moderate contusion model) on satellite cell activity in a slow-twitch extensor and a fast-twitch flexor muscle in the rat and 2) to examine the influenc e of one week of locomotor training on satellite cell activity in these muscles in spinal cord-injured animals. Satellite cell activity was monitored by measuring IGF-1 and by using various molecular makers. 8.2 Methods 8.2.1 Animals Twenty-four SpragueDawley rats (femal e, 228 g, weighing 250-290gms; Charles River, NJ, USA) were used in this study. Si x rats per group were assigned to either a SCI-

PAGE 145

145 training group, a SCI-no training group, a contro l group, a control traini ng group. Six of the injured rats received treadmill locomotor training (TM) starting 1 week after SCI, when the surgical staples were removed and soft tissue had healed sufficiently to to lerate training without increasing the risk of trauma at the incision si te. Training in the TM group was implemented for 5 consecutive days, 20 min/trial, 2 trials/day. Th e additional 8 injured rats received no exercise intervention (no TM). The rats were housed in a temperature-controlled room at 21 C and were provided unrestricted access to f ood and water. All procedures we re approved by the Institutional Animal Care and Use Committee at the University of Florida 8.2.2 Contusion Spinal Cord Injury Spinal cord contusion injuries were produced using a prot ocol described previously. A NYU (New York University) impactor was used to produce the injuries. Br iefly, a 10g weight was dropped from a 2.5-cm height onto the T8 se gment of the spinal cord which was exposed by laminectomy. The entire procedure was carried out under sterile conditions. All injuries were performed under ketamine (100mg/kg)-xylazine (6 .7mg/kg) anesthesia. Animals received two doses of Ampicillin (100mg/kg) per day for 5 days starting on the day of surgery. To prevent dehydration, subcutaneous lactated Ringers solution (5 ml) was administered after completion of the surgery. Animals were given Buprenophine (0.05 mg/kg) and Ketoprofen (5.0 mg/kg s.c.) for pain and inflammation over the first 36 hours after SCI. The anim als were kept under vigilant postoperative care, including daily examination for signs of distress, weight loss, dehydration, and bladder dysfunction. Manual expression of bladders was performed 2-3 times daily until spontaneous voiding returned (~2 weeks), and an imals were monitored for the possibility of urinary tract infection. Animals were housed in pairs with the excepti on of the first few hours following surgery.

PAGE 146

146 8.2.3 Locomotor Treadmill Training Animals with spinal cord injury were exposed to treadmill locomotor training. Training was started on post-operative day 7. There were two reasons for this First, on day 8 the surgical staples were removed and soft ti ssue had healed sufficiently so th at trauma could be avoided at the incision site. Second, red por phyrin expression around the eyes, a symptom associated with stress, disappeared within a week post SCI. Therefore, animals could be trained without apparent discomfort and stress at this time. Animals assigned to the treadmill training group were given five minutes to explore the treadmill on the first training day and then encouraged to walk on the moving treadmill (11 meter/minute) for a series of four, five-minut e bouts. A minimum of five minutes rest was provided between bouts. On the second day of training, animals completed two bouts of ten minutes each, twice a day. Starting on day 3, an imals trained continuously for 20 minutes with a minimum interval between training sessions of 2 hours. Training consisted of quadrapedal treadmill stepping. Body weight support was prov ided manually by the trainer as necessary. The level of body weight support was adjusted to ma ke sure that the animals hind limbs did not collapse and was gradually removed as locomotor capability improved. Typically, when all rats had profound paraplegia, assistance was provided to place the rat hind paws in plantar stepping position during training. 8.2.4 Tissue Harvest At the time points indicated above, the soleus and TA muscles of both legs were dissected and snap-frozen at restin g length in isopentane, pre-cooled in liquid nitrogen and stored at 0C.

PAGE 147

1478.2.5 Determination of IGF-I Protein Concentration Frozen soleus and TA muscles were rinsed with PBS to remove excess blood, homogenized in 20 mL of PBS and stored overnight at -200C. The homogenates were then centrifuged for 5 minutes at 5000 x g. The supernatan ts were utilized for measurements of total IGF-1 in a commercially available ELISA kit specific for rodent IGF-I (R&D Systems, Minneapolis, MN). IGF-I concentration was calcula ted based on a standard curve generated from recombinant rat IGF-I. This kit detects total rode nt IGF-I, and the measurements are not affected by the presence of IGF-I binding proteins or IGF-II.227 This kit has been validated for the determination of rat IGF-I at 303000 pg/ml with an intra-assay precision of ~4.3% and an interassay precision o f ~6.0%.227 All samples were measured on a micro-plate reader at 450nm in duplicate. 8.2.6 Immunohistochemistry Measurements Cryostat sections (10 m) in a transverse plan e were prepared from the central portion of each muscle taken from both legs and mounted serially on gelatin-coated glass slides. Immunocytochemical reactions were performed on cryostat sections with anti-laminin and antiPax-7 or anti-embryonic myosin antibody at various dilutions. Rabbit anti-laminin (Neomarker,Labvision, Fremont, CA) was used to outline the muscle fibers. Sections were incubated with rabbit anti-laminin and the anti-P ax-7 (1:300) and anti-embryonic myosin (1:10) antibodies (4C over night), followed by incuba tion with rhodamine-conjugated anti-rabbit IgG and Fitc-conjugated anti-mouse IgG (Nordic Immunological La boratories, Tilburg, The Netherlands). Stained sections were mounted in mounting me dium for fluorescence (Vector Laboratories, Burlingame, CA) and kept at 4C to diminish fading. Stained cross-sections were photographed (10 x magnification) by using a Leica fluorescence microscope (Leica Microsystems, Bannockburn, IL) with a digital camer a. Regions of the stained sections from

PAGE 148

148 each muscle were randomly selected for positiv e pax-7 and embryonic myosin. The proportions of each fiber type were determined from a samp le of 150 fibers across the entire section of each muscle. The pixels setting used for conversion of pixels to micrometers was 1.5 pixels to 1 m2 for a 10 x objective. 8.2.7 Western Blot Analysis Quantification and expression of MyoD, My f5 and Myogenin will be measured using Western blot analysis. Muscles will be homogenized in a lysis buffer with Fast-Prep homogenizer machine at 13,000 RPM at 40C for five minutes. The supernatan t will be preserved for protein assay. Protein will be denatured by heating samples to 95-100 0C for 5 minutes. Protein will be measured using BCA protein assay kit from Pi erce. Electrophoresis will be performed by mixing 40-50 g protein with 5X loading buffer and loading it to 4-15% SDS page gel from Bio-Rad. Protein will then be transferred from gel to nitrocellulose membrane. Blocking will be conducting using 5% non fat dry milk in TBS/T (Tris Buffer Saline, Tween20). Blot with be incubated with primary anti body overnight at 40C according to manufacturers instruction. Blot will then be incubated with HRP-conjugate secondary antibody for 40 minutes to one hour at room temperature. Finally pr otein will be detected using Western Blotting Luminal Reagent from Santa Cruz. 8.2.8 Data Analysis All statistical analyses were performed with SPSS, Version 13.0.1. Tests for normality will be performed on all of the measured variables before proceeding with tests of statistical inference. Results are expressed as mean standard error of mean One-way ANOVA was used to test for differences among the four experiment al groups. In an effort to control for multiple comparisons, post-hoc analysis was implemented. For all analyses, significance was established when p< 0.05.

PAGE 149

1498.3 Results 8.3.1 Effects of Incomplete Spina l cord Injury and Locomoto r Training on Insulin-Like Grow th Factor-1 (IGF-1) Expression Activation and regulation of satellite cells seem s to require IGF-1. Therefore, an enzymelinked immuno sorbent assay (ELISA) was used to quantify IGF-1 levels in the slow-twitch soleus and fast-twitch tibialis anterior (TA) muscles. Measurements were made in animals two weeks after SCI (SCI group) and in animals with one week of lo comotor training one week after SCI (SCI +locomotor training group). The IGF-1 le vels in the soleus muscle were approximately four-fold higher in the SCI group in comparison to the control group (p<0.01) (Fig.8-6A). Locomotor training lead to an approximately 23-fold increase in soleus IGF-1 levels in comparison to the untrained SCI group. SCI or locomotor training did not affect IGF-1 levels in the TA muscle (Fig.8-6B). These results indicate that there are significant increases in IGF-1 protein levels following SCI in the slow-twitch extensor soleus muscle. In addition, locomotor training results in additional increases in soleus IGF-1 protein levels in spinal cord-injured animals. 8.3.2 Effects of Incomplete Spinal Cord In jury and Locomotor Training on Pax-7 Pax-7 is a transcription fact or, and its expression is upregulated in activated and proliferating satellite cells. Th erefore, immunohistochemistry was used to study the frequency of Pax-7-positive myonuclei in transver se sections of the soleus and TA muscles (Figs 8-1 A& B). Although the number of Pax-7-positive myonuclei seem ed to be increased in the soleus and TA muscles two weeks after SCI, these increases were not significant. However, locomotor training lead to an approximately 2-fold increase in Pa x-7 positive-myonuclei in the soleus muscle and an approximately 50% increase in the TA muscle in comparison to the untrained SCI group. These results indicate that locomotor training leads to significant incr eases in Pax-7-positive myonuclei

PAGE 150

150 in both the slowtwitch extensor soleus and fast-twitch flexor TA muscles in spinal cord-injured animals. Furthermore, it is interesting to note that the number of Pax-7-positive fibers in the soleus muscle was almost 2-fold higher compared to the TA muscle in the SCI and locomotor training groups (p<0.05). 8.3.3 Effects of Incomplete Spi nal Cord Injury and Locomotor Training on Myogenic Regulatory Factors (MyoD, Myf5 and Myogenin) Myogenic regulatory factors (M RFs) are known to regulate sa tellite cell activity as the cells pass through the different stages of muscle regeneration. Upon sate llite cell activation, MyoD and Myf5 are thought to be involved in promoting satellite cell proliferation and progression toward terminal differentiation. Ther efore, western blot an alysis was used to quantify MyoD and Myf5 protein levels in the sl ow-twitch soleus and fast-twitch TA muscles. Two weeks after SCI, there was no significant differe nce in the MyoD or Myf5 protein levels in both the soleus and TA muscles in comparison to the control group. Although we saw increases in MyoD and Myf5 protein levels in both musc les following locomotor training in spinal cordinjured animals, the results were not significant. (Fig.8-2 & 3). Myogenin levels are known to be upregulated when satellite cells begin their terminal differentiation program. Therefore, we measured myogenin protei n levels using western blot analysis. Two weeks after SCI, th e myogenin levels in the soleus muscle were approximately 23-fold higher in the SCI group in comparison to the control group (p<0.05) (Fig.8-4A). However, locomotor training did not result in any change in myogenin leve ls in the soleus muscle in comparison to the SCI untrained group. SCI or locomotor training did not affect myogenin protein levels in the TA muscle (Fig.8-4B). Over all, these results show that following SCI soleus myogenin levels were increased, but MyoD and Myf5 levels were not significantly altered. In

PAGE 151

151 addition, locomotor training did not significantly impact any of the MRF protein levels in either of the muscles. 8.3.4 Effects of Incomplete Spi nal Cord Injury and Locomotor Training on Embryonic Myosin Expression of embryonic myosin indicates ne w fiber formation. Therefore we used immunohistochemistry to study the frequency of embryonic myosin-positive muscle fibers in transverse sections of the soleus and TA mu scles (Figs 8-5 A& B). Although the number of embryonic myosin-positive fibers seemed to be increased in the soleus muscle two weeks after SCI, the increase was not significant. However, one week of locomotor training lead to an approximately 3-fold increase in embryonic myosin -positive in the soleus in comparison to the untrained SCI group (p<0.001). In contrast, SCI resulted in a significa nt increase in the embryonic myosin-positive-fibers in the TA muscle, while locomotor training in spinal cordinjured animals had no effect (p<0.01). It is also interesting to not e that the numbers of embryonic myosin positive fibers in the soleus mu scle was almost 6-fold higher compared to the TA in the SCI and locomotor training group (p<0 .05). These results indicate that SCI alone without training results in signi ficant increases in embryonic myosin in the TA, while locomotor training in combination with SCI results in signif icant embryonic myosin levels in the soleus. 8.4 Discussion Atrophy in skeletal muscle has been shown to be associated with a loss of myonuclei independent of the manner the at rophy was induced. Satellite cells once activated proliferate and migrate to the site of muscle fi ber atrophy and then differentiate to either form a new fiber or help repair the damaged fiber122,128.228-230 To better understand rec overy from contusion-SCI induced atrophy and loss of myonucle i, we studied satelli te cell activity in two different muscles, the soleus and the TA. In our current study, we found that there was an in crease in satellite cell

PAGE 152

152 activity following contusion SCI. In addition, locomotor training initiated one-week following contusion-SCI further substantially increased satellite cell activity. Furthermore, we found the increase in satellite cell activity to be different in the slow-extensor soleus compared to the fastflexor tibialis anterior (TA). Satellite cell activation requires the influence of growth factors.122,231,232 In our study, we saw significant increases in IGF-1 levels in the soleus following contusion SCI, which was further significantly increased with locomotor tr aining. However, IGF-1 levels remained stable in the TA muscle. We studied the growth factor IGF-1 as it has been shown to stimulate satellite cell activation, proliferation and diff erentiation in the rat muscle and also to increase myonuclei number and myofiber size.233,234 In addition, exercise results in elevated IGF-I levels, which could result in an increase in sa tellite cell activation and a compensatory hypertrophy of skeletal muscle, thereby making it relevant to study IGF-1 levels following SCI and locomotor training.223,235,236 There are a few studies which involve d SCI and IGF-1. In the first study by Resnick et al.237 2004, IGF-1 levels were up-regulated with in the spinal cord following contusion injury. Even though the regions of IGF-1 measur ement were different in our studies, we felt since IGF-1 presence is systemic in nature our studies related and both st udies reported increased IGF-1 levels following contusion SCI. However, Versteegden et al. 2000238 found no changes in IGF-1 mRNA levels 30 days follo wing transection SCI and cycling training which is different to our results. In retrospect, the authors of the st udy felt that they waited too long to measure IGF-1 levels and transient increase in IGF could have occurred at an earlier tim e point. Interestingly, based on our results we feel the increases in IGF-1 following SCI and locomotor training could have triggered satellite cell activity in the soleus while no significant changes in IGF-1 levels in the TA mirrors the significant lack of satellite cell activity in this muscle.

PAGE 153

153 In the present study, SCI resu lted in stimulating similar increases in pax-7-positive myonuclei in both soleus and TA muscles. In ad dition, locomotor training lead to a further substantial increase in pax-7-positive myonuclei in both soleus a nd TA, with the soleus almost having the twice the number of Pax-7-positive myonuclei compar ed to the TA. Pax-7 was quantified in the current study because act ivated satellite ce lls express Pax7. 125,229Most activated satellite cells then prolifer ate, thereby down regulating Pax7 and then differentiate.125,239,240 Furthermore, treadmill training is known to stimul ate satellite cell activity in skeletal muscle, thereby making it relevant to study Pax-7 activit y following SCI and locomotor training. In our results, the soleus and TA had similar levels of Pax7 following contusion SCI. These results are different from that seen in ot her types of SCI like isolation we re they found the slow twitch muscle to have higher satellite cell expression compared to the fast twitch.216 We however feel the contusion SCI presenting itself with spared spinal trac ts and varied activation levels could be the reason for the similar expression of Pax-7 in both slow and fast muscle. However, locomotor training following SCI resulted in twice the number of Pax-7 positives compared to the TA. The reason for this may be due to differences in muscle activity, with the slow soleus being 20-times more active and frequently recruited than the fast flexor TA muscle during locomotor tasks.241 To summarize, both SCI and locomotor training resulted in activating satellite cells in both the soleus and TA, with the soleus having higher leve ls of satellite cell activ ation compared to the TA due to its higher recru itment in loading conditions. Surprisingly in this study we did not find any significant changes in myogenic regulatory factor (MRF) protein levels in both muscles following SCI or SCI + locomotor training except for myogenin in the soleus. Myogenin protein levels in the soleus were significantly elevated following SCI and SCI + locomotor training in comparison to c ontrols. We studied MRF

PAGE 154

154 proteins as they are transcription factors th at influence and modulate the proliferation and differentiation of the satellite cells.23,113,122,216,238 Specifically, Myogenin is a MRF protein that regulates the terminal differentiation of satell ite cells to myoblasts. Based on our myogenin results we suggest that following SCI and locomotor training one-week-post SCI leads to significant terminal differentiation of satellite cells into myoblasts.128,242 However there was no difference in myogenin levels betw een the SCI trained and un-train ed group. These results were similar to a study by Versteegden et al. 1999113, who observed similar si gnificant increases in soleus myogenin expression 10 days after transe ction SCI and cycling tr aining 5 days post-SCI with no differences in myogenin levels betw een the trained and untrained SCI groups. We suggest it could be because myogenin proteins levels may have reached their maximum levels following SCI and the training does not cause any further increase. In summary, we feel that even though there were no increases in MyoD a nd Myf5 protein levels, significant increases in myogenin following SCI and SCI + locomotor trai ning indicate terminal differentiation of satellite cells only in the soleus. Although after SCI there were a few embryonic m yosin positive muscle fibers in the both the soleus and TA compared to no positives in the controls, their numbers were very minimal. However following locomotor training we noticed significant increases in embryonic myosin numbers in the soleus muscle after locomotor training, while in the TA the numbers remained insignificant. In the present study we quantified the developmental isoform of the myosin heavy chains termed as embryonic myosin because it se quentially precedes the appearance of definitive adult myosin heavy chains in rats and is an indicator of new fiber formation.63,91,213 Based on our results we can suggest that locomotor training following SCI resulted in new fiber formation in the soleus muscle. A point of interest in th is study which is similar to a study by Yablonka-

PAGE 155

155 Reuveni et al. 1994 239is that the numbers of embryonic myos in positive cells in our study were less than half the number of positive satellite ce lls, indicating that not all the satellite cell descendants entered the phase of terminal differentiation, sugge sting muscle plasticity through regeneration does directly rela ted to activated satellite cell numbers especially following contusion SCI. So in conclusion were satel lite cells involved in the exer cise induced maintenance of muscle fiber size following contusion SCI? Even though there are lim itations in study and alternate theories, we suggest that satellite cell ac tivation to form new fibers could be one of the pathways in which the soleus recovers after co ntusion SCI and locomotor training. One of the limitations of the study was that MRF levels of MyoD and Myf5 which indicate satellite cell proliferation did not significan tly change in both the SCI and locomotor training group in both muscles. A plausible explanation we feel is that these proteins are transiently expressed, and the time points we used only provide snapshots of sate llite cells or MRF activity through their entire cycle and hence we might have missed the expres sion of these proteins. We also have a few suggestions regarding the lack of satellite cell activity in the fast TA. First, there was very less atrophy in the TA to start of with and hence there was less muscle plasticity required to recover from the atrophy. Also, in a fast muscle like the TA, myonuclear numbe r is significantly high and therefore satellite cells may not have been required to restore myonuclear levels. In summary, atrophy following contusion SCI may be associated with myonuclear loss. Increase in satellite cell activity and new fiber formation might be potential mechanisms to compensate for atrophy and myonuclear loss in the soleus and locomotor training might accelerate the recovery of the soleus muscle th rough muscle regeneration as a response to increased activity, while in the TA; it might be an order of events which need further

PAGE 156

156 investigation. Overall th is study provides more information on the exercise induced contribution of satellite cells towards muscle plasticity thr ough regeneration after mode rate contusion SCI.

PAGE 157

157 0 5 10 15 20 25 30 Soleusn/100 fibers CON CON+TM SCI SCI+TM A 0 2 4 6 8 10 12 14 16Tibialis Anteriorn/100 fibers CON CON+TM SCI SCI+TM B Figure 8-1 Pax-7 staining for the (A) so leus and (B) tibialis anterior muscle. Significant difference between SCI+TM group fr om the other groups (p<0.05)

PAGE 158

158 Soleus0 0.2 0.4 0.6 0.8 1 1.2controlcontrol+TMSCISCI+TMMyoD/GAPDH rati o control control+TM SCI SCI+ T M A TA0 0.2 0.4 0.6 0.8 1 1.2 1.4controlcontrol+TMSCISCI+TMMyoD/GAPDH rati o control control+TM SCI SCI+ T M B Figure 8-2 MyoD protein levels in the (A) soleus and (B) TA muscle.

PAGE 159

159 Soleus0 0.2 0.4 0.6 0.8 1 1.2 1.4controlcontrol+TMSCISCI+TMMyf5/GAPDH ratio control control+TM SCI SCI+ T M A TA0 0.2 0.4 0.6 0.8 1 1.2 1.4controlcontrol+TMSCISCI+TMMyf5/GAPDH ratio control control+TM SCI SCI+ T M B Figure 8-3 Myf5 protein levels in the (A) soleus and (B) TA muscle.

PAGE 160

160 Soleus0 0.2 0.4 0.6 0.8 1 1.2 1.4controlcontrol+TMSCISCI+TMMyogenin/GAPDH rati o control control+TM SCI SCI+ T M *# A TA0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6controlcontrol+TMSCISCI+TMMyogenin/GAPDH rati o control control+TM SCI SCI+ T M B Figure 8-4 Myogenin protein in the (A) soleus and (B) TA muscle.* Significant difference between the SCI no training group and the control groups (p<0.05). # Significant difference between SCI+TM group fr om the other groups (p<0.05).

PAGE 161

161 0 1 2 3 4 5 6 7 8 91Soleusn/100 fibers CON CON+TM SCI SCI+TM A 0 0.2 0.4 0.6 0.8 1 1.2 Tibilais Anteriorn/100 fibers SCI+TM SCI control control + TM B Figure 8-5 Embryonic myosin positives. (A) soleus and (B) TA muscle.* Significant difference between the SCI group and th e other groups (p<0.05).

PAGE 162

162 Soleus (IGF-1 )0 20 40 60 80 100 120controlcontrol+TMSCI SCI+TMng/g control control+TM SCI SCI+TM *# A TA (IGF-1)-10 5 20 35 50controlcontrol+TM SCI SCI+TMng/g control control+TM SCI SCI+TM B Figure 8-6 IGF-1 levels. (A) soleus and (B) TA muscle. Significant difference between the SCI no training group and the control groups. # Significant difference between SCI+TM group from the other groups (p<0.05).

PAGE 163

163 LIST OF REFERENCES 1. www.spinalcord.uab.edu (National Spinal Cord Injury Datab ase), accessed April 2007. 2. http://www.ninds.nih.gov (National Institute of Neur ological Disorders and Stroke), accessed April 2007. 3. Fuller KS. Traumatic spinal cord injury. Pathology for Physical Therapists 2003; 10861097. 4. Somers MF. Spinal cord injury: Functional Rehabilitation 2001. 5. Lieber RL. Skeletal muscle adaptability II: Muscle properties following spinal-cord injury. Dev Med Child Neurol 1986; 28: 533-542. 6. Dittuno PL, Dittuno Jr JF, Jr. Walking index for spinal cord injury (WISCI II): scale revision. Spinal Cord 2001; 39: 654-656. 7. Mahoney FI, Barthel DW. Functional evaluation: the barthel index. Md State Med J 1965; 14: 61-65. 8. Collen FM, Wade DT, Robb GF, Bradshaw CM. The Rivermead mobility index: a further development of the rivermead motor assessment. Int Disabil Stud 1991; 13: 5054. 9. Keith RA, Granger CV, Hamilton BB, Sherwin FS. The functional independence measure: a new tool for rehabilitation. Adv Clin Rehabil 1987; 1: 6-18. 10. Catz A, Itzkovich M, Agranov E, Ring H, Tamir A. SCIM--spinal cord independence measure: a new disability scale for patients with spinal cord lesions. Spinal Cord 1997; 35: 850-856. 11. Itzkovich M, Tamir A, Philo O, Steinberg F, Ronen J, Spasser R, Gepstein R, Ring H, Catz A. reliability of the catz-itzkovich sp inal cord independence measure assessment by interview and comparison with observation. Am J Phys Med Rehabil 2003; 82: 267-272. 12. Alaimo MA, Smith JL, Roy RR, Edgerton VR. EMG activity of slow and fast ankle extensors following spinal cord transection. J Appl Physiol 1984; 56: 1608-1613. 13. Albin MS, White RJ, Yashon D, Massopust LC, Jr. Functional and electrophysiologic limitations of delayed spinal cord cooling after impact injury. Surg Forum 1968; 19: 423424. 14. de Leon RD, Hodgson JA, Roy RR, Edgerton VR. Locomotor capacity attributable to step training versus spontaneous recove ry after spinalization in adult cats. J Neurophysiol 1998; 79: 1329-1340.

PAGE 164

164 15. de Leon RD, London NJ, Roy RR, Edgerton VR Failure analysis of stepping in adult spinal cats. Prog Brain Res 1999; 123: 341-348. 16. Edgerton VR, Roy RR, Hodgson JA, Prober RJ, de Guzman CP, de Leon R. Potential of adult mammalian lumbosacral spinal cord to execute and acquire improved locomotion in the absence of supraspinal input. J Neurotrauma 1992; 9 Suppl 1: S119-128. 17. Gregory CM, Vandenborne K, Castro MJ, D udley GA. Human and rat skeletal muscle adaptations to spinal cord injury. Can J Appl Physiol 2003; 28: 491-500. 18. Gruner JA. A monitored contusion model of spinal cord injury in the rat. J Neurotrauma 1992; 9: 123-126; discussion 126-128. 19. Houle JD, Morris K, Skinner RD, Garcia-Rill E, Peterson CA. Effects of fetal spinal cord tissue transplants and cycling exercise on the soleus muscle in spinalized rats. Muscle Nerve 1999; 22: 846-856. 20. Hutchinson KJ, Linderman JK, Basso DM. Sk eletal muscle adaptations following spinal cord contusion injury in rat and the relati onship to locomotor function: a time course study. J Neurotrauma 2001; 18: 1075-1089. 21. Eldridge L, Liebhold M, Steinbach JH. Alterations in cat skeletal neuromuscular junctions following prolonged inactivity. J Physiol 1981; 313: 529-545. 22. Pierotti DJ, Roy RR, Bodi ne-Fowler SC, Hodgson JA, Edgerton VR. Mechanical and morphological properties of chronically inac tive cat tibialis anterior motor units. J Physiol 1991; 444: 175-192. 23. Hyatt JP, Roy RR, Baldwin KM, Edgerton VR. Nerve activity-independent regulation of skeletal muscle atrophy: role of MyoD and myogenin in satellite cells and myonuclei. Am J Physiol Cell Physiol 2003; 285: C1161-1173. 24. Lieber RL, Friden JO, Hargens AR, Feri nga ER. Long-term effects of spinal cord transection on fast and slow rat skeletal muscle. II. Morphometric properties. Exp Neurol 1986; 91: 435-448. 25. Lieber RL, Johansson CB, Vahlsing HL, Ha rgens AR, Feringa ER. Long-term effects of spinal cord transection on fast and slow rat skeletal muscle. I. Contractile properties. Exp Neurol 1986; 91: 423-434. 26. Roy RR, Acosta L, Jr. Fibe r type and fiber size changes in selected thigh muscles six months after low thoracic spinal cord tran section in adult cats: exercise effects. Exp Neurol 1986; 92: 675-685. 27. Kwon BK, Oxland TR, Tetzlaff W. Animal mo dels used in spinal cord regeneration research. Spine 2002; 27: 1504-1510. 28. Taoka Y, Okajima K. Spinal cord injury in the rat. Prog Neurobiol 1998; 56: 341-358.

PAGE 165

165 29. Albin MS, White RJ, Acosta-Rua G, Yashon D. Study of functiona l recovery produced by delayed localized coo ling after spinal cord injury in primates. J Neurosurg 1968; 29: 113-120. 30. Koozekanani SH, Vise WM, Hashemi RM, McGhee RB. Possible mechanisms for observed pathophysiological variability in expe rimental spinal cord injury by the method of Allen. J Neurosurg 1976; 44: 429-434. 31. Parker AJ, Smith CW. Functional recove ry from spinal cord trauma following dexamethazone and chlorpromazine therapy in dogs. Res Vet Sci 1976; 21: 246-247. 32. Basso DM, Beattie MS, Bresnahan JC, A nderson DK, Faden AI, Gr uner JA, Holford TR, Hsu CY, Noble LJ, Nockels R, Perot PL, Sa lzman SK, Young W. MASCIS evaluation of open field locomotor scores: effects of experience and teamwork on reliability. Multicenter Animal Spinal Cord Injury Study. J Neurotrauma 1996; 13: 343-359. 33. Bresnahan JC, Beattie MS, Stokes BT, Conway KM. Three-dimensional computerassisted analysis of graded contusion le sions in the spinal cord of the rat. J Neurotrauma 1991; 8: 91-101. 34. Guizar-Sahagun G, Grijalva I, Madrazo I, Fr anco-Bourland R, Salgado H, Ibarra A, Oliva E, Zepeda A. Development of post-traumatic cysts in the spinal cord of rats-subjected to severe spinal cord contusion. Surg Neurol 1994; 41: 241-249. 35. Noble LJ, Wrathall JR. Spinal cord contusion in the rat: morphometric analyses of alterations in the spinal cord. Exp Neurol 1985; 88: 135-149. 36. Osterholm JL, Mathews GJ. Treatment of se vere spinal cord injuries by biochemical norepinephrine manipulation. Surg Forum 1971; 22: 415-417. 37. Shah PK, Stevens JE, Gregory CM, Pathare NC, Jayaraman A, Bickel SC, Bowden M, Behrman AL, Walter GA, Dudley GA, Vanden borne K. Lower-extremity muscle crosssectional area after incomplete spinal cord injury. Arch Phys Med Rehabil 2006; 87: 772778. 38. Adams MM, Ditor DS, Tarnopolsky MA, Phillips SM, McCartney N, Hicks AL. The effect of body weight-supported treadm ill training on muscle morphology in an individual with chronic, motor-complete spinal cord injury: A case study. J Spinal Cord Med 2006; 29: 167-171. 39. Stewart BG, Tarnopolsky MA, Hicks AL McCartney N, Mahoney DJ, Staron RS, Phillips SM. Treadmill training-i nduced adaptations in muscle phenotype in persons with incomplete spinal cord injury. Muscle Nerve 2004; 30: 61-68. 40. Edgerton VR, Leon RD, Harkema SJ, H odgson JA, London N, Reinkensmeyer DJ, Roy RR, Talmadge RJ, Tillakaratne NJ, Timoszyk W, Tobin A. Retraining the injured spinal cord. J Physiol 2001; 533: 15-22.

PAGE 166

166 41. Martin TP, Stein RB, Hoeppner PH, Reid DC Influence of electrical stimulation on the morphological and metabolic properties of paralyzed muscle. J Appl Physiol 1992; 72: 1401-1406. 42. Rochester L, Chandler CS, Johnson MA, Sutton RA, Miller S. Influence of electrical stimulation of the tibialis anterior muscle in paraplegic subjects. 1. Contractile properties. Paraplegia 1995; 33: 437-449. 43. Reeves ND, Maganaris CN, Narici MV. U ltrasonographic assessmen t of human skeletal muscle size. Eur J Appl Physiol 2004; 91: 116-118. 44. Engstrom CM, Loeb GE, Reid JG, Forrest WJ, Avruch L. Morphometry of the human thigh muscles. A comparison between anatom ical sections and computer tomographic and magnetic resonance images. J Anat 1991; 176: 139-156. 45. Dudley GA, Castro MJ, Rogers S, Apple DF, Jr. A simple means of increasing muscle size after spinal cord injury: a pilot study. Eur J Appl Physiol Occup Physiol 1999; 80: 394-396. 46. Castro MJ, Apple DF, Jr., Staron RS, Cam pos GE, Dudley GA. Influence of complete spinal cord injury on skeletal muscle within 6 mo of injury. J Appl Physiol 1999; 86: 350-358. 47. Mahoney ET, Bickel CS, Elder C, Black C, Slade JM, Apple D, Jr., Dudley GA. Changes in skeletal muscle size and glucose toleran ce with electrically stimulated resistance training in subjects with ch ronic spinal cord injury. Arch Phys Med Rehabil 2005; 86: 1502-1504. 48. Talmadge RJ, Castro MJ, Apple DF, Jr., Dudley GA. Phenotypic adaptations in human muscle fibers 6 and 24 wk after spinal cord injury. J Appl Physiol 2002; 92: 147-154. 49. Castro MJ, Apple DF, Jr., Rogers S, Dudley GA. Influence of complete spinal cord injury on skeletal muscle mechanics within the first 6 months of injury. Eur J Appl Physiol 2000; 81: 128-131. 50. Gerrits HL, De Haan A, Hopman MT, van Der Woude LH, Jones DA, Sargeant AJ. Contractile properties of the qua driceps muscle in individuals with spinal cord injury. Muscle Nerve 1999; 22: 1249-1256. 51. Gerrits HL, Hopman MT, Sargeant AJ, de Haan A. Reproducibility of contractile properties of the human paralysed and non-paralysed quadriceps muscle. Clin Physiol 2001; 21: 105-113. 52. Shields RK. Muscular, skeletal, and neural adaptations following spinal cord injury. J Orthop Sports Phys Ther 2002; 32: 65-74.

PAGE 167

167 53. Jayaraman A, Gregory CM, Bowden M, St evens JE, Shah P, Behrman AL, Vandenborne K. Lower extremity skeletal muscle function in persons with incomplete spinal cord injury. Spinal Cord 2006; 44: 680-687. 54. Noreau L, Vachon J. Comparison of thr ee methods to assess muscular strength in individuals with spinal cord injury. Spinal Cord 1998; 36: 716-723. 55. Schwartz S, Cohen ME, Herbison GJ, Shah A. Relationship between two measures of upper extremity strength: manual muscle te st compared to hand-held myometry. Arch Phys Med Rehabil 1992; 73: 1063-1068. 56. Drolet M, Noreau L, Vachon J, Moffet H. Muscle strength changes as measured by dynamometry following functional rehabilitation in individuals with spinal cord injury. Arch Phys Med Rehabil 1999; 80: 791-800. 57. West SP, Roy RR, Edgerton VR. Fiber type and fiber size of cat ankle, knee, and hip extensors and flexors following low thoracic sp inal cord transection at an early age. Exp Neurol 1986; 91: 174-182. 58. Roy RR, Pierotti DJ, Flores V, Rudolph W, Edgerton VR. Fi bre size and type adaptations to spinal isolation and cyclical passive stretch in cat hindlimb. J Anat 1992; 180 ( Pt 3): 491-499. 59. Termin A, Staron RS, Pette D. Myosin hea vy chain isoforms in histochemically defined fiber types of rat muscle. Histochemistry 1989; 92: 453-457. 60. Peterson CA, Murphy RJ, Dupont-Versteegden EE, Houle JD. Cycling exercise and fetal spinal cord transplantation act synergisti cally on atrophied muscle following chronic spinal cord injury in rats. Neurorehabil Neural Repair 2000; 14: 85-91. 61. Talmadge RJ, Roy RR, Edgerton VR. Persiste nce of hybrid fibers in rat soleus after spinal cord transection. Anat Rec 1999; 255: 188-201. 62. Talmadge RJ, Roy RR, Chalmers GR, E dgerton VR. MHC and sarcoplasmic reticulum protein isoforms in functionally overl oaded cat plantaris muscle fibers. J Appl Physiol 1996; 80: 1296-1303. 63. Zhong H, Roy RR, Woo J, Kim JA, Edgert on VR. Differential m odulation of myosin heavy chain phenotype in an inactive extens or and flexor muscle of adult rats. J Anat 2007; 210: 19-31. 64. Roy RR, Sacks RD, Baldwin KM, Short M, Edgerton VR. Interrelationships of contraction time, Vmax, and myosin ATPase after spinal transection. J Appl Physiol 1984; 56: 1594-1601. 65. Stevens JE, Liu M, Bose P, O'Steen WA, Thompson FJ, Anderson DK, Vandenborne K. Changes in soleus muscle function and fiber morphology with one week of locomotor training in spinal cord contusion injured rats. J Neurotrauma 2006; 23: 1671-1681.

PAGE 168

168 66. Barbeau H, Wainberg M, Finch L. Descrip tion and application of a system for locomotor rehabilitation. Med Biol Eng Comput 1987; 25: 341-344. 67. Visintin M, Barbeau H. The effects of parallel bars, body weight support and speed on the modulation of the locomotor pattern of spastic paretic gait. A preliminary communication. Paraplegia 1994; 32: 540-553. 68. Wolpaw JR, Tennissen AM. Activity-dependent spinal cord plasticity in health and disease. Annu Rev Neurosci 2001; 24: 807-843. 69. Edgerton VR, Tillakaratne NJ, Bigbee AJ, de Leon RD, Roy RR. Plasticity of the spinal neural circuitry after injury. Annu Rev Neurosci 2004; 27: 145-167. 70. Rossignol S, Brustein E, Bouyer L, Barthelemy D, Langlet C, Leblond H. Adaptive changes of locomotion after cen tral and peripheral lesions. Can J Physiol Pharmacol 2004; 82: 617-627. 71. Dietz V, Harkema SJ. Locomotor activity in spinal cord-injured persons. J Appl Physiol 2004; 96: 1954-1960. 72. De Leon RD, Hodgson JA, Roy RR, Edgerton VR. Retention of hindlimb stepping ability in adult spinal cats after th e cessation of step training. J Neurophysiol 1999; 81: 85-94. 73. Lovely RG, Gregor RJ, Roy RR, Edgerton VR. Weight-bearing hindlimb stepping in treadmill-exercised adult spinal cats. Brain Res 1990; 514: 206-218. 74. Chau C, Barbeau H, Rossignol S. Early loco motor training with clon idine in spinal cats. J Neurophysiol 1998; 79: 392-409. 75. Barbeau H, Basso M, Behrman A, Harkema S. Treadmill training after spinal cord injury: good but not better. Neurology 2006; 67: 1900-1901; author reply 1901-1902. 76. Barbeau H, McCrea DA, O'Donovan MJ, Rossignol S, Grill WM, Lemay MA. Tapping into spinal circuits to restore motor function. Brain Res Brain Res Rev 1999; 30: 27-51. 77. Dobkin B, Barbeau H, Deforge D, Ditunno J, Elashoff R, Apple D, Basso M, Behrman A, Harkema S, Saulino M, Scott M. The evol ution of walking-rela ted outcomes over the first 12 weeks of rehabilitation for incomplete traumatic spinal cord injury: the multicenter randomized Spinal Co rd Injury Locomotor Trial. Neurorehabil Neural Repair 2007; 21: 25-35. 78. Wernig A, Nanassy A, Muller S. Maintena nce of locomotor abiliti es following Laufband (treadmill) therapy in paraand tetraplegic persons: follow-up studies. Spinal Cord 1998; 36: 744-749. 79. Behrman AL, Harkema SJ. Loco motor training after human spinal cord injury: a series of case studies. Phys Ther 2000; 80: 688-700.

PAGE 169

169 80. Dobkin B, Apple D, Barbeau H, Basso M, Behrman A, Deforge D, Ditunno J, Dudley G, Elashoff R, Fugate L, Harkema S, Saulino M, Scott M. Weight-supported treadmill vs over-ground training for walking after acute incomplete SCI. Neurology 2006; 66: 484493. 81. Shurrager PS, Dykman RA. Walking spinal carnivores. J Comp Physiol Psychol 1951; 44: 252-262. 82. Gordon T, Mao J. Muscle atrophy and proced ures for training after spinal cord injury. Phys Ther 1994; 74: 50-60. 83. Stein RB. Functional electrical stim ulation after spinal cord injury. J Neurotrauma 1999; 16: 713-717. 84. Scremin AM, Kurta L, Gentili A, Wiseman B, Perell K, Kunkel C, Scremin OU. Increasing muscle mass in spinal cord injured persons with a functional electrical stimulation exercise program. Arch Phys Med Rehabil 1999; 80: 1531-1536. 85. Stein RB, Chong SL, James KB, Kido A, Be ll GJ, Tubman LA, Belanger M. Electrical stimulation for therapy and mobility after spinal cord injury. Prog Brain Res 2002; 137: 27-34. 86. Gerrits HL, Hopman MT, Sargeant AJ, Jones DA, De Haan A. Effects of training on contractile properties of pa ralyzed quadriceps muscle. Muscle Nerve 2002; 25: 559-567. 87. Crameri RM, Weston AR, Rutkowski S, Mi ddleton JW, Davis GM, Sutton JR. Effects of electrical stimulation leg trai ning during the acute phase of spinal cord injury: a pilot study. Eur J Appl Physiol 2000; 83: 409-415. 88. Shields RK, Dudley-Javoroski S. Musculoskele tal plasticity after acute spinal cord injury: effects of long-term neuromuscular electrical stimulation training. J Neurophysiol 2006; 95: 2380-2390. 89. Harridge SD, Andersen JL, Hartkopp A, Zhou S, Biering-Sorensen F, Sandri C, Kjaer M. Training by low-frequency stimul ation of tibialis anterior in spinal cord-injured men. Muscle Nerve 2002; 25: 685-694. 90. Bajd T, Kralj A, Stefancic M, Lavrac N. Us e of functional electrical stimulation in the lower extremities of incomplete spinal cord injured patients. Artif Organs 1999; 23: 403409. 91. Modlin M, Forstner C, Hofer C, Mayr W, Richter W, Carraro U, Protasi F, Kern H. Electrical stimulation of dene rvated muscles: first results of a clinical study. Artif Organs 2005; 29: 203-206. 92. Ragnarsson KT, Pollack S, O'Daniel W, Jr ., Edgar R, Petrofsky J, Nash MS. Clinical evaluation of computerized functional electrical stimulation after spinal cord injury: a multicenter pilot study. Arch Phys Med Rehabil 1988; 69: 672-677.

PAGE 170

170 93. Baldi JC, Jackson RD, Moraille R, Mysiw WJ. Muscle atrophy is prevented in patients with acute spinal cord injury using functional electrical stimulation. Spinal Cord 1998; 36: 463-469. 94. Crameri RM, Weston A, Climstein M, Da vis GM, Sutton JR. Effects of electrical stimulation-induced leg training on skeletal mu scle adaptability in spinal cord injury. Scand J Med Sci Sports 2002; 12: 316-322. 95. Postans NJ, Hasler JP, Granat MH, Maxw ell DJ. Functional electric stimulation to augment partial weight-bearing supported tr eadmill training for patients with acute incomplete spinal cord injury: A pilot study. Arch Phys Med Rehabil 2004; 85: 604-610. 96. Mohr T, Andersen JL, Biering-Sorensen F, Galbo H, Bangsbo J, Wagner A, Kjaer M. Long-term adaptation to electri cally induced cycle training in severe spinal cord injured individuals. Spinal Cord 1997; 35: 1-16. 97. Andersen JL, Mohr T, Biering-Sorensen F, Galbo H, Kjaer M. Myosin heavy chain isoform transformation in single fibres from m. vastus lateralis in spinal cord injured individuals: effects of long-term functional electri cal stimulation (FES). Pflugers Arch 1996; 431: 513-518. 98. Gerrits HL, de Haan A, Sargeant AJ, Da llmeijer A, Hopman MT. Altered contractile properties of the quadriceps muscle in people with spinal cord injury following functional electrical stimulated cycle training. Spinal Cord 2000; 38: 214-223. 99. Chilibeck PD, Syrotuik DG, Bell GJ. The e ffect of strength training on estimates of mitochondrial density and distri bution throughout muscle fibres. Eur J Appl Physiol Occup Physiol 1999; 80: 604-609. 100. Nash MS, Jacobs PL, Montalvo BM, Klos e KJ, Guest RS, Needham-Shropshire BM. Evaluation of a training program for persons with SCI paraplegia using the Parastep 1 ambulation system: part 5. Lower extrem ity blood flow and hyperemic responses to occlusion are augmented by ambulation training. Arch Phys Med Rehabil 1997; 78: 808814. 101. van der Ploeg HP, van der Beek AJ, van der Woude LH, van Mechelen W. Physical activity for people with a disa bility: a conceptual model. Sports Med 2004; 34: 639-649. 102. Kjaer M. Why exercise in paraplegia? Br J Sports Med 2000; 34: 322-323. 103. Gellman H, Sie I, Waters RL. Late comp lications of the weight-bearing upper extremity in the paraplegic patient. Clin Orthop Relat Res 1988; 132-135. 104. Silfverskiold J, Waters RL. Shoulder pain and functional disability in spinal cord injury patients. Clin Orthop Relat Res 1991; 141-145. 105. Nilsson S, Staff PH, Pruett ED. Physical work capacity and the effect of training on subjects with long-standing paraplegia. Scand J Rehabil Med 1975; 7: 51-56.

PAGE 171

171 106. Cooney MM, Walker JB. Hydrau lic resistance exercise benefits cardiovascular fitness of spinal cord injured. Med Sci Sports Exerc 1986; 18: 522-525. 107. Dallmeijer AJ, Hopman MT, van As HH, van der Woude LH. Physical capacity and physical strain in persons with tetraplegia; the role of sport activity. Spinal Cord 1996; 34: 729-735. 108. Janssen TW, van Oers CA, Hollander AP, Veeger HE, van der Woude LH. Isometric strength, sprint power, and aerobic power in individuals with a spinal cord injury. Med Sci Sports Exerc 1993; 25: 863-870. 109. Giangregorio LM, Webber CE, Phillips SM, Hicks AL, Craven BC, Bugaresti JM, McCartney N. Can body weight supporte d treadmill training increase bone mass and reverse muscle atrophy in individuals with chronic incomplete spinal cord injury? Appl Physiol Nutr Metab 2006; 31: 283-291. 110. Giangregorio LM, Hicks AL, Webber CE, Phillips SM, Craven BC, Bugaresti JM, McCartney N. Body weight supported treadmill training in acute spinal cord injury: impact on muscle and bone. Spinal Cord 2005; 43: 649-657. 111. Roy RR, Talmadge RJ, Hodgson JA, Zhong H, Baldwin KM, Edgerton VR. Training effects on soleus of cats spinal co rd transected (T12-13) as adults. Muscle Nerve 1998; 21: 63-71. 112. Dupont-Versteegden EE, Houle JD, Gurley CM, Peterson CA. Early changes in muscle fiber size and gene expression in response to spinal cord transection and exercise. Am J Physiol 1998; 275: C1124-1133. 113. Dupont-Versteegden EE, Murphy RJ, Houle JD, Gurley CM, Peterson CA. Activated satellite cells fail to restore myonuclear numbe r in spinal cord transected and exercised rats. Am J Physiol 1999; 277: C589-597. 114. Liu M, Bose P, Walter GA, Anderson DK, Thompson FJ, Vandenborne K. Changes in muscle T2 relaxation properties following sp inal cord injury and locomotor training. Eur J Appl Physiol 2006; 97: 355-361. 115. Lieber RL. Skeletal muscle structure and function. Implications for rehabilitation and sports medicine; 1992. 116. Schmalbruch H, Lewis DM. Dynamics of nuc lei of muscle fibers and connective tissue cells in normal and denervated rat muscles. Muscle Nerve 2000; 23: 617-626. 117. Robertson TA, Maley MA, Grounds MD, Papadimitriou JM. The role of macrophages in skeletal muscle regeneration with particular reference to chemotaxis. Exp Cell Res 1993; 207: 321-331. 118. Orimo S, Hiyamuta E, Arahata K, Sugita H. Analysis of inflammatory cells and complement C3 in bupivacaine-induced myonecrosis. Muscle Nerve 1991; 14: 515-520.

PAGE 172

172 119. MacIntyre DL, Reid WD, McKenzie DC. De layed muscle soreness. The inflammatory response to muscle injury a nd its clinical implications. Sports Med 1995; 20: 24-40. 120. Blaveri K, Heslop L, Yu DS, Rosenblatt JD, Gross JG, Partridge TA, Morgan JE. Patterns of repair of dystrophic mouse muscle: studies on isolated fibers. Dev Dyn 1999; 216: 244-256. 121. Bourke DL, Ontell M. Branched myofibers in long-term whole mu scle transplants: a quantitative study. Anat Rec 1984; 209: 281-288. 122. Charge SB, Rudnicki MA. Cellular and mol ecular regulation of muscle regeneration. Physiol Rev 2004; 84: 209-238. 123. Holterman CE, Rudnicki MA. Molecular regulation of satellite cell function. Semin Cell Dev Biol 2005; 16: 575-584. 124. Gibson MC, Schultz E. The distribution of sate llite cells and their rela tionship to specific fiber types in soleus and extensor digitorum longus muscles. Anat Rec 1982; 202: 329337. 125. Seale P, Sabourin LA, Girgis-Gabardo A, Mansouri A, Gruss P, Rudnicki MA. Pax7 is required for the specification of myogenic satellite cells. Cell 2000; 102: 777-786. 126. Olguin HC, Olwin BB. Pax-7 up-regulat ion inhibits myogenesis and cell cycle progression in satellite cells: a potential mechanism for self-renewal. Dev Biol 2004; 275: 375-388. 127. Oustanina S, Hause G, Braun T. Pax7 di rects postnatal renewal and propagation of myogenic satellite cells bu t not their specification. Embo J 2004; 23: 3430-3439. 128. Zammit PS, Relaix F, Nagata Y, Ruiz AP, Collins CA, Partridge TA, Beauchamp JR. Pax7 and myogenic progression in sk eletal muscle satellite cells. J Cell Sci 2006; 119: 1824-1832. 129. Sabourin LA, Girgis-Gabardo A, Seale P, Asakura A, Rudnicki MA. Reduced differentiation potential of primary MyoD-/myogenic cells derived from adult skeletal muscle. J Cell Biol 1999; 144: 631-643. 130. Megeney LA, Kablar B, Garrett K, Anderson JE, Rudnicki MA. MyoD is required for myogenic stem cell function in adult skeletal muscle. Genes Dev 1996; 10: 1173-1183. 131. Kablar B, Rudnicki MA. Skeletal muscle development in the mouse embryo. Histol Histopathol 2000; 15: 649-656. 132. Shi X, Garry DJ. Muscle stem cells in development, regeneration, and disease. Genes Dev 2006; 20: 1692-1708.

PAGE 173

173 133. Ehrhardt J, Morgan J. Regene rative capacity of skeletal muscle. Curr Opin Neurol 2005; 18: 548-553. 134. Tomczak KK, Marinescu VD, Ramoni MF Sanoudou D, Montanaro F, Han M, Kunkel LM, Kohane IS, Beggs AH. Expression prof iling and identification of novel genes involved in myogenic differentiation. Faseb J 2004; 18: 403-405. 135. Peault B, Rudnicki M, Torrente Y, Cossu G, Tremblay JP, Partridge T, Gussoni E, Kunkel LM, Huard J. Stem and progenitor cel ls in skeletal muscle development, maintenance, and therapy. Mol Ther 2007; 15: 867-877. 136. Bischoff R. Chemotaxis of sk eletal muscle satellite cells. Dev Dyn 1997; 208: 505-515. 137. Floss T, Arnold HH, Braun T. A role fo r FGF-6 in skeletal muscle regeneration. Genes Dev 1997; 11: 2040-2051. 138. Modlesky CM, Slade JM, Bickel CS, Meyer RA, Dudley GA. Deteriorated geometric structure and strength of th e midfemur in men with complete spinal cord injury. Bone 2005; 36: 331-339. 139. Mangold S, Keller T, Curt A, Dietz V. Transcutaneous functional electrical stimulation for grasping in subjects with cervical spinal cord injury. Spinal Cord 2005; 43: 1-13. 140. Skold C, Harms-Ringdahl K, Seiger A. Movement-provoked muscle torque and EMG activity in longstanding motor complete spinal cord injured individuals. J Rehabil Med 2002; 34: 86-90. 141. Waters RL, Adkins RH, Yakura JS, Sie I. Motor and sensory recovery following incomplete tetraplegia. Arch Phys Med Rehabil 1994; 75: 306-311. 142. Muslumanoglu L, Aki S, Ozturk Y, Soy D, Filiz M, Karan A, Berker E. Motor, sensory and functional recovery in patients with spinal cord lesions. Spinal Cord 1997; 35: 386389. 143. Herbison GJ, Isaac Z, Cohen ME, Ditunno JF, Jr. Strength post-spinal cord injury: myometer vs manual muscle test. Spinal Cord 1996; 34: 543-548. 144. Field-Fote EC. Combined use of body weight support, functional elect ric stimulation, and treadmill training to improve walking ability in individuals with chronic incomplete spinal cord injury. Arch Phys Med Rehabil 2001; 82: 818-824. 145. Kim CM, Eng JJ, Whittaker MW. Level walking and ambulatory capacity in persons with incomplete spinal cord injury: re lationship with muscle strength. Spinal Cord 2004; 42: 156-162. 146. Gregory CM, Vandenborne K, Huang HF, Ottenweller JE, Dudley GA. Effects of testosterone replacement therapy on skelet al muscle after spinal cord injury. Spinal Cord 2003; 41: 23-28.

PAGE 174

174 147. Chilibeck PD, Jeon J, Weiss C, Bell G, Burnham R. Histochemical changes in muscle of individuals with spinal cord injury followi ng functional electrical stimulated exercise training. Spinal Cord 1999; 37: 264-268. 148. Sloan KE, Bremner LA, Byrne J, Day RE, Scull ER. Musculoskeletal effects of an electrical stimulation induced cyclin g programme in the spinal injured. Paraplegia 1994; 32: 407-415. 149. Donaldson N, Perkins TA, Fitzwater R, Wood DE, Middleton F. FES cycling may promote recovery of leg function afte r incomplete spinal cord injury. Spinal Cord 2000; 38: 680-682. 150. Maynard FM, Jr., Bracken MB, Creasey G, Ditunno JF, Jr., Donovan WH, Ducker TB, Garber SL, Marino RJ, Stover SL, Tator CH, Waters RL, Wilberger JE, Young W. International Standards for Neurological a nd Functional Classification of Spinal Cord Injury. American Spinal Injury Association. Spinal Cord 1997; 35: 266-274. 151. Dobkin BH, Apple D, Barbeau H, Basso M, Behrman A, Deforge D, Ditunno J, Dudley G, Elashoff R, Fugate L, Harkema S, Sauli no M, Scott M. Methods for a randomized trial of weight-supported treadmill training vers us conventional training for walking during inpatient rehabilitation after incomple te traumatic spinal cord injury. Neurorehabil Neural Repair 2003; 17: 153-167. 152. Shield A, Zhou S. Assessing voluntary musc le activation with the twitch interpolation technique. Sports Med 2004; 34: 253-267. 153. Todd G, Gorman RB, Gandevia SC. Measurement and reproducibility of strength and voluntary activation of lower-limb muscles. Muscle Nerve 2004; 29: 834-842. 154. Pap G, Machner A, Awiszus F. Strength and voluntary activati on of the quadriceps femoris muscle at different severities of osteoarthritic knee joint damage. J Orthop Res 2004; 22: 96-103. 155. Norregaard J, Bulow PM, Vestergaard-Poul sen P, Thomsen C, Danneskiold-Samoe B. Muscle strength, voluntary activ ation and cross-sectional muscle area in patients with fibromyalgia. Br J Rheumatol 1995; 34: 925-931. 156. Allen GM, Middleton J, Katrak PH, Lord SR, Gandevia SC. Prediction of voluntary activation, strength and e ndurance of elbow flexors in postpolio patients. Muscle Nerve 2004; 30: 172-181. 157. Stevens JE, Mizner RL, Snyder-Mackler L. Quadriceps strength and volitional activation before and after total knee arthroplasty for osteoarthritis. J Orthop Res 2003; 21: 775779. 158. Binder-Macleod SA. Variable-frequency stim ulation patterns for the optimization of force during muscle fatigue. Muscle wi sdom and the catch-like property. Adv Exp Med Biol 1995; 384: 227-240.

PAGE 175

175 159. Neptune RR, Kautz SA, Zajac FE. Contributions of the individual ankl e plantar flexors to support, forward progression and swing initiation during walking. J Biomech 2001; 34: 1387-1398. 160. Aagaard P, Simonsen EB, Andersen JL, Ma gnusson P, Dyhre-Poulsen P. Increased rate of force development and neural drive of human skeletal muscle following resistance training. J Appl Physiol 2002; 93: 1318-1326. 161. Bemben MG, Tuttle TD, Bemben DA, Kneh ans AW. Effects of creatine supplementation on isometric force-time curve characteristics. Med Sci Sports Exerc 2001; 33: 1876-1881. 162. Gerrits HL, de Haan A, Hopman MT, van der Woude LH, Sargeant AJ. Influence of muscle temperature on the contractile properties of the quadriceps muscle in humans with spinal cord injury. Clin Sci (Lond) 2000; 98: 31-38. 163. Shields RK. Fatigability, re laxation properties, and electr omyographic responses of the human paralyzed soleus muscle. J Neurophysiol 1995; 73: 2195-2206. 164. Ostchega Y, Dillon CF, Lindle R, Carroll M, Hurley BF. Isokinetic leg muscle strength in older americans and its relationship to a sta ndardized walk test: data from the national health and nutrition examination survey 1999-2000. J Am Geriatr Soc 2004; 52: 977-982. 165. Mueller MJ, Minor SD, Schaaf JA, Strube MJ, Sahrmann SA. Relationship of plantarflexor peak torque and dorsiflexion range of motion to kinetic variables during walking. Phys Ther 1995; 75: 684-693. 166. Muller R, Dietz V. Neuronal function in ch ronic spinal cord injury: divergence between locomotor and flexionand H-reflex activity. Clin Neurophysiol 2006; 117: 1499-1507. 167. Huang H, He J, Herman R, Carhart MR. Modulation effects of ep idural spinal cord stimulation on muscle ac tivities during walking. IEEE Trans Neural Syst Rehabil Eng 2006; 14: 14-23. 168. Behrman AL, Lawless-Dixon AR, Davis SB, Bowden MG, Nair P, Phadke C, Hannold EM, Plummer P, Harkema SJ. Locomotor training progression and outcomes after incomplete spinal cord injury. Phys Ther 2005; 85: 1356-1371. 169. Wirz M, Zemon DH, Rupp R, Scheel A, Co lombo G, Dietz V, Hornby TG. Effectiveness of automated locomotor training in patients with chronic incomplete spinal cord injury: a multicenter trial. Arch Phys Med Rehabil 2005; 86: 672-680. 170. Hicks AL, Adams MM, Martin Ginis K, Giangregorio L, Latimer A, Phillips SM, McCartney N. Long-term body-weight-suppor ted treadmill training and subsequent follow-up in persons with chronic SCI: e ffects on functional walking ability and measures of subjective well-being. Spinal Cord 2005; 43: 291-298. 171. Giangregorio LM, McCartney N. Reduced lo ading due to spinal-c ord injury at birth results in "slender" bones: a case study. Osteoporos Int 2007; 18: 117-120.

PAGE 176

176 172. Nadeau S, Gravel D, Arsenault AB, Bour bonnais D. Plantarflexor weakness as a limiting factor of gait speed in stroke subjects and the compen sating role of hip flexors. Clin Biomech (Bristol, Avon) 1999; 14: 125-135. 173. Franco JC, Perell KL, Gregor RJ, Scremi n AM. Knee kinetics during functional electrical stimulation induced cycling in subjects with spinal cord injury: a preliminary study. J Rehabil Res Dev 1999; 36: 207-216. 174. Gregory CM, Bowden MG, Jayaraman A, Sh ah P, Behrman A, Kautz SA, Vandenborne K. Resistance training and locomotor recovery after incomplete spinal cord injury: a case series. Spinal Cord 2007; 45: 522-530. 175. Stevens JE, Pathare NC, Tillman SM, Scarborough MT, Gibbs CP, Shah P, Jayaraman A, Walter GA, Vandenborne K. Relative contributio ns of muscle activa tion and muscle size to plantarflexor torque during rehabilitation after immobilization. J Orthop Res 2006; 24: 1729-1736. 176. Marino RJ, Barros T, Biering-Sorensen F, Burns SP, Donovan WH, Graves DE, Haak M, Hudson LM, Priebe MM. International standards for neurologi cal classification of spinal cord injury. J Spinal Cord Med 2003; 26 Suppl 1: S50-56. 177. Marino RJ, Ditunno JF, Jr., Donovan WH, Mayn ard F, Jr. Neurologic recovery after traumatic spinal cord injury: data from the Model Spinal Cord Injury Systems. Arch Phys Med Rehabil 1999; 80: 1391-1396. 178. Geisler FH, Coleman WP, Grieco G, Poonian D. Measurements and recovery patterns in a multicenter study of acut e spinal cord injury. Spine 2001; 26: S68-86. 179. Newton RU, Kraemer WJ, Hakkinen K. Effects of ballistic training on preseason preparation of elite volleyball players. Med Sci Sports Exerc 1999; 31: 323-330. 180. Cronin J, Sleivert G. Challenges in understanding the influe nce of maximal power training on improving athletic performance. Sports Med 2005; 35: 213-234. 181. Myer GD, Ford KR, Palumbo JP, He wett TE. Neuromuscular training improves performance and lower-extremity biomechanics in female athletes. J Strength Cond Res 2005; 19: 51-60. 182. Saunders PU, Telford RD, Pyne DB, Pelto la EM, Cunningham RB, Go re CJ, Hawley JA. Short-term plyometric training improves r unning economy in highly trained middle and long distance runners. J Strength Cond Res 2006; 20: 947-954. 183. Herrero JA, Izquierdo M, Maffiuletti NA, Garcia-Lopez J. Electromyostimulation and plyometric training effects on jumping and sprint time. Int J Sports Med 2006; 27: 533539. 184. Behm DG, St-Pierre DM, Perez D. Muscle in activation: assessment of interpolated twitch technique. J Appl Physiol 1996; 81: 2267-2273.

PAGE 177

177 185. Teixeira-Salmela LF, Nadeau S, McBride I, Olney SJ. Effects of muscle strengthening and physical conditioning training on tempor al, kinematic and kinetic variables during gait in chronic stroke survivors. J Rehabil Med 2001; 33: 53-60. 186. Ferri A, Scaglioni G, Pousson M, Capodag lio P, Van Hoecke J, Na rici MV. Strength and power changes of the human plantar flexors an d knee extensors in response to resistance training in old age. Acta Physiol Scand 2003; 177: 69-78. 187. Stevens JE, Walter GA, Okereke E, Scar borough MT, Esterhai JL, George SZ, Kelley MJ, Tillman SM, Gibbs JD, Elliott MA, Frim el TN, Gibbs CP, Vandenborne K. Muscle adaptations with immobilization and re habilitation after ankle fracture. Med Sci Sports Exerc 2004; 36: 1695-1701. 188. Perry J, Mulroy SJ, Renwick SE. The relationship of lower extremity strength and gait parameters in patients with post-polio syndrome. Arch Phys Med Rehabil 1993; 74: 165169. 189. Canning CG, Ada L, O'Dwyer N. Slowness to develop force cont ributes to weakness after stroke. Arch Phys Med Rehabil 1999; 80: 66-70. 190. Pohl PS, Duncan P, Perera S, Long J, Liu W, Zhou J, Kautz SA. Rate of isometric knee extension strength development a nd walking speed after stroke. J Rehabil Res Dev 2002; 39: 651-657. 191. Barbeau H. Locomotor training in neuroreh abilitation: emerging re habilitation concepts. Neurorehabil Neural Repair 2003; 17: 3-11. 192. Levy CE, Nichols DS, Schmalbrock PM, Keller P, Chakeres DW. Functional MRI evidence of cortical reorga nization in upper-limb stroke hemiplegia treated with constraint-induced movement therapy. Am J Phys Med Rehabil 2001; 80: 4-12. 193. Love FM, Son YJ, Thompson WJ. Activity al ters muscle reinnervation and terminal sprouting by reducing the number of Schwann cell pathways th at grow to link synaptic sites. J Neurobiol 2003; 54: 566-576. 194. Kim CM, Eng JJ. The relationship of lowe r-extremity muscle torque to locomotor performance in people with stroke. Phys Ther 2003; 83: 49-57. 195. Neptune RR, Zajac FE, Kautz SA. Muscle force redistributes segmental power for body progression during walking. Gait Posture 2004; 19: 194-205. 196. Barbeau H, Ladouceur M, Norman KE, Pepin A, Leroux A. Walking after spinal cord injury: evaluation, treatment, and functional recovery. Arch Phys Med Rehabil 1999; 80: 225-235. 197. Edgerton VR, de Leon RD, Tillakaratne N, Recktenwald MR, Hodgson JA, Roy RR. Use-dependent plasticity in spinal stepping and standing. Adv Neurol 1997; 72: 233-247.

PAGE 178

178 198. Edgerton VR, Kim SJ, Ichiyama RM, Gera simenko YP, Roy RR. Rehabilitative therapies after spinal cord injury. J Neurotrauma 2006; 23: 560-570. 199. Basso DM, Beattie MS, Bresnahan JC. Graded histological and locomotor outcomes after spinal cord contusion using the NYU we ight-drop device versus transection. Exp Neurol 1996; 139: 244-256. 200. Rosenzweig ES, McDonald JW. Rodent models for treatment of spinal cord injury: research trends and progress toward useful repair. Curr Opin Neurol 2004; 17: 121-131. 201. Gazula VR, Roberts M, Luzzio C, Jawad AF Kalb RG. Effects of limb exercise after spinal cord injury on motor neuron dendrite structure. J Comp Neurol 2004; 476: 130145. 202. Thomason DB, Booth FW. Atrophy of th e soleus muscle by hindlimb unweighting. J Appl Physiol 1990; 68: 1-12. 203. Basso DM, Beattie MS, Bresnahan JC. De scending systems contributing to locomotor recovery after mild or moderate spinal cord injury in rats: experimental evidence and a review of literature. Restor Neurol Neurosci 2002; 20: 189-218. 204. Dietz V, Wirz M, Jensen L. Locomoti on in patients with spinal cord injuries. Phys Ther 1997; 77: 508-516. 205. Barbeau H, Rossignol S. Enhancement of locomotor recovery following spinal cord injury. Curr Opin Neurol 1994; 7: 517-524. 206. Edgerton VR, Roy RR. Paralysis rec overy in humans and model systems. Curr Opin Neurobiol 2002; 12: 658-667. 207. de Leon RD, Roy RR, Edgerton VR. Is the recovery of stepping following spinal cord injury mediated by modifying existing neural pathways or by generating new pathways? A perspective. Phys Ther 2001; 81: 1904-1911. 208. Liu M, Bose P, Walter GA, Thompson FJ, Vandenborne K. A longitudinal study of skeletal muscle following spinal co rd injury and locomotor training. Spinal Cord 2008. 209. Roy RR, Zhong H, Siengthai B, Edgerton VR Activity-dependent influences are greater for fibers in rat medial gastrocnemius than tibialis anterior muscle. Muscle Nerve 2005; 32: 473-482. 210. Roy RR, Zhong H, Monti RJ, Vallance KA, Kim JA, Edgerton VR. Mechanical properties and fiber type composition of chronically inactive muscles. J Gravit Physiol 2000; 7: P103-104. 211. Roy RR, Zhong H, Monti RJ, Vallance KA, Edgerton VR. Mechanical properties of the electrically silent adult rat soleus muscle. Muscle Nerve 2002; 26: 404-412.

PAGE 179

179 212. Roy RR, Zhong H, Bodine SC, Pierotti DJ, Talmadge RJ, Barkhoudarian G, Kim J, Fanton JW, Kozlovskaya IB, Edgerton VR. Fiber size and myosin pheno types of selected rhesus lower limb muscles af ter a 14-day spaceflight. J Gravit Physiol 2000; 7: S45. 213. Talmadge RJ. Myosin heavy chain isofor m expression following reduced neuromuscular activity: potential regulatory mechanisms. Muscle Nerve 2000; 23: 661-679. 214. Lee YS, Lin CY, Caiozzo VJ, Robertson RT Yu J, Lin VW. Repair of spinal cord transection and its effects on muscle mass and myosin heavy chain isoform phenotype. J Appl Physiol 2007; 103: 1808-1814. 215. Grossman EJ, Roy RR, Talmadge RJ, Zhong H, Edgerton VR. Effects of inactivity on myosin heavy chain composition an d size of rat soleus fibers. Muscle Nerve 1998; 21: 375-389. 216. Hyatt JP, Roy RR, Baldwin KM, Wernig A, Edgerton VR. Activity-unrelated neural control of myogenic fact ors in a slow muscle. Muscle Nerve 2006; 33: 49-60. 217. Talmadge RJ, Roy RR, Edgerton VR. Myosin heavy chain profile of cat soleus following chronic reduced activity or inactivity. Muscle Nerve 1996; 19: 980-988. 218. Talmadge RJ, Garcia ND, Roy RR, Edgerton VR. Myosin heavy chain isoform mRNA and protein levels after long-term paralysis. Biochem Biophys Res Commun 2004; 325: 296-301. 219. Behrman AL, Harkema SJ. Physical rehabilitation as an agent for recovery after spinal cord injury. Phys Med Rehabil Clin N Am 2007; 18: 183-202, v. 220. Harkema SJ, Hurley SL, Patel UK, Requejo PS, Dobkin BH, Edgerton VR. Human lumbosacral spinal cord interprets loading during stepping. J Neurophysiol 1997; 77: 797-811. 221. Hopman MT, Dueck C, Monroe M, Ph ilips WT, Skinner JS. Limits to maximal performance in individuals with spinal cord injury. Int J Sports Med 1998; 19: 98-103. 222. Machida S, Booth FW. Insulin-like growth factor 1 and muscle gr owth: implication for satellite cell proliferation. Proc Nutr Soc 2004; 63: 337-340. 223. Adams GR. Role of insulin-like growth fact or-I in the regulation of skeletal muscle adaptation to increased loading. Exerc Sport Sci Rev 1998; 26: 31-60. 224. Darr KC, Schultz E. Exercise-induced sate llite cell activation in growing and mature skeletal muscle. J Appl Physiol 1987; 63: 1816-1821. 225. Cabric M, James NT. Morphometric analyses on the muscles of exercise trained and untrained dogs. Am J Anat 1983; 166: 359-368.

PAGE 180

180 226. Allen DL, Monke SR, Talmadge RJ, Roy RR, Edgerton VR. Plasticity of myonuclear number in hypertrophied and atrophie d mammalian skeletal muscle fibers. J Appl Physiol 1995; 78: 1969-1976. 227. Barton ER. Viral expression of insulin-like growth factor-I isoforms promotes different responses in skeletal muscle. J Appl Physiol 2006; 100: 1778-1784. 228. Scime A, Rudnicki MA. Anabolic potential a nd regulation of the skel etal muscle satellite cell populations. Curr Opin Clin Nutr Metab Care 2006; 9: 214-219. 229. Kuang S, Charge SB, Seale P, Huh M, R udnicki MA. Distinct roles for Pax7 and Pax3 in adult regenerative myogenesis. J Cell Biol 2006; 172: 103-113. 230. Grenier G, Rudnicki MA. The potential us e of myogenic stem cells in regenerative medicine. Handb Exp Pharmacol 2006; 299-317. 231. Grounds MD. Muscle regeneration: molecu lar aspects and therapeutic implications. Curr Opin Neurol 1999; 12: 535-543. 232. Wagers AJ, Conboy IM. Cellular and molecu lar signatures of muscle regeneration: current concepts and controversies in adult myogenesis. Cell 2005; 122: 659-667. 233. Caroni P. Activity-sensitive signaling by mu scle-derived insulin-like growth factors in the developing and regenerating neuromuscular system. Ann N Y Acad Sci 1993; 692: 209-222. 234. Allen RE, Boxhorn LK. Regulation of skeletal muscle satellite cell proliferation and differentiation by transforming growth factor-b eta, insulin-like growth factor I, and fibroblast growth factor. J Cell Physiol 1989; 138: 311-315. 235. Adams GR, Haddad F. The relationships among IGF-1, DNA content, and protein accumulation during skeletal muscle hypertrophy. J Appl Physiol 1996; 81: 2509-2516. 236. Adams GR, Caiozzo VJ, Haddad F, Baldwin KM. Cellular and molecular responses to increased skeletal muscle loading after irradiation. Am J Physiol Cell Physiol 2002; 283: C1182-1195. 237. Resnick DK, Schmitt C, Miranpuri GS, Dhodda VK, Isaacson J, Vemuganti R. Molecular evidence of repair and plasticity following spinal cord injury. Neuroreport 2004; 15: 837839. 238. Dupont-Versteegden EE, Murphy RJ, Houle JD, Gurley CM, Peterson CA. Mechanisms leading to restoration of muscle size with ex ercise and transplantation after spinal cord injury. Am J Physiol Cell Physiol 2000; 279: C1677-1684. 239. Yablonka-Reuveni Z, Rivera AJ. Temporal expression of regulatory and structural muscle proteins during myogenesis of sate llite cells on isolated adult rat fibers. Dev Biol 1994; 164: 588-603.

PAGE 181

181 240. Cornelison DD, Wold BJ. Single-cell analysis of regulatory gene e xpression in quiescent and activated mouse skeletal muscle satellite cells. Dev Biol 1997; 191: 270-283. 241. Hawke TJ, Garry DJ. Myogenic satellite cells: physiology to molecular biology. J Appl Physiol 2001; 91: 534-551. 242. Olguin HC, Yang Z, Tapscott SJ, Olwin BB. Reciprocal inhibition between Pax7 and muscle regulatory factors modulates myogenic cell fate determination. J Cell Biol 2007; 177: 769-779.

PAGE 182

182 BIOGRAPHICAL SKETCH Arun Jayaram an was born in Chennai, India. He received his bachelors in physical therapy from Dr. MGR Medical University in 2000 and his masters in hospital management from Loyola Institute of Business Administration in 2001. He also worked as an in-patient physical therapist in cardiac rehab in the Institut e of Cardio-Pulmonary Diseases in Chennai till the year 2001. He received his advanced masters of science in physical therapy from Georgia State University, Atlanta, GA, in the year 2003. He joined the doctoral program in rehabilitation science at the Univ ersity of Florida in the fall of 2003.