<%BANNER%>

Survivin Expression after Traumatic Brain Injury: Potential Roles in Neuroprotection

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 E20110115_AAAACH INGEST_TIME 2011-01-15T14:24:01Z PACKAGE UFE0008337_00001
AGREEMENT_INFO ACCOUNT UF PROJECT UFDC
FILES
FILE SIZE 6016 DFID F20110115_AABOCH ORIGIN DEPOSITOR PATH johnson_e_Page_58thm.jpg GLOBAL false PRESERVATION BIT MESSAGE_DIGEST ALGORITHM MD5
64d112b7b8775dd2c2d1249433e7f35e
SHA-1
b167d51e0e68ad61425b6644b462b67444a13841
90648 F20110115_AABNLP johnson_e_Page_82.jpg
af23c32ea6e9f5709dfc2decd3be1913
82f45c2d38f4e776fa9cf5a83d58a9ab763d5096
2102 F20110115_AABNVK johnson_e_Page_23.txt
ddbfc1ca5acb698494c967c14e580a05
e841092112c655e35fdf36c7f7f85254e4760168
51114 F20110115_AABNGS johnson_e_Page_21.pro
40b62729d8b3617383572e16c57b4ecd
7e6dce78529cedbec430f240f79ffd14b15fa353
25271604 F20110115_AABNQN johnson_e_Page_58.tif
44d650d9c77bd61c854a3e40017e7405
15f6a79b7237017067cf3091c3e2cbb828e636db
20294 F20110115_AABOCI johnson_e_Page_59.QC.jpg
4ed798675f1c4215e398613bc8c60938
adc792a84b3f55dc6729bbeaf4ad96aa18a7f22c
101204 F20110115_AABNLQ johnson_e_Page_84.jpg
b54c27225e3ec427727be494ed91790b
a054cb2e53fb54e47e91cb7f6dc112a49908446b
345 F20110115_AABNVL johnson_e_Page_24.txt
9dbab1baaf21e9f8b151fbd75a58ed89
d78f9b2f0327b443232cee2b771c2d760adf4cff
8423998 F20110115_AABNGT johnson_e_Page_36.tif
4c260412b57f8ade3f2bc2c821493cea
68ed620db7af6b39b66ab0b23f5d5d934610ed66
5744 F20110115_AABOCJ johnson_e_Page_59thm.jpg
31f82b0e9953b047c67faea3568fcc05
36c5afee481d5d44e32ed857e85384ab897349c3
94149 F20110115_AABNLR johnson_e_Page_85.jpg
584318ce224e08a0c0bd13ec2c66ed9c
1ab71f00926f0c8f0a7e9651209ee86a501957d2
1817 F20110115_AABNVM johnson_e_Page_25.txt
771a19bfe37a4ea1fd1fbd40e4147e27
bd3bf58986acfb14ed241cb4cfe0c6267ca9b938
5767 F20110115_AABNGU johnson_e_Page_11thm.jpg
75051f69fc8b57c74d70e40d6d1c936e
254e69e581a1b4a1d8c615114e55d1f4751cb13a
F20110115_AABNQO johnson_e_Page_59.tif
8264bda4fcda6e0f1b0524f6fcc9d942
ede6da303067d7f6c3ba18b3b90fa58ccb60b48c
23652 F20110115_AABOCK johnson_e_Page_60.QC.jpg
a90dc60163a28560f46542473ae3e48b
db31a5c24460d9cfd2a077d326ae624bf7f5ff66
63221 F20110115_AABNLS johnson_e_Page_86.jpg
ec2820b6a9b17120f764ce4d5a7fdad0
20639a9ca41514acf8b4bab34f81254592e24d83
1936 F20110115_AABNVN johnson_e_Page_26.txt
8d4ad667877aa0f77dac71b5bd53bb33
198d164919a8641bd6c08a2d652751ebde16b7ab
84308 F20110115_AABNGV johnson_e_Page_73.jpg
4a25880b0614aab206ad8db31bced983
7e8abfe4af84142c015875d9c6b29ad84e1f12b8
1053954 F20110115_AABNQP johnson_e_Page_60.tif
7229b446a365af6823927fc85433a641
0e16b9279a30da57b1ef8e72fff0aace4d480182
23039 F20110115_AABOCL johnson_e_Page_61.QC.jpg
60ffe103536541c201aee5de3a1ef8a7
7bcbe4645f34195d56054039ac582007ed9205e4
42374 F20110115_AABNLT johnson_e_Page_87.jpg
6a122dd0e4d34f9c3f5c9e30c098462d
356454d1ac424233e7450530f675d90c3cd1d4b9
1913 F20110115_AABNVO johnson_e_Page_27.txt
5d6d2870311870d86d33fa9d00d673db
1d39a2e88689848305a12d45ee1620be37357565
72921 F20110115_AABNGW johnson_e_Page_51.jpg
a9aa96a8132bcfdf0cee65ac202ae261
fe72a46394edbfb8dab8fb37001076a45ebac6a2
F20110115_AABNQQ johnson_e_Page_61.tif
075949f1e7fa9bdb2203705ac708dbad
2d8c37b490c0b3458fe6b24407102e60918ecbfd
5423 F20110115_AABOCM johnson_e_Page_63thm.jpg
f5881154e12eb828e6bb931c8f1725a0
2906661052f78c246d743746e37e9c931405ea83
24773 F20110115_AABNLU johnson_e_Page_01.jp2
f7250c7d3e4ea3cea503b37ed385d2be
7219a5dac19e9ccec8651265fc50d2335e5d29b5
1907 F20110115_AABNVP johnson_e_Page_28.txt
03f5d515830641d6e829476cba7eaa86
25e1f1453309ed21d3339d1fa3540da11322d0e7
F20110115_AABNGX johnson_e_Page_64.tif
750c7de8a468a2175b7bf05cec1963e6
17eec1ee4847d341dc8c34b7bfd86a00e4cb077c
F20110115_AABNQR johnson_e_Page_62.tif
ef4c00d02315b2d6652cab089b986ba8
4d28b02473651d29e9171d5383182bac3ef1f2bc
6321 F20110115_AABOCN johnson_e_Page_64thm.jpg
b2997e8bbd02a4c9cb0e7f54221d45c3
d42644b4f86c82f6cbd32f7d5db95c82baabee0d
5880 F20110115_AABNLV johnson_e_Page_02.jp2
d7db3c2c0d7d04031779126c55f2008a
b5d7c97f421512b691a4fa0e9eba832628814922
1587 F20110115_AABNVQ johnson_e_Page_29.txt
6dcd081f5ffd9178f22fb530219e6d1a
ec30c711a91bb12dee2d60f962e7d161b6a344f9
90528 F20110115_AABNGY johnson_e_Page_44.jpg
568813a028c32dcfd976410600086c7d
dcc6cd138c0f94b06b62a12991d26b5c39469e2e
F20110115_AABNQS johnson_e_Page_63.tif
991433fd2744a62f8216e8b680864085
41c3265fa164b6b685582ee123cac713eefd86b8
24003 F20110115_AABOCO johnson_e_Page_65.QC.jpg
81d70dbc4029aafea9617bb7f9bda8f6
0fae63895bdc5596a2b651425c2b8637eba09fb6
83752 F20110115_AABNLW johnson_e_Page_04.jp2
f7edc0a082574cf7e55a695f5a90fa79
99f78b6b9628381b9e170d43adf1c69b2d52ef58
1780 F20110115_AABNVR johnson_e_Page_30.txt
9d063f3b05b4e5f6a154397cf3247169
72bccecf3674cb6e845f3d813c01b4601ea0a860
61051 F20110115_AABNGZ johnson_e_Page_63.jpg
800947ba636bfabacb280a2c92c9337a
5371ebba7e82914ba699a749a9672311c560494e
F20110115_AABNQT johnson_e_Page_65.tif
7603a45ee521e4762bfb0ea2fce475fe
4c47d23fc72745cee532614a78503dcbea8190cb
6586 F20110115_AABOCP johnson_e_Page_65thm.jpg
66e473a4e9f0f5399fe2fd83bf3bd36f
bdcd656fa7db84134a6d740fc453448106f2fd49
1051985 F20110115_AABNLX johnson_e_Page_05.jp2
588891eceb8fbf0d7a3ccc52a312fae5
212bd41aa2804037f15d4561a63d9b5a4f3cac5d
1978 F20110115_AABNVS johnson_e_Page_31.txt
28de8a50a8b42d1dcfe860f3aa5502ec
443ad074b7657d662e62758cc349937d6c135f20
F20110115_AABNQU johnson_e_Page_66.tif
a155fdedfde3c15366fd21fec6507c95
d5437c03f96160da5da96a322f5408551ef36d12
24436 F20110115_AABOCQ johnson_e_Page_66.QC.jpg
5af67f13e73ffc43d15b4e5206055f73
d48f6ff3440cd66b3ed8d768a995e1513530f9a7
1051916 F20110115_AABNLY johnson_e_Page_06.jp2
b62952559bb9a54f3b641048512886e8
fe8d6b3b14a0e1a066eefb2dbb3144ea02d475ab
F20110115_AABNQV johnson_e_Page_67.tif
e74292cb081913976a83f3f956de28ca
ad5225d5bcb44a06aba74c5d020a2fb1c4eef72d
12856 F20110115_AABNJA johnson_e_Page_03.jpg
52c4010cc26f8b7fc9502cc78e22f4f0
64c65ad310e8a38bdd470ac8d1451b7db4eb16ab
F20110115_AABOCR johnson_e_Page_66thm.jpg
e96bc06c1ea58ebaf7fdbbeb6823c5a4
8a2b4b4840e82b388c20bdd9f446f8137cce809e
1820 F20110115_AABNVT johnson_e_Page_32.txt
c22a6cf72d21814e33b4686d54c3b7ca
5f836bfa0cdbc32be0389ba6964125e9a3be6393
1051957 F20110115_AABNLZ johnson_e_Page_07.jp2
dd460221413bfadcd2050b623331c06e
20c004926fabb9c2fc76677fcc8e47bc3439bd84
F20110115_AABNQW johnson_e_Page_68.tif
36c309b05ab6c51c2559be1f6637cf0a
16bf8bda271da8a25ce35f2dda79805c31c4bd0b
58428 F20110115_AABNJB johnson_e_Page_04.jpg
ed7dacc8b3fb9d695330191b196a5b7c
3c76611f1d260ed57af477d6fd99200c11153686
23228 F20110115_AABOCS johnson_e_Page_67.QC.jpg
645540f07ecb6caa0811121b183acf3d
6a7963c7c7841e6ba12f844deddc6e44f6832f7c
1185 F20110115_AABNVU johnson_e_Page_34.txt
03dc328d4a38d79fd0f58a2bd294002e
aea2af0effc5b5efa4af21d6cd583ba8d4987b70
F20110115_AABNQX johnson_e_Page_69.tif
85ae9f112ca0df52f77f7cea8a66e30e
4161423f2342d5fb14143739c3ff30f2dab835ba
79292 F20110115_AABNJC johnson_e_Page_05.jpg
63abd7a799f1cdaf4c3f4c8c637e7e85
90d180f2d9cfa74231fe9ba280e5d286fc2c59f8
6442 F20110115_AABOCT johnson_e_Page_67thm.jpg
9baf08049a7636143d96c3c87b9c8729
a043be73ce807a9861ca54620d06e6156ed3ad81
1664 F20110115_AABNVV johnson_e_Page_35.txt
b0d2bbbbb32095223c66e0e3b714fdda
0f15e3c4a6103e3dbc5bd38c99ba9c0b4414636a
131681 F20110115_AABNOA johnson_e_Page_73.jp2
bbd8ebe8a56281046efbac8a49472a9e
fe8287aead617e6d20e46036617948a720eade2a
F20110115_AABNQY johnson_e_Page_70.tif
e7d5e65425a2a685a182477c1ca858bc
97e8d6d62492b5e2e11ae85cc61f5788eb0a4e8c
56084 F20110115_AABNJD johnson_e_Page_06.jpg
6247f60edee2077c15d083d5c5d614b2
c18fad1c1b5061052c8c901787148ae766ba0387
23654 F20110115_AABOCU johnson_e_Page_68.QC.jpg
c48dc19880095c72bb858df5f8a77f3f
317ef1489d3f73e605e0e15341518f43c0ee314c
1910 F20110115_AABNVW johnson_e_Page_36.txt
d0f5225f490c6103dd1f4b71e88c7e7f
8874ac63582ce89d95ada78a1ef00de51572a31c
119458 F20110115_AABNOB johnson_e_Page_74.jp2
e7432ad110cb53513f093616ae9ea16e
b4585fb7dbd07fd22485cef23101718506caf84d
F20110115_AABNQZ johnson_e_Page_71.tif
d9f3850dc2b760d02822c3057f7342cc
33d9c2b7caa1feb9f05b9d9c60e62ccd15d65e40
66839 F20110115_AABNJE johnson_e_Page_07.jpg
ef0e22f0002e598ca7283b2fe9c1ef10
bd13fbc0295eeaafbb800107aa5f9b84a64b9357
F20110115_AABOCV johnson_e_Page_68thm.jpg
00703324d88b4476f7f719b36484bb6e
1d6b35bde14df800ae6e59fada6f8951fead6014
1683 F20110115_AABNVX johnson_e_Page_37.txt
075fb87b3b6d00c35fb6b7897f187d6d
09376e7169f3515e01dcd7732f10a1e64ad83e04
132155 F20110115_AABNOC johnson_e_Page_75.jp2
6dc8d26d6a457e87c478b4cfbee35ec4
f07475058c31b16b96c27a8cc6e7e4d1c8ae909c
14578 F20110115_AABNJF johnson_e_Page_08.jpg
88ef854de2504a4f1dea31f93cc334b0
22a2cbce7e6349a7356afa4ed513959077fffb95
16696 F20110115_AABOCW johnson_e_Page_69.QC.jpg
3669e38f7237344a5f371888a0929d3d
48e70626b96c2512d6682c0d9b4741c3cc7a5b2f
1777 F20110115_AABNVY johnson_e_Page_38.txt
42529eebb29e8ad6fd062dbb2e7f52a1
8845a6a970d53df22c5521d17caa6cba25fc3e55
130607 F20110115_AABNOD johnson_e_Page_76.jp2
57f3200c1b07a1c887f15c87b2185dfa
174d0aee5434e6bf4a7d5a5512930e4044079926
59485 F20110115_AABNJG johnson_e_Page_09.jpg
358e46d9e8be41dc98c16d66564158f7
2f89394d21452c49b3842a9b79e0cf08e8e7a80f
56107 F20110115_AABNTA johnson_e_Page_44.pro
dd4563d0a8d49a5f144b36d96d2d7678
175c561178acb0009e104af573d6dc5a27c3326d
4784 F20110115_AABOCX johnson_e_Page_69thm.jpg
4b68058a04b738878d37e6f2c91cdd84
07b758efa24834a128b59081fcb6f9ea19ccb985
1476 F20110115_AABNVZ johnson_e_Page_40.txt
f8aa63dd662f49417f9cff7349f9d28f
acc3783f577bf7afe281110837a9da95b3a4e468
132331 F20110115_AABNOE johnson_e_Page_77.jp2
bb7916e03a3fe0e26d6fa3020581eda2
4d40c9b1ebdad665e9475abb9aad94455d9492a4
39622 F20110115_AABNTB johnson_e_Page_45.pro
eb61b0babc83ca2da82cb761ae80f2b6
4767eb127df5bd24a525588d12dc09ad354fe796
21455 F20110115_AABOCY johnson_e_Page_70.QC.jpg
6f78df94989dcfb5d361c5dfbc9a15cf
815133c788612de7d00e0e3f900e332ecc4221f4
131079 F20110115_AABNOF johnson_e_Page_78.jp2
8c57834812a67b7ba3cb5fc693a2edb6
d8677a5197b1bbd9b8a20fb6b1bf925866a8ab7d
51722 F20110115_AABNJH johnson_e_Page_10.jpg
c1646e82d1fe87be488515a346bc112c
888aa1de3520fb3a20c60ff8fa0138bff271924f
52990 F20110115_AABNTC johnson_e_Page_46.pro
05fa2b2486ff02c0695d216b1940311d
61466b48c0e45f74adcc10d47b3c8ba4aad98f1b
7063 F20110115_AABOCZ johnson_e_Page_71thm.jpg
cd028aa2c4d95f8f54baef68afeae0cf
5310408e1687aeb1809716a6000a0be19a7c52bb
20731 F20110115_AABOAA johnson_e_Page_11.QC.jpg
c5e1c8fd6745f26319e6356399d115f0
c5741160effff53de9b1c60611aac8b671b56067
19975 F20110115_AABNYA johnson_e_Page_07.QC.jpg
fba5c3e514a45ea0cbe60a4a4b0f1278
df9018e0a328be2e3c5375aff74ad0922b1d9dc5
127041 F20110115_AABNOG johnson_e_Page_80.jp2
fabcc5e8b2caa94e922aae2d7469097b
9190ee991d47b714fad7a6376bc074ec60ae47dd
65033 F20110115_AABNJI johnson_e_Page_11.jpg
2905267740e80b959965093495a2e011
30d7214cbbfb18f48fd8d13f515148224b5efb29
49250 F20110115_AABNTD johnson_e_Page_47.pro
a6cc736dc108c7a9275629dafb9fb30b
4ebf88760725cc735d4f5cb1c14f3d8efc188a40
22909 F20110115_AABOAB johnson_e_Page_12.QC.jpg
e52768ceb238a153117eab054112a88b
d68ad49bbee58c6f1f5bad8c5bd33ec2ccd583e9
3762 F20110115_AABNYB johnson_e_Page_03.QC.jpg
aaae271d8d7fe0d42c66f078733550ed
dec5b45b36e8ca9e246b162917e6e5ae823b385d
129887 F20110115_AABNOH johnson_e_Page_81.jp2
44ecfe6e556b2e9c4533914df1a8f00b
d9c3ce1c35e4bc68540cb72945b5544a749c84fd
68872 F20110115_AABNJJ johnson_e_Page_12.jpg
7cbf792dc95bc0bcb88f265273bfb9ee
43582688eb3728440e91991b9c4991d8772fa774
52807 F20110115_AABNTE johnson_e_Page_48.pro
6da35489bbd3ba6b1454fe323ede2f81
b40348cf8fcb5d01b4c60b14a0b35bd7f2ce9b5d
6384 F20110115_AABOAC johnson_e_Page_12thm.jpg
065df2440f198bf91ac9b018917f715c
fa6fdc0bc1f355d8c72be2cdc17672126b18fd14
6047 F20110115_AABNYC johnson_e_Page_24.QC.jpg
a46aec9fac06b53454dfbb3d8e9e1a11
10b1608f44afd73fa7353db32b759a1e4d831d7c
133568 F20110115_AABNOI johnson_e_Page_82.jp2
496bef6f5a366efc175d2835be676ecd
6de0ecb5fc2fc6d7e0348b6cc51b89e0ab2e6527
75190 F20110115_AABNJK johnson_e_Page_13.jpg
e14e94c4cda838b5b212f9da7b8dde0e
49f35fd89fda4d43f59f622b2a6930d38ffb1d80
42162 F20110115_AABNTF johnson_e_Page_50.pro
a7c68bffcc0e60a75d4c791fe245bdfb
cc95f684848bf891fa53ecf72829d3cd22d19d93
6778 F20110115_AABOAD johnson_e_Page_13thm.jpg
be8c464233359fc3229724cffe662e0e
8c92b8590fde9e267ed72ddb13da85dd6e301777
20312 F20110115_AABNYD johnson_e_Page_43.QC.jpg
89571ac93c962234492c7291f167eaaa
076fa5b18993f7d8085fd5950d164bbc631c10c1
130221 F20110115_AABNOJ johnson_e_Page_83.jp2
025562d8b18a1ce9ebe79115e8ae1d7f
3f8928abf8235704f8230ceed3bb920f09e7c239
67078 F20110115_AABNJL johnson_e_Page_14.jpg
c3a441b081c4cb5065c1c1a61bf8e6bb
bca4c6d6c95c6be613561625d0182c14e68a9867
42322 F20110115_AABNTG johnson_e_Page_51.pro
3651f70db6b035f7d8062e2cc600bdeb
8b91ba85fab15d39f0ef4915903dabaffbbefed2
6083 F20110115_AABOAE johnson_e_Page_14thm.jpg
a724fdb50fa67c1af1d83f5d3bc7905e
17ad10fccef7fd4061277f76783855c2e63d6217
23519 F20110115_AABNYE johnson_e_Page_29.QC.jpg
ee652698899eca883a924533e47bbb4d
9f2e4db5b96888800188bb11197c83978196af72
136318 F20110115_AABNOK johnson_e_Page_85.jp2
7f7a96f959e4016a64f3d811d44a10e1
dc7bde7ce6a0f3c95a6a6f4663ec408c1ec40ba5
68922 F20110115_AABNJM johnson_e_Page_16.jpg
1b30a3a52dc5c092df8652f7ed222a86
54c03f15faf25c3d3d176609ce85e78163f72555
41286 F20110115_AABNTH johnson_e_Page_52.pro
fca538a15c54d0d3dbce4186fbb59568
8e4cb74f5ad61c9273256a45b42dfbe65d001f62
22035 F20110115_AABOAF johnson_e_Page_16.QC.jpg
c7dbac04b722a623936fa24e20cd01e1
2255b449ff8ff2970593cfcc721498dc0e0d5db4
6006 F20110115_AABNYF johnson_e_Page_70thm.jpg
048da8e3470d4d12accbf21e99ff80d4
85826ffee616c499db42063d930e40cfd58be341
89974 F20110115_AABNOL johnson_e_Page_86.jp2
11ab6ea188e98867eff018971232cbda
0f598ad59e187fd82be3d8cc49c9417da415afb0
74543 F20110115_AABNJN johnson_e_Page_17.jpg
d598badb25c8676ff12dcfaa2413e5b1
a0982b85e0eef526c44c7d124471dcd4b62628df
39788 F20110115_AABNTI johnson_e_Page_53.pro
0c32ee7e4ee298a222f1c3c476b45f7c
287027c668d79f2f779368d3be31d42dfc37c3ac
6177 F20110115_AABOAG johnson_e_Page_16thm.jpg
f1aa308469cc0f3270d924119b763332
37e773ff230ee3ac096fdd875f57b827796a88de
7044 F20110115_AABNYG johnson_e_Page_41thm.jpg
200ebd1a199a58d9ec35236b472a6328
03ae6bbe314e29dd51166c6e6322bb642862660a
71564 F20110115_AABNJO johnson_e_Page_18.jpg
6f7e118080696c79e61e6ca1f771a842
c47f5413258c37ee51091a7e600c1ced975e5cf8
50168 F20110115_AABNTJ johnson_e_Page_54.pro
87190ce98f082b7ee5194d48950b41f2
30478afa18d51f8d51e52cbce2ee0936f9fab7c6
23966 F20110115_AABOAH johnson_e_Page_17.QC.jpg
26f5b9af23cc9625996f9b7ad00f4d2e
ff8daf81df7c41aed47ee4562f665cf185940b7b
4104 F20110115_AABNYH johnson_e_Page_87thm.jpg
74a43a90fa1daa31dc97c1c95fd68998
ad63f47c2e7103ca8decb7810235b98ead72ee9d
58742 F20110115_AABNOM johnson_e_Page_87.jp2
1b2b1aedb15f6782413299cd13662c26
2ffda7840f67e3d7ba7c9925f2cfcd799f9d9c8c
71350 F20110115_AABNJP johnson_e_Page_19.jpg
16b259722624b607a8076595952e0502
e9417299b48fb273402ec7a5ba30d6c022ea5c8c
55085 F20110115_AABNTK johnson_e_Page_55.pro
4e2caa137ac6bcca2f219dba42bef60f
75078dfc8061984860d39553d5ba9dbdbf2c5a75
23914 F20110115_AABOAI johnson_e_Page_19.QC.jpg
a6c8e6418bc08a40eddf16d0d1ecba73
4419f8a4d7c98330f53cbdd0ea6b002786d0aed4
6824 F20110115_AABNYI johnson_e_Page_76thm.jpg
ad14fccb1533561a57b8a34dd288a09d
eae581af0283fc0722e3b7ecd191e5a39250a19f
F20110115_AABNON johnson_e_Page_01.tif
5debd9b8fe57eba00e39d9769fa8e2ad
03199df5c80714137709d84b6ece2aa86ba8fc64
72041 F20110115_AABNJQ johnson_e_Page_20.jpg
5e089edd027e4148df784357c683e0f9
59139a2fd723b96121a0dcc54a52a7049965098c
36894 F20110115_AABNTL johnson_e_Page_56.pro
e3b7d66357062cce760b30b82d3369f5
73aad3e686ba67cf8622327c9568974cfb59b06f
6487 F20110115_AABOAJ johnson_e_Page_21thm.jpg
7f624bc0a52dc9567db9ba71dc2cb156
84fb630c37341e5d77f2342f24164e584b0a8311
1785 F20110115_AABNYJ johnson_e_Page_08thm.jpg
a95d0e81d753fb2a6c60e66cfd2e13fb
e4c1079ed7354a2abf944f6edb52a3474e1c7c9f
F20110115_AABNOO johnson_e_Page_03.tif
f5978293100a268d4ef1e9a7b9fb1424
ef32117fa7ef0a32c135b43942bcd29896a381ef
73151 F20110115_AABNJR johnson_e_Page_21.jpg
762c3bd45b9c709dd78dd84cb7cf4ee1
22b12c56bd109f6f652f79f223e3cce2e2622038
37765 F20110115_AABNTM johnson_e_Page_57.pro
9a57c4608bd46700208b05c884f34a1e
0e552a3763dba91d1efcc1702af43b8485fdcc09
22920 F20110115_AABOAK johnson_e_Page_22.QC.jpg
f94734868687f2ef77ca44896a9963b7
c858cdd4928dffadea44861a703f5d46f6164411
1554 F20110115_AABNYK johnson_e_Page_62thm.jpg
e1c68e01049f38d119e21a17a3ee65af
0efb47925326ac20fcde0c229d37ae5bf5e1d6ea
F20110115_AABNOP johnson_e_Page_04.tif
d2fdc536f63e7b8b532b7590826479a5
30a2b9c3a893e8b53ce4da4c94c53a3b7f392e1f
72603 F20110115_AABNJS johnson_e_Page_22.jpg
fba1c422d786cc8f21d76bd4910247c2
528078029cf101f9c453a25b941cc9e5c2799a75
42241 F20110115_AABNTN johnson_e_Page_58.pro
7b1af3984ef2fa8c2292455976d17d52
57d9401f7d90d0492b8f58a4cb207f80a773275c
6312 F20110115_AABOAL johnson_e_Page_22thm.jpg
c519bd1be6dd150dc4a4b264b2fe8cf8
ab422f8d3e90ae2859590cc804c19bc5849383ab
6141 F20110115_AABNYL johnson_e_Page_40thm.jpg
158b12e893c204dab97ac85750ec7019
b2aa012ffbc54ce6cee6f7702ab34a790ef034b5
F20110115_AABNOQ johnson_e_Page_05.tif
6a90853316696505929a2add0a3b8196
5c53b52970117c1046ccb80abd1567902d59cd22
73610 F20110115_AABNJT johnson_e_Page_23.jpg
6145ca77313feb21d48cfa281ddb4eaa
282269d2e211adb736a1b7954aa31e1e12cbc6ab
34348 F20110115_AABNTO johnson_e_Page_59.pro
841fc1751e80f660dad8c0d3e8e9065f
9009ca66540d040ab4729d59f44fa680ccf37b65
23981 F20110115_AABOAM johnson_e_Page_23.QC.jpg
ddcc497a0c437227f7896fb6602c936c
8b1524a84f4b865e2464ae70f5d7fcf82e8dee3c
24720 F20110115_AABNYM johnson_e_Page_79.QC.jpg
2e3b84dc70bf12b5d41a5ebf4004a94e
c488c07dfcf117983ead56bfca7c5d361311824a
F20110115_AABNOR johnson_e_Page_06.tif
a4e784bbfb87ea63babacaedc07f33f8
4e191bf36cb4943a8d09efb96163a69d759a5d8b
17739 F20110115_AABNJU johnson_e_Page_24.jpg
480d5d680ed42c5445850e9eb7171dc3
219d88423c9c82882547eba33442ddd841d7dbb5
50519 F20110115_AABNTP johnson_e_Page_60.pro
81822ce7967a23160ae9cb1f589972fc
172d55768d7c1dabccf504bf86c1866224058e17
6705 F20110115_AABOAN johnson_e_Page_23thm.jpg
30a1db55850a19160436b3e4f45f3a60
c44092ff7717b7d3bd3b5f9073684b2a314a215e
1391 F20110115_AABNYN johnson_e_Page_02thm.jpg
200ec4712418631fe3c9649e5763f836
e9a882ccdae289f46a47754f6f75f631f669161f
F20110115_AABNOS johnson_e_Page_07.tif
518c991704213cd94d3bda3d58c3f40c
1fe8e6dc5d062b7d9a50caf8b54af9bfbdaec533
74205 F20110115_AABNJV johnson_e_Page_26.jpg
4d5b8e78c783cabf16a0e53062330f65
5f1b9a770de3a3bc7d0aa20dd329f4944f1ec070
49919 F20110115_AABNTQ johnson_e_Page_61.pro
45913039e515e7948105a98630c7d477
15e7ebd150a3f3da66238b40186380243c6515c3
2100 F20110115_AABOAO johnson_e_Page_24thm.jpg
b9170b0fd0607200eedcdfdafa2e0e6f
1f02eac356296b46aeb0aebed87c461ef1440462
26229 F20110115_AABNYO johnson_e_Page_71.QC.jpg
cf665f5557eb3461b9ac06511161d55e
4cb00d3b1a49f8b06ae2df72e8335463f4054f02
F20110115_AABNOT johnson_e_Page_08.tif
e16b4450c5968a235f952b0a998258b7
d7f4170e0492e2f45c3da16d37fedbaefaac2a92
70233 F20110115_AABNJW johnson_e_Page_27.jpg
ed3fb064fc84063b0fd8709d7f7de813
09dbb4b52959d93e79d9eeca37bfef42faee5907
21775 F20110115_AABOAP johnson_e_Page_25.QC.jpg
7e048fed93d4d04a295a31ae12341653
555cd887bc6ec51c996f4465559b50837487d2fa
24596 F20110115_AABNYP johnson_e_Page_13.QC.jpg
26671586c535d6b5980de762ada9c176
f6dc1a9e46e9844adaf34dd5f8829f7a6e45199b
F20110115_AABNOU johnson_e_Page_09.tif
8d55055a0e16b1133f67cbe67de2d7ca
11fa188e74e48a9dc2986d8e4e218905a9e6bc68
70129 F20110115_AABNJX johnson_e_Page_28.jpg
123ebe671e03ca5dc2b9a2055079f899
0eec8cb7cc2cf57103ccdb23c44c9a2ff8051e04
2810 F20110115_AABNTR johnson_e_Page_62.pro
829062e62e6ee20c8d481a02cd64a239
55f79dcc1939cc5c06de489c44b7581f9da96df6
6036 F20110115_AABOAQ johnson_e_Page_25thm.jpg
f1fa44e54e3f8175e4ccf3857f516350
7e8fc3cd83475e14243d3d6e012977b32cd78462
6461 F20110115_AABNYQ johnson_e_Page_29thm.jpg
f9eb8e3e4f0151d3bb9d5d599acb384f
29ff54fb57458378b9cfbd9f284e236ff4a940d2
F20110115_AABNOV johnson_e_Page_10.tif
150f5d2b0698a7e24e309ccc16868c6d
0ee9afa83d79a918d6e2c4851fe172c64b6390e4
2488 F20110115_AABNHA johnson_e_Page_79.txt
522ba7649412357d9b7844f2348b3195
00dcf8556aac48aa7f5f88f9b06e2d5a53abce6b
77465 F20110115_AABNJY johnson_e_Page_29.jpg
628587f5f90a1a3108a2ad761e46274e
f0c57c2c27b6a17d4a8458f5d40c30066a74960d
41445 F20110115_AABNTS johnson_e_Page_63.pro
a8ab29b51b3f80d94febd40c68c4e2a0
b4d88b2ae6aa5becc231fc863a5465a6412b0830
24092 F20110115_AABOAR johnson_e_Page_26.QC.jpg
74fe09270ecbfa918ad3416fef036246
0bf83a503982529dc802355b6b27bfbac8833f8f
17870 F20110115_AABNYR johnson_e_Page_86.QC.jpg
553ec4b7d3b34b311583f4c231f7419b
db358e3a2cd2f8aa94bd8ab5c965ec1d4acf93d7
F20110115_AABNOW johnson_e_Page_11.tif
3f8ee526de680f695b70b285c8b7d796
7bf61150c27d2c86abffe3c44fe37346401047cb
24124 F20110115_AABNHB johnson_e_Page_54.QC.jpg
a9e2f5d772e71230c7e11aa76381f20f
4ddf7b37ca9cf019d8ed6bbf00fd11fc4ec077b8
67250 F20110115_AABNJZ johnson_e_Page_30.jpg
aae732564ffa2b8b665ea2b3c6c19a1c
0b6933c115bb8d8b23d8084725b021e11d4c0b28
49805 F20110115_AABNTT johnson_e_Page_64.pro
25080f5bad4bd209f65788c0e4ae289a
f70cb3b872bf18870c75578e86b30d927aff877c
22893 F20110115_AABOAS johnson_e_Page_27.QC.jpg
0327cd8ad9ccda93f215c35d1ac78546
2c72557fb405269cf1e7abca4c752fbfa4226537
6084 F20110115_AABNYS johnson_e_Page_30thm.jpg
3d1b1a44a7ccda461463ac61151e64af
4c6fcaca2b558a834f6a7ef4f16080908e7b3cfb
F20110115_AABNOX johnson_e_Page_12.tif
6f49f02aa3e168954cf30708ec1ebc32
81c60ab91c7230c986e54c7c2182c10bcf16ccdc
72231 F20110115_AABNHC johnson_e_Page_67.jpg
5fd34e3a5f386c51c42d3eb75895caa1
eec4395ef78e20eb277c821f54a43b7583a51f1a
51912 F20110115_AABNTU johnson_e_Page_65.pro
d9982e2df86168c7a43508b5e78ca263
32c8e51eef48f5c3f69d409d924fe423afa82348
6446 F20110115_AABOAT johnson_e_Page_27thm.jpg
43a00e316c4ed094f251b95f352c8d80
5424185c169c970cabccf0c83360aeec7f9dc962
6528 F20110115_AABNYT johnson_e_Page_31thm.jpg
2beb69164e03ff0cfd46e97eaadf53c8
1eb91640d7fc18be7b0d098537d71c81df052f6d
F20110115_AABNOY johnson_e_Page_14.tif
402d673a0779b9cd765b9f53adcb9401
03a2a6b1961aa7a2b664d43fbd5f2273dbf7efc3
923332 F20110115_AABNHD johnson_e_Page_35.jp2
36b79114d720cb3f61aef2ab7ad9d1e3
3662bc2e30cde27409c68e6a9b6759fcfc518717
52132 F20110115_AABNTV johnson_e_Page_66.pro
f8aea7cf13dec05a0b569db28d5dcf89
7b3cc3d90ee4eb9155560a3edf5c984f487d4f6e
219104 F20110115_AABNMA johnson_e_Page_08.jp2
0a4fe5dfcd32434501f119fc96e8baf5
90232a8850ec6881cfe368862e71031c89d64b42
22958 F20110115_AABOAU johnson_e_Page_28.QC.jpg
c797435f18be5ad65ba74fe89bc6e1d0
d255e1b73d3585f549222692555c5795fc451b7f
4646 F20110115_AABNYU johnson_e_Page_08.QC.jpg
dd58d2c0d2e7f651a2e8c21c91e4a0e2
5d9ea55501d2579eb6d7a6fda619eb5bbe618f85
F20110115_AABNOZ johnson_e_Page_15.tif
ffd77a28248e0e0f70eb4987a76f78fb
299066eca3719b75ba697f16c92b8b8ca9014a57
72112 F20110115_AABNHE johnson_e_Page_68.jpg
535dce8fcb50a697d44587966ddc5617
3be4a0f779300c67fb66b037fef1ac53cf8cd8f4
50548 F20110115_AABNTW johnson_e_Page_67.pro
2f15a6ecc7e656eba1835cbbe90b190d
f17579da24863235a7bdf77db834c003a63e014d
115257 F20110115_AABNMB johnson_e_Page_13.jp2
448c4ee2c77b95512fbaaac42ca34937
4d0c7b1b9c396b1200660c04d1b269021c6432c3
6156 F20110115_AABOAV johnson_e_Page_28thm.jpg
677c648938622d7f58b26b8640790849
9f2f8d20954ad663f7d16065d8ad269564a70069
23897 F20110115_AABNYV johnson_e_Page_21.QC.jpg
e238719ea4aadac8f4349ca8894bb977
149cf7d779f6092cdbcb4135c0e62ff48815f81c
50567 F20110115_AABNTX johnson_e_Page_68.pro
2875a52b1fe5458e2090d04e6fbcf18d
2b1c64f3c45a76fa44b2badd6f635fca0eedce0a
1051965 F20110115_AABNMC johnson_e_Page_15.jp2
0ca2cd2799e5833f4c1eab57fccc7d2f
fda9f51978bcf1d3d75f076f41faa7f2664a7780
21580 F20110115_AABOAW johnson_e_Page_32.QC.jpg
2cadb18fe3467de8433ca274376a7baa
c45e5e168c8644c3d2da1eb47cec7c02374b1845
44597 F20110115_AABNHF johnson_e_Page_32.pro
10d961fe8a6da88ec117ccc9451a70bc
34dbd106a7c10953bf5f098cc99e76deead61f24
F20110115_AABNRA johnson_e_Page_72.tif
d8caebb029559f4d436be60c8068fb0a
e994508ceaa4e73bd683811e1fd00556f772e699
32909 F20110115_AABNTY johnson_e_Page_69.pro
90ca084bd559a5ee88a8f743d49c8ac1
8c847411ac6918576dcc5c0a017752cf41bce3c7
103261 F20110115_AABNMD johnson_e_Page_16.jp2
433b6be263252c313d2c384f60513c47
ad75dfd1be2b4cee172c279502bb0c179a41d010
6235 F20110115_AABOAX johnson_e_Page_32thm.jpg
f6a0a1b8116e7cfb6e511a069f8fab77
8d36081bb2b2e90ca51875a46939feaa52355189
20373 F20110115_AABNYW johnson_e_Page_05.QC.jpg
af00d136a8ab94345794c97541d0af2c
5beb41b5270e09369b38ee7faa181aa2146ac1ae
127433 F20110115_AABNHG johnson_e_Page_79.jp2
808e81c61365e7e10f6c2cda0ff43505
c01b8390a500bd5323470e021b73b9ffb763d172
F20110115_AABNRB johnson_e_Page_73.tif
6f773f880c4f5816acebd1340a2e175e
0567e5441d6b9966edd3c611e1a4bf9aa1dbe14c
49606 F20110115_AABNTZ johnson_e_Page_70.pro
745237974f412974f4627ed13e670b6e
a0abae944d8d939a8c79168ae8c363b2834e7432
111915 F20110115_AABNME johnson_e_Page_17.jp2
360ac643acc64d409fb9e80417cf2984
25d4a660f3b7ddbbd72635ea0d0b2866b1133c47
25470 F20110115_AABOAY johnson_e_Page_33.QC.jpg
2f746779b1faeba6d246f3084aa46384
ceed72cc2f8f8eb6018489be7554da1ee12e2811
23331 F20110115_AABNYX johnson_e_Page_18.QC.jpg
0acc44a28ca4268746c4fc7a8f8ccd01
9d7f8eba6615a26de0a33c303e23a64474a8c596
107269 F20110115_AABNHH johnson_e_Page_27.jp2
4d8246324a1129d9c16e81e026ff0d69
4f40c1e50131dee42d0ba23e15f818c1dc014af2
F20110115_AABNRC johnson_e_Page_74.tif
3875312a2f218d1aebb61e7110597c18
01ef869b8f2d63e4bb0556d1cee56b77167e0283
106131 F20110115_AABNMF johnson_e_Page_19.jp2
2a90259fc260e34750eb89546166958d
12f6f455b1ca9f77013d3eff671049f95f38b9e7
6610 F20110115_AABOAZ johnson_e_Page_33thm.jpg
a6cbd2d277f5e0b17514271e5a6a6102
1cf576ea5300a39e1bdc1cd2ed43c9aeaec93049
14137 F20110115_AABNYY johnson_e_Page_06.QC.jpg
3a34f5e8ab066681c443cf5c7fce8a91
7928507fcf508bb216f898737d2d4953cf7e95c2
1754 F20110115_AABNWA johnson_e_Page_41.txt
f534f8dc7cb051b2c68292ba0638388d
6484c99759fb4204a2e0f14f57b15d7071725216
105967 F20110115_AABNHI johnson_e_Page_12.jp2
38aeada698ef7151aba8dc06f51b0cb9
ffc8172bf58ec82ba92bfca59144007bbfbcf9a9
F20110115_AABNRD johnson_e_Page_75.tif
58bd63b48e986251ebb79e38e8e74218
b77b8d30329b587eab198282e3e69123e3179370
108039 F20110115_AABNMG johnson_e_Page_20.jp2
1a73b214319e333b87a432f37bdd9cae
c77c4c9c4b17d3197ea78b534c7bc2282e1e0aba
24198 F20110115_AABNYZ johnson_e_Page_46.QC.jpg
76cc86dd2d2ebd0018d6f7c82885de60
6a06f0a1c8ddebd4eada9efcfa8254b0520a65f3
1418 F20110115_AABNWB johnson_e_Page_42.txt
07faae4a80cdef5ec99ad37ea505062a
fd9ee8abaea4606c51c45eab01e59181156e6656
F20110115_AABNHJ johnson_e_Page_02.tif
7b9d41adf77b41775491c74b97187a5a
b7807822d3b1594d4077d69968b09e91f207f6aa
F20110115_AABNRE johnson_e_Page_76.tif
0034f4884a804ef45877b0fbaff7dd23
e6fbf6a7c3049d049adac1453a859b71a3b8f71a
109651 F20110115_AABNMH johnson_e_Page_21.jp2
f705aae45308694df01a55d667297d34
7a4c3eb1ee872a40c65fdb8b5845dc0e05b1a302
942 F20110115_AABNWC johnson_e_Page_43.txt
476e3ef6379ce55de922d370b3214909
c78c1176cbe43d0a1b4bf5123d6ea5358758d23a
70863 F20110115_AABNHK johnson_e_Page_15.jpg
dbdf415757345ef002a3de2afdfa7560
9b04b678a6bedbac235af3ee5ce8db4ab85e0e8b
F20110115_AABNRF johnson_e_Page_77.tif
da10bbb3e4144339317be7a1dc287239
c3e4bc16fe75bc63e207450f135f2a74b0f827a1
1051949 F20110115_AABNMI johnson_e_Page_22.jp2
e525717e963f6104d223ebe07b4592af
3a4bc379b1a7eab8d142ffed24d5eade3133723c
24185 F20110115_AABODA johnson_e_Page_72.QC.jpg
421316fb54d53654f479087e92d7b08d
e1653510da0f4f81163f87b6dcc725a11fed3dc2
1684 F20110115_AABNWD johnson_e_Page_45.txt
f82d2214e3df4b949012e2d2e71bc887
6fd3471edb28aa7b2720841728084245a1f8270f
6483 F20110115_AABNHL johnson_e_Page_60thm.jpg
943ea9c424b75e453947ba83c6325e4e
40a95ddc453f329eb363194252fdfb5c75d70346
F20110115_AABNRG johnson_e_Page_78.tif
e1943950a88c0c5e112dc5f960b0ca56
0a05484aa4c5713554a4fed64434d2798193e7ad
110969 F20110115_AABNMJ johnson_e_Page_23.jp2
333c6c8f058ebaea84980e07d0019e21
f280e85edb5059924172931b205ec40dae2237f3
6812 F20110115_AABODB johnson_e_Page_72thm.jpg
2861b3eb79985aed6c3520d57fc78545
500fecffb75bd97082c79042f6fb4bac1a326d2c
2117 F20110115_AABNWE johnson_e_Page_46.txt
7433855627c16b7610a7826b68b6a123
51869551da3232265a09ea319a95aa8e70d6a87a
2155 F20110115_AABNHM johnson_e_Page_07.txt
a5f9cac1e3d5b91ac455be74dfdf06d3
55a08a1a19c06c7dc0840728c3255776390f9d3b
F20110115_AABNRH johnson_e_Page_79.tif
1f5b0f15675e032d9fbc91cfe5db25c1
d30d6fd9848b28a7b49a9ca0bf89aa4e3ca2f8e3
6821 F20110115_AABODC johnson_e_Page_73thm.jpg
2ed8f922c065e26ca355e02c05641b41
f6988bb6ab9f776f482d863ea2021ceb742bafdb
1940 F20110115_AABNWF johnson_e_Page_47.txt
412b75e1230bb31f6c12a313cb9d33cb
a1610a04153e956636661512b338c95d497d409e
F20110115_AABNRI johnson_e_Page_80.tif
b99dca3c5faa4dbc0776d233edb7e6fa
d8bd418a9a0f797f86d856ae75618744b1f61de5
19891 F20110115_AABNMK johnson_e_Page_24.jp2
06bdc57d91e083253ae45648f570e1fe
0bb42ff0ce4ef045968da61f56e728afa56ee270
49236 F20110115_AABNHN johnson_e_Page_26.pro
55d03298645d5ed92fde85855b264fef
c986aa1adcc07d911a544fe95f9ba41a89b7e3ac
6708 F20110115_AABODD johnson_e_Page_74thm.jpg
d557f31f9c00cd510ca5b883bbb1a3fd
cc52e813f1ca7c6a66cf3ddee5e75f0abcc64099
2076 F20110115_AABNWG johnson_e_Page_48.txt
d9e7470a657b19c4a71e32261e1746f9
6c229910e46263e6101d4c97b92be7c1a2eaf520
F20110115_AABNRJ johnson_e_Page_81.tif
6934f66669e3036670b8c91d2353313b
deec104fcfdc0d81269ddeb9f7b9130d62eb1a96
96316 F20110115_AABNML johnson_e_Page_25.jp2
4402a03024dfb1ebfa7d13128798c478
c964481e3653eb10a560cb7dc919f0ff6a197a58
98912 F20110115_AABNHO johnson_e_Page_32.jp2
e07624f0afa3b678a1a6335e98c78ae4
27d56070c5ecb29ba5b588ad0efc5ce4c4e56c8c
6990 F20110115_AABODE johnson_e_Page_75thm.jpg
cec56b4c0dc3f40f995a7aa7aaeb50c2
ab1f7caad9b4af3a802e1442537ec876b6cf4229
1259 F20110115_AABNWH johnson_e_Page_49.txt
73558973a7d3ea7a5e089fe123a0f9f4
fd9c4ec423b2ad016cafd6f41cec2bb33028c4b7
F20110115_AABNRK johnson_e_Page_82.tif
83e16b766fe7f3a80cc973f876346707
436046a9a5b00937119e95e7bd9b5232337101f5
103753 F20110115_AABNMM johnson_e_Page_28.jp2
e1d8af2c30253f17f8fa127a87d36da9
de29509a45a3240fa432d47c046ac3060b67f69f
61871 F20110115_AABNHP johnson_e_Page_77.pro
eee9532428cf9855082add2d7ed3a90e
b858bbd9535ee4d82c7570ce162a2c110fa5a8e7
25639 F20110115_AABODF johnson_e_Page_76.QC.jpg
ca4f76d554ce76813037a8b34092a942
44833f4e583b9d52951cd9eaeaf7572e2e4581ff
1788 F20110115_AABNWI johnson_e_Page_50.txt
85115f98fbe25d9a1734046538dec2b7
17cb61ec73c94f3c200a03c6f2e304d70a366eeb
F20110115_AABNRL johnson_e_Page_83.tif
ba5d4e2c0996750e04db3257403f5b16
2d0d9c2bc3333d0ffd05c34052157abe1ee4407e
1051948 F20110115_AABNMN johnson_e_Page_29.jp2
9f0dd149bdfa60b096fd4f908a40e633
cadf5002e77438773cac3ef8dc60074ca31d3f1e
109138 F20110115_AABNHQ johnson_e_Page_26.jp2
ca13a29ecc3f52f9892d98e173f96f64
e361b6a7db113955851bd6fd67eccc431e8b6f95
25356 F20110115_AABODG johnson_e_Page_77.QC.jpg
0fa0723b8695a19c9d0224cd3b54a886
434efd3994647cf4284fd420409130616c2b4751
1925 F20110115_AABNWJ johnson_e_Page_51.txt
08e58fe20b6f1ffed04d3e86e6e64b7b
e68673208fd6a31b366aca2b3be9daa462a70d0e
F20110115_AABNRM johnson_e_Page_84.tif
0ceec96c835e7e75f6b061cc348e6e8f
84955750ee8aa94359828f81ac458e4c7fe3ae20
99909 F20110115_AABNMO johnson_e_Page_30.jp2
5f8454dc1e35fc7bf3f14cebf8e58c6e
16ff372310d86aed6d2d1acb837e84a5a391264f
86325 F20110115_AABNHR johnson_e_Page_09.jp2
4d6f9626f2e209cd294ca4235f07393c
a4f911224ebcca56cb02d8a51c7cec345461f4b7
6828 F20110115_AABODH johnson_e_Page_77thm.jpg
e126c933c208fed60b7a1b86a6fa8c9f
8ac2948bebceffbf375bae6ceeb1f61789b718b5
1728 F20110115_AABNWK johnson_e_Page_52.txt
d10ff02deb6cd796a8bade73c895cfcf
71bd3eab06b8d0b7fda2ed4566e670250284fac2
F20110115_AABNRN johnson_e_Page_85.tif
302d007c0ea95dd2b07e79e7e688c3ff
fa7bc47f86695d5dffae89013321f3a1fa929742
108757 F20110115_AABNMP johnson_e_Page_31.jp2
2d2d4ca203c36311e822039d1dcd2893
731fbcf5686dd614f9dd353991fd71d47466a7e2
21783 F20110115_AABNHS johnson_e_Page_14.QC.jpg
0afcd8e854cd1d338a39d28e787ba2b6
7ae4280b9f61b4af7e5f5f4570a04a3e3cfc4175
25287 F20110115_AABODI johnson_e_Page_78.QC.jpg
c89fb175e20d1abb1ad6e2e3f88c9681
020bfb73b49e0c7c13de0f1abb1d1cabaf2223a9
1899 F20110115_AABNWL johnson_e_Page_53.txt
57289d0e601ba8eb347f6b03b4abd16c
6644a69f199e19260b502268df255ca77982b315
F20110115_AABNRO johnson_e_Page_86.tif
8a579ca75370a3d4400790fb3063baaa
ea922418ccb9697d4a3fb58834e00be5cfb47a50
1051925 F20110115_AABNMQ johnson_e_Page_33.jp2
e424708f61d56c8a0af89898ce6ecf75
e306849eca03e5a4e569436194bf7289e824254f
F20110115_AABNHT johnson_e_Page_13.tif
63d3431423818e887cee9a839f751a3b
84ee96fac5d9348d9b53b41b6754bf717cd94f6b
6739 F20110115_AABODJ johnson_e_Page_78thm.jpg
f398b6b957def8e9fc0db97751589b1f
0901c965531295d23e141f99843dd8c199459a60
2011 F20110115_AABNWM johnson_e_Page_54.txt
cd9a0ad2feaf5060978cc24dc1165356
550699ce7da8b00b554142f03822f6695c94ba3a
66288 F20110115_AABNMR johnson_e_Page_34.jp2
ea83a60e897b2d37e24c521b2da095bc
8ec5e989748d2ca7d0008de737fd95d498b2a4a4
1965 F20110115_AABNHU johnson_e_Page_61.txt
a27e7d39150efb04592c35c7f630df20
77249a811d91acc4f20b2d3c97c03f6538831e92
6655 F20110115_AABODK johnson_e_Page_79thm.jpg
15f0f1aebe8faca2c5d3b8af54de6820
97aa1e43ea68b3863c151523711ebd80d1027428
2463 F20110115_AABNWN johnson_e_Page_55.txt
9dd0b978cb4d96afe5ff96918b1ca05d
9d544f4317e23e19e9bc7887b0a24bfb2370a121
F20110115_AABNRP johnson_e_Page_87.tif
a40ca5ef8be7c6d6113b0eed075d3277
1bd209c149fc95d6ff996cc3f51e017776ea1715
983609 F20110115_AABNMS johnson_e_Page_36.jp2
3fc87e3fc556a8bc82f78785f1bd5327
7b3a04d440d3707c3be30c9d6c5787de794fda38
22950 F20110115_AABNHV johnson_e_Page_38.QC.jpg
e900715865495973b9a5f13343a6fccd
18226e9558f6506c43f086918e96dd539ca611d4
F20110115_AABODL johnson_e_Page_80thm.jpg
c918e9dd6f31dfe19044e04d74f70cfb
97eb6721e2590237cceae840756ddff5a0e1f674
1545 F20110115_AABNWO johnson_e_Page_56.txt
e7bba799bda58d6e37e83396ac4a8221
019402ad60a3dfb495e56d8fadc66e8d45132b06
8502 F20110115_AABNRQ johnson_e_Page_01.pro
03da8e7dc2f422dd4ca29a11d668cb8b
4aaec60067710c92b35563a4928fb6053205238b
801910 F20110115_AABNMT johnson_e_Page_37.jp2
5ab0e19438152991032f0ab5628b8de0
b059fc8615562bca4e6bca814a36653f6bffe18a
6511 F20110115_AABNHW johnson_e_Page_17thm.jpg
756fa4c9b17557e396e87b05ba00293e
2e0e0092fa1fea3cfbb59f395650444387986b0e
25540 F20110115_AABODM johnson_e_Page_81.QC.jpg
a3797f2f7b3b69423e890e952c4f8ee1
74dd15844e644ad6261aa1495f1f79e9e005423f
1626 F20110115_AABNWP johnson_e_Page_57.txt
c6f2fd6c5c9f9645fae4fcc79ce9481a
5cadb3fecccfda43425e210153426000c41ff06e
1279 F20110115_AABNRR johnson_e_Page_02.pro
f39f2fb371492187a871c6c7d8134a1a
edd5b25ccfddfe5d19c52da3db2ac75a0f63f5de
1020204 F20110115_AABNMU johnson_e_Page_38.jp2
5a49786381548a93f54b81f46a2fdeba
fad4812062189e2e7485815c78e8170b069a88ef
21933 F20110115_AABNHX johnson_e_Page_30.QC.jpg
b20ed4124fea918cf859603ba665dc3c
fdb57833fa9b6aed0a4721c2d6fc5acc135716cd
6986 F20110115_AABODN johnson_e_Page_81thm.jpg
fe164f4988802e3eb4933c34e2d78922
7ab14ca0c72cd1586bb3804d918cdfd10b39438c
2045 F20110115_AABNWQ johnson_e_Page_58.txt
739a543abde9d13048a6901770ea2a97
cd514c7c8ef0ae662a54631f2ba1fb9396b4e9cb
3006 F20110115_AABNRS johnson_e_Page_03.pro
6bc12cf1ee77bee177cc48fb96872a34
c7f22476012a658ced59b96d6a5c1d6649a980a7
639644 F20110115_AABNMV johnson_e_Page_39.jp2
30f4b7282ee23dba374cbac70ac46086
fb739c98292381753950c591e73c59a3a4b090cb
F20110115_AABNHY johnson_e_Page_47.tif
95c42ada351112a290aea20fe1e42d6f
0f8219d7777f4288196120c022779edf44b9c22d
25288 F20110115_AABODO johnson_e_Page_82.QC.jpg
06cbdace548eb15b08deb539dc0631cd
4fac8320d0b6d67ffbc841d776d493c1ee1c2d3a
1646 F20110115_AABNWR johnson_e_Page_59.txt
866d222f9d614470f6306d1289820a9d
6d4561cc87afebffe2ec145a160ccfce0bb29acd
38872 F20110115_AABNRT johnson_e_Page_04.pro
d53e63948e04dcc47aae31afd547baa4
463c696c6a5eac8cbedc112bb718f7134078466e
1051962 F20110115_AABNMW johnson_e_Page_40.jp2
f8eb47c8d4a1b1f94939b4c6e0ba4b92
bf704946b355a7375327f92d5357bfb40146be18
83892 F20110115_AABNHZ johnson_e_Page_74.jpg
1a5836523b9dcff7208a21f506386a1b
9f5f89c5a386f2eb3138dd11857a1ca83f2ee0cd
7034 F20110115_AABODP johnson_e_Page_82thm.jpg
8a88fd396275af09fb5211733b93833c
d0a1c9ab2196e0e5418be25d02a2a9af15605aaf
1983 F20110115_AABNWS johnson_e_Page_60.txt
aa6e2813660e56ebfb8792379bc56ac9
bc38daa8971e29859c3dfd241b789b02fc23b210
76749 F20110115_AABNRU johnson_e_Page_05.pro
53ff8ffec3a2ef9d9728ee219db08afb
fcc61eeee6f0c3146805456a14e348a11f5927ba
1051960 F20110115_AABNMX johnson_e_Page_41.jp2
981ed5fadbd4bc6d6802e63233c5c1a2
07f2a29202141eba85946c5cf2e4d4fa93b9ab20
6763 F20110115_AABODQ johnson_e_Page_83thm.jpg
e73dfd90ecd7c5bcb2042e80484c7cbe
87de5fb258190fd260cd008542de26491fb73401
159 F20110115_AABNWT johnson_e_Page_62.txt
7277485474ec6076fb3aef49ab4ca67d
aa20819d50a08d76d8ff687c311511aabc56459e
44791 F20110115_AABNRV johnson_e_Page_06.pro
8436abdb702b2b993fc932abdb8766c8
7b3b8475225d0023c4a83a701756ca5a2412675e
73816 F20110115_AABNKA johnson_e_Page_31.jpg
fcbab6139d4cdffb0ac9f20199f3fe87
6f3ad7520cef77a90debb83ff6754d709d0afdda
1051974 F20110115_AABNMY johnson_e_Page_42.jp2
deb3952409a824cf0774f9956ac43e7b
8b8dd316918424e48353862da5e50f5649a9c9ee
27478 F20110115_AABODR johnson_e_Page_84.QC.jpg
fd5cdce327d15c13ba0930ad63737f5b
e839fc88c5cca1eeedd56ac13eafe29a48665dce
67222 F20110115_AABNKB johnson_e_Page_32.jpg
22b78e836794e232a9e324c4799f0d2f
e9fdd2953dbc4d9fa37bc83d7d21e5448340d5e6
1051956 F20110115_AABNMZ johnson_e_Page_43.jp2
c590d40024d3d0a546e03067bad7b8cf
7527528320cbce6b8b9ae0649eaa6a74f6684a77
53508 F20110115_AABNRW johnson_e_Page_07.pro
0a66243fd2f97fb4f77cdac72c8f40ea
bbb7ed7ac1d2f3b35ae0fdd1dbd83055592d94d0
7204 F20110115_AABODS johnson_e_Page_84thm.jpg
bf679b0cbf8f314604d483fe675c9839
ed522cb7c1bb54d044dd8eef696a46606ed2889e
1745 F20110115_AABNWU johnson_e_Page_63.txt
22662de533e33a0207f397aa3b03cdd2
72d42b0058ae7324b8722df171649483ba5f6db4
91368 F20110115_AABNKC johnson_e_Page_33.jpg
714e1547361554fe333367cb1210301f
f8e17610ac2de6c434f4db654dbaffb98970c49d
5192 F20110115_AABNRX johnson_e_Page_08.pro
588a78c21ebf343a8dbd873166f401c7
070a448a52ac25c452a36d8bcf8071b37ee8f218
26296 F20110115_AABODT johnson_e_Page_85.QC.jpg
c65414530c6b04a1d0ef183645e91e3c
c8fa3a448a1a422637d584a81101e8947e509500
1966 F20110115_AABNWV johnson_e_Page_64.txt
22404d60a895ab0dd2f80e55ebefd828
ba98d475db9bddee2f24f15341ab3402f994f345
68714 F20110115_AABNKD johnson_e_Page_35.jpg
5cdf36da8cdc1024b7f16d43fa1fc967
c7bdbf3e877f25ce004e6f25a448f6f6cb0039ba
F20110115_AABNPA johnson_e_Page_16.tif
7ebd5a53722b9a4fce4d86af30f77d37
82764be5c3a7c2c3bd6d2307bfcb255b5fef40f9
39433 F20110115_AABNRY johnson_e_Page_09.pro
2b3a5cf5bcbaa359f86a9b15914908e1
6e69e668c4e39354bc5ccf010cbc33c4e6ffc1f8
6962 F20110115_AABODU johnson_e_Page_85thm.jpg
e1e01a7de2618d4af21231afa53edcec
f725bc62f869e760851a3c82edee7746bf32c47c
2058 F20110115_AABNWW johnson_e_Page_66.txt
36903450a123ee8a8a3c6483ed0d2fbc
a8e8d4f30f6bf0abf4cdd1dcef90a6a696fa1702
68656 F20110115_AABNKE johnson_e_Page_36.jpg
f8b8a0f21437b382d694b6df6c343407
9f46968ced33dc8e604bd766e7dbf03c47bf7a8b
F20110115_AABNPB johnson_e_Page_17.tif
375e59621a8abb5ccb673241016b7955
541d0c4cb3fd23a7da97a86fab8e10601c993cbd
34417 F20110115_AABNRZ johnson_e_Page_10.pro
a47ccdd56a0662b9c0aa709db0d7d3b5
8fea63be66b385e2224582cfc63c76820e9ea04a
5216 F20110115_AABODV johnson_e_Page_86thm.jpg
ebf70e0133c9fdfd92ef6abce6fcfccb
3cb83de994b82a512c20b8181d49846f4c1878cd
1988 F20110115_AABNWX johnson_e_Page_67.txt
10bc7edd6e74b5ebae58a1ab3046eefd
ca0d9637aa63aa370b9b80b5e7f8c54de2302783
61190 F20110115_AABNKF johnson_e_Page_37.jpg
217ae39e21b5d67b2b0a6e6ad06277c3
5effc44942f5f3d823ac7ed96d3f39a4da163f95
F20110115_AABNPC johnson_e_Page_18.tif
9aae07a76351002f0df1133d09ef4df3
e33630273a49134b5565eeba014fdc64394c7744
13708 F20110115_AABODW johnson_e_Page_87.QC.jpg
6687d3418db011f7756580a864da2177
74187b8ae8525dba6a708004ef575ac84e285f88
2019 F20110115_AABNWY johnson_e_Page_68.txt
142b74d24783b82b59186f23bd42ee24
9bea8d88974adf313bd1201b01c18c99a092f8e8
F20110115_AABNKG johnson_e_Page_38.jpg
2166ce60f780b3c8785e486ee4b3485f
cc3596846451e34f14ffe3038378505ef63c60fb
64295 F20110115_AABNUA johnson_e_Page_71.pro
f26253c8439a436c25fa9d052a4bdafa
01e1e91b5278c77a591d9f4a8ce975284b34cbea
F20110115_AABNPD johnson_e_Page_19.tif
42ba104d4912a1b2a5c2d187a43be5da
3889a6734869ed987147ff3342ae395d2c16c3a3
1320 F20110115_AABNWZ johnson_e_Page_69.txt
9d8d834dc87a754530fcaae26eed1c4d
848a7282c44c005afd5ec3228fa1ab65a9278058
49974 F20110115_AABNKH johnson_e_Page_39.jpg
63eeabbb1f14ebbf82bf47c8aeb6fac1
1fc6dfacb723cefdf8b7cbc76b651fa2f03fe462
58915 F20110115_AABNUB johnson_e_Page_72.pro
25e1d3738b60aee63b8ca2a819289fc2
65b6fef0067471f36db41979231bd86026437824
F20110115_AABNPE johnson_e_Page_20.tif
b122f9bdbe7735e19586b1b20ae7592b
b1fae4d2c2eb99d1555c91771278849a64cb6700
60692 F20110115_AABNUC johnson_e_Page_73.pro
03d48603c20b769280adf1953898be23
65cfaa248f718c7233290f64621b79e216034e26
F20110115_AABNPF johnson_e_Page_21.tif
1a2386b38b0b06c78c6a6ba1d18b5813
4f5d6e1a7151d3f956f162d860296a7682a79616
15247 F20110115_AABOBA johnson_e_Page_34.QC.jpg
c85e78adf2e520bf2c9b5b07dcb3c63d
b101524b5f3f93b063291f1ccce0a01b3bc06d04
26431 F20110115_AABNZA johnson_e_Page_75.QC.jpg
5ba686dc1b02b4e9a068d18f55ce06a4
4a01607753a718343a2df48223790d9e2ca2fc5b
73963 F20110115_AABNKI johnson_e_Page_40.jpg
b08dfe3e594f6b808ac1013c33b96857
e6dbc0c908825b88f8a6f6ac35dd6311eefc47b9
55050 F20110115_AABNUD johnson_e_Page_74.pro
329e5610fd3cfa40c23831388d3c4193
59b51c60079ada220240d0d2dc3af51ba6473afb
F20110115_AABNPG johnson_e_Page_22.tif
23ab8d8b3d6b0dff0cf6e367b676cd0f
959afcd93bd30f10d594cc3b3de1ac9308b80f97
4328 F20110115_AABOBB johnson_e_Page_34thm.jpg
ab466a9fd32b5d595404c2f58979d414
68d1d5cdf34ce735ddd0ca41d79015dfe041e037
22245 F20110115_AABNZB johnson_e_Page_40.QC.jpg
1c05ece74739ba3a3a57d5b6f898b9fd
a800a4a9a0899bd2e7330583c4e9bfdce3b34893
62548 F20110115_AABNUE johnson_e_Page_75.pro
21680888a853b319f701dd9237f38449
3426fba1beda817516f99d061a6335ca4e596a5e
F20110115_AABNPH johnson_e_Page_23.tif
0ac277161ef6fc0751bb890313f2be08
0a93f3f130f35f73b925d2e430ff2681cd81081b
85101 F20110115_AABNKJ johnson_e_Page_41.jpg
00cfdd5765b92a7acd4a5789d8589332
e4d72ff2ed895a7d3922b5a0fd27847b0e0f1288
21905 F20110115_AABOBC johnson_e_Page_35.QC.jpg
24295dc79e8881639c66bbec46360e99
35badbdfb5e9ff3f7b1962ec4fe8c17e35458f93
7205 F20110115_AABNZC johnson_e_Page_44thm.jpg
b30827e769c38901f9a34062385c6445
6ed32b1c028753912e1881c39eebc77d888e2c6c
61825 F20110115_AABNUF johnson_e_Page_76.pro
da5744089b6f539836f769ba144324f7
685895125e6e6819f83e847a768bb91aa0285e70
F20110115_AABNPI johnson_e_Page_24.tif
340b488496f06dbeb872a23c024b7ca1
b80e1cdd4eb2d11e7fb77fc4dd2dc955644281cc
76309 F20110115_AABNKK johnson_e_Page_42.jpg
34fa8cad7120f9c1513d5f2a62991aeb
f1fd9c6c0dbfc7aed31bdfc53e12143b88ff13b9
5922 F20110115_AABOBD johnson_e_Page_35thm.jpg
a7d9a85d7d03be93fd2dea235bf7d2e3
05dc024f699a1e5193e462e6a3f35824279ccd76
6105 F20110115_AABNZD johnson_e_Page_15thm.jpg
2247307f929f3cf3222f0a437038265b
8560d1091c675a7f585302ac6a501be092a7d242
61639 F20110115_AABNUG johnson_e_Page_78.pro
612eaadc49a062c5a084baa7c0da6c78
4d4451d62463a3d4642a0706b30534e56830cbcc
F20110115_AABNPJ johnson_e_Page_25.tif
4815dd313555c357bbbf5a3da882f18f
07f7a85d66661474f97553285da66169971bfbd0
65308 F20110115_AABNKL johnson_e_Page_43.jpg
b173c094cffc90eaa5b76168b9ca76c7
eaea70b1ab78f4f9b58daa85bee25d9e7d9944a5
20601 F20110115_AABOBE johnson_e_Page_36.QC.jpg
649aae1e878f87003b75a44a6f960890
72aefac713a5c619946d4f373e799c81647878dd
24364 F20110115_AABNZE johnson_e_Page_31.QC.jpg
ee5d5a9b05c206aafa88d59d7ed571df
808dd618e2ab79222cb69c0f1f5c6f4ef114fdf8
60217 F20110115_AABNUH johnson_e_Page_79.pro
c23122cd078d712a6178e5a5dc3d9035
966716efc61d8398e99a4a64adcb61bc58bd3322
F20110115_AABNPK johnson_e_Page_26.tif
60a40f7bb8b93b1d15bb379b05f3de45
2959e3b75a5b4ccab98967e9a862dd0d29301f69
83056 F20110115_AABNKM johnson_e_Page_45.jpg
24ba4ea87b915ca166f2fade3288bb47
f65da74713ab20dd39596ac781815b84ecd880b9
5574 F20110115_AABOBF johnson_e_Page_36thm.jpg
95397580aa8594b9211330f0a47576c5
d56f1b8277e0396e8f680e9c7f1a157c0c9d1f62
15601 F20110115_AABNZF johnson_e_Page_49.QC.jpg
66cd8b8bfa188474a0fe3441b959a418
c8ab66b88867e2b4616acbb7cbccc315fa948c38
59084 F20110115_AABNUI johnson_e_Page_80.pro
c04e3fac6ede35013a3e61c77adfe1f7
c8014d73258a26da55b0ee7685eb8f7df55845fc
F20110115_AABNPL johnson_e_Page_27.tif
d6ccf2f18f4116f5aae199dfbac3147a
d0acb77fd4b460f774bb5044933a78c92f150c16
71189 F20110115_AABNKN johnson_e_Page_47.jpg
c9f8c08911dac2e02dbee7da2af1a966
a6abca4346c9cc4046438c9212ce6c2dc8ce8387
16526 F20110115_AABOBG johnson_e_Page_37.QC.jpg
8d0e8bab0110288406b20fcba37c10a3
feb00f60dbc1cc1f91a5c7eea4fea14d80e0e319
6531 F20110115_AABNZG johnson_e_Page_42thm.jpg
355e27271f1c1537acac08be284006f3
42d885d89a49da74acbbab47cd4920bb7a095a4b
60436 F20110115_AABNUJ johnson_e_Page_81.pro
647d8367b0435b512367ee9f69eec327
2d29df814665a85b40eb6561cfd8488c50d2ce7d
F20110115_AABNPM johnson_e_Page_28.tif
cba3b0c9c460e2dd5a7ae3d4473ee8da
4ffe19f9503ef6092a0201065027e1b4d840e055
74327 F20110115_AABNKO johnson_e_Page_48.jpg
adae8a249463704c9aca2c2cbc2d0727
4275df416d3adfe30bd0d392924ec5ab39354ba0
4431 F20110115_AABOBH johnson_e_Page_37thm.jpg
466a0cfc8036e83243aecfadc17d6f54
b94b36a82ac3ee3ca8b8b5f700fccbedf70ff5ab
6492 F20110115_AABNZH johnson_e_Page_18thm.jpg
313a4e03af529bcad967d56f2bc453dc
d973a01ae7f0d1ea901cf5e5e6d308213a93accd
62909 F20110115_AABNUK johnson_e_Page_82.pro
420cebff1058627d3df17a915ec636e2
701b3966d28f3d3d11401b7f213c160ee7cf9c1a
47878 F20110115_AABNKP johnson_e_Page_49.jpg
eb964b78e8383a7e9d086ca6d05cc8c3
98ad24446f0ec14f5d515f071970ec2cd0ce4046
6501 F20110115_AABOBI johnson_e_Page_38thm.jpg
29b7935b56d2cb972c64f786fbc68287
eb57841340410df0dceb164ffbc04bff2dfac045
20836 F20110115_AABNZI johnson_e_Page_50.QC.jpg
228a2e05d1fbf836072ac8afffb6c68c
2fe9105ef207943e45e9c17b7406a8f51e1ebc10
60660 F20110115_AABNUL johnson_e_Page_83.pro
538de16589fc854dbc65edb10bdc5a3b
9e9e8db24cc9c5f44498b85b6c2832fef991b02f
F20110115_AABNPN johnson_e_Page_29.tif
2ea043d11fefa90f043e1ca7f5924c23
2de798fb4631e1504410fd34f999570df3c9bf47
63468 F20110115_AABNKQ johnson_e_Page_50.jpg
02ad50301b1166bcdb36d2bed0b15ce4
adbaf80fb5b1f5e05a16fbb9f57edb962996b105
14852 F20110115_AABOBJ johnson_e_Page_39.QC.jpg
19ad8224ec4d67195f27773173553d49
9efac6afcff895d1084e11a8a8f81780f6dd5ed5
3957 F20110115_AABNZJ johnson_e_Page_62.QC.jpg
7ac24055b3dde4091e7c83ca03c53d22
4b8177a1d1bae2a5ea1338add511d7c0a5c6da2e
62715 F20110115_AABNUM johnson_e_Page_84.pro
554b4f7d13298f7c84969ef0467a8a92
b5a107fed117cb5b741aa0b23b1285bd8342a7c6
F20110115_AABNPO johnson_e_Page_30.tif
6e9a513627d0dc100e64db116a99b260
81cf08fa9981d92dcac87c7d871eab4cb36ce942
88830 F20110115_AABNKR johnson_e_Page_52.jpg
a4d2b2e079582e3306c87af54f1ef860
7e2ffd947afad2dd48956d0e1a46a9dfc8ca54a0
4485 F20110115_AABOBK johnson_e_Page_39thm.jpg
4397b1e1bf5a4fa8793dab5a28fd1a27
922ace1d9ee17cc1823fcc0c266610ee97938b86
24244 F20110115_AABNZK johnson_e_Page_80.QC.jpg
75af9274b093f240121f919375ecf149
404e87901abd59b3195fedd2235f3842bb41327a
63571 F20110115_AABNUN johnson_e_Page_85.pro
2907a61df87920af9498c3da69bd5160
0af26b586f5d67b9aa50a76211447a1425a3f7c6
F20110115_AABNPP johnson_e_Page_31.tif
1ecec15df41710f843707eda6a207ce8
c3d852c4c6dff0f3ac4b8b1d1d90b740c06426b4
74112 F20110115_AABNKS johnson_e_Page_54.jpg
8dc8b1fd88c188ef15f2812b14c47efe
736f4e88d5ac94bef91c5889ae6be431eec848a4
25208 F20110115_AABOBL johnson_e_Page_41.QC.jpg
b3701ebdcfa891c7052213c58e78c9d7
9034913401b9bd41cc0365deb4983bfdd8ef3898
20839 F20110115_AABNZL johnson_e_Page_15.QC.jpg
1ef8b8c9efc21acf909d482d5d2a1bfe
f65d4abc9754be3229a885db9087d0a932f1e931
40335 F20110115_AABNUO johnson_e_Page_86.pro
e99d6773b87b200dbddff4d176db7e8d
0adf97f6f4a7ae5dcf038e7c02d1318df6377e17
F20110115_AABNPQ johnson_e_Page_32.tif
601a24337ca7efb1e43cb62854f0b3ea
c4860df17525a91bf421b235435bdb485a1ae1d1
88248 F20110115_AABNKT johnson_e_Page_55.jpg
bb2a3aa1d7a251bb7c27271b9393ec94
8c06d5cb6c7dc4f152798554eb553da0e45acab5
22344 F20110115_AABOBM johnson_e_Page_42.QC.jpg
1115368a7c7576fad4ff3065a4fa362a
6af53c0661719f600e87f71c0eddc7494edd78c9
18857 F20110115_AABNZM johnson_e_Page_09.QC.jpg
43104fe75eb9013583574785ec095f54
65bd40c87d617b86af63bc3e5c5d3889820c6732
25272 F20110115_AABNUP johnson_e_Page_87.pro
56e10f588bc5cfa0cfc44a9247618d84
93d8b0334ce33e834a627125e2273770c143c632
F20110115_AABNPR johnson_e_Page_33.tif
a2a4c9715b5a0646357537bfeee519d7
8b4c38e851b75f1dd29a62c6ead391db224d64aa
87241 F20110115_AABNKU johnson_e_Page_56.jpg
9b2e7b0530e03b047d1f928a9a1fe251
9fdd8fff05a35440a7d492e3b01f58961399c7a6
6148 F20110115_AABOBN johnson_e_Page_43thm.jpg
994dc09ad5a679da3e14867a86597be3
48c931ec518d3db7bfa28c292f34539fd0d4bfe6
131839 F20110115_AABNZN UFE0008337_00001.xml FULL
05c61a62ffb135839962508d1b1c98da
b2c63607385b93ece6c7a4bd3646c2ef7bb511c5
468 F20110115_AABNUQ johnson_e_Page_01.txt
03c91c3b9ea205501be636b9286c91c6
4054046ce914ba67f68ea90ddead0b028f4ceaa1
F20110115_AABNPS johnson_e_Page_34.tif
46550a7b6eb5ad3d03972d397f98d798
339f0db8432674d4642ad7baaf4857e9546f2e26
80396 F20110115_AABNKV johnson_e_Page_57.jpg
da8776c35176835679f9e5714104bb43
595a13cc4b46e940f66b96eceedbae32ba7375fe
26947 F20110115_AABOBO johnson_e_Page_44.QC.jpg
ffd49f8ab79a5b4d37cb0e5bec40ca33
5eba063231833592d67e631696f6b99458256095
7550 F20110115_AABNZO johnson_e_Page_01.QC.jpg
f1ccb9acba9a89019f91808175821a9a
c9d29d39a84bd95ec9933f126414c04831c193ea
119 F20110115_AABNUR johnson_e_Page_02.txt
add366a6ea0d4a792acfb5ca5cd52321
f266b918367eeda0afde4830cd9c33bdac4d6e00
F20110115_AABNPT johnson_e_Page_35.tif
5b289573a9999b091bf389e54b72e457
9bad0a4a3790f237209409f44d43956a4e04fc76
71355 F20110115_AABNKW johnson_e_Page_58.jpg
0f5f4edef309b40939e2888a32d47ecd
0f15701077dfed48cd153e2f64e11742d60066e4
6567 F20110115_AABOBP johnson_e_Page_46thm.jpg
4ff6e5d2387f148d6f95821762e39394
e1202e1c27561250d84a533378814fcc94d89174
2458 F20110115_AABNZP johnson_e_Page_01thm.jpg
7a6a7a4a8784333c8882a544f7d03a65
0f3deb9aeba4442326b6510face34f637a74ac98
F20110115_AABNPU johnson_e_Page_37.tif
a4a7e183bc35e35ba61e318d35dbc487
70fcb33fb2b79a67186f7570dad2ce37364651b3
67122 F20110115_AABNKX johnson_e_Page_59.jpg
47f0dc421f9b08abdc1a22ae5ce3dbc2
5670cc8a40225a6edf0658e3447ef6b2b67922d4
6465 F20110115_AABOBQ johnson_e_Page_47thm.jpg
16ec9329cf0903bcaea0a66b5f3633d1
dd3a356c8d5ff5513e9fb5bbfc1988d6c157a4da
3360 F20110115_AABNZQ johnson_e_Page_02.QC.jpg
abad9c61c6fb143bcf1e397d97d0e4c9
564e468e51df822c5aa167528ae0576c7390a7e6
F20110115_AABNPV johnson_e_Page_38.tif
255d7be5d5c9fd2f8bdd10435140227d
02ae013fd7be96c6c97fa8c933283a81f6d0c195
87941 F20110115_AABNIA johnson_e_Page_83.jpg
ffa138b0ab0a06db140de764e4a32e3e
7b88ed90957126ee7ef53f5984dbb57f0693e694
73067 F20110115_AABNKY johnson_e_Page_60.jpg
1441f0a83626d4995bc3baed5c960e51
bfab9f598e62bab88993317f6473af78b85960c1
200 F20110115_AABNUS johnson_e_Page_03.txt
cce8acafe5b21612f7ddd682bfc9041e
6e53cc18bcb4e627d03e1c7fcaf8f93f149e2393
24297 F20110115_AABOBR johnson_e_Page_48.QC.jpg
ceb5f8083e2bb2a7e1d452ba96df6425
ba0bdc201bd134bb6d6aa6b584036ea45da7899a
1581 F20110115_AABNZR johnson_e_Page_03thm.jpg
bc28ebca2f1ca031a37e9143de1c21a3
14ac3672e5eb5bebc58d51ec265ece0bcbc45c36
F20110115_AABNPW johnson_e_Page_39.tif
fc3744d7635eb3c51ec064b872961cea
f806b5db2fa3fc078ef756cf6215248180cf01cd
66528 F20110115_AABNIB johnson_e_Page_25.jpg
cd5816112f5a3e5172f8dd2b64f27a97
01b9f4958b5f476569c9fd2935fef631696d2e7a
70760 F20110115_AABNKZ johnson_e_Page_61.jpg
a65072f0f362e907f79840eeec2bb54d
a783be6c547acec268592bb01d52dc0cf8f83821
1586 F20110115_AABNUT johnson_e_Page_04.txt
39d4864097439b85de5f90217820f6d4
54cae0a78c11f3af672ac25b225ff897e9a6a325
6614 F20110115_AABOBS johnson_e_Page_48thm.jpg
2fdcf8ce65ed9bd71b459f632f4a69a8
db58e9846bb9d06a69aee599cb960bcb22548465
19315 F20110115_AABNZS johnson_e_Page_04.QC.jpg
eabca9cf8d2d9df5e23cb4c7f709a5af
2f145034bcb6326e51070cea14685feba9aed317
F20110115_AABNPX johnson_e_Page_40.tif
6af082d8e4fe265491b2be32821ec84c
9e1bbdb3495151c5db475c3a819fe3298a4e9637
F20110115_AABNIC johnson_e_Page_16.txt
5673ae4a91dfe990e3b8590525b8d36f
2b6c47cebad16c3e706c7f6edb5a950fd59bbb06
3148 F20110115_AABNUU johnson_e_Page_05.txt
3a49e6086980b5e1ca1bfc53ba2f8bf1
4028fbe412d2f877a9175b5d0e58a73f42072206
4570 F20110115_AABOBT johnson_e_Page_49thm.jpg
b69e616f548fe76a052ce16f17d0597d
3fec38d3751ae5570958d009970141a9a42fb68f
5459 F20110115_AABNZT johnson_e_Page_04thm.jpg
3fa64b4fac17ea3a1f356a8f25a9d95e
2de1bee1270c264a84780c6fb776814991d4a3dd
1051945 F20110115_AABNNA johnson_e_Page_44.jp2
a16a7f6711577aae14f56da157a4a61f
9a43cbb1ad8400c710fdd8f6d5ca56df3c81af63
F20110115_AABNPY johnson_e_Page_41.tif
6b51e25d5154fc339dd125d9b4154e1b
ce3ba5b9dccfcbae640857ea14d9aeb461569598
108099 F20110115_AABNID johnson_e_Page_61.jp2
fc6770c4de7351ad788b42808c973075
78b60f20c6d267ce107ed3e6148f8e07134d5d20
1811 F20110115_AABNUV johnson_e_Page_06.txt
b4c15225eaebccb733ebf8c4d2001790
5a80dab256a20a307d05441fc20862e7fc5db578
5793 F20110115_AABOBU johnson_e_Page_50thm.jpg
cd2cd4193c324030a351f860b9d3b71e
f90b3b158b0310fb337cb08080d2d42349266977
5218 F20110115_AABNZU johnson_e_Page_05thm.jpg
0eea22b602e50743e6b1a9e2c732afed
c2632997259b0c6127f055acf0b1d1fbbe709ee0
1051970 F20110115_AABNNB johnson_e_Page_45.jp2
b16c4d695fa0b687f0846da18ce81254
56ba0eb6e82954ff632341af327b742bdcb16f2a
F20110115_AABNPZ johnson_e_Page_42.tif
6367fb48110bd4f719d9b603e136a436
027c24240d81fdf56225aed971630a8eeee5a26c
39974 F20110115_AABNIE johnson_e_Page_36.pro
1dc4bced18394ff9a3207b3ec1c92033
a5392fc52ffe6afbf36b5fa394b8a0586ef1064f
211 F20110115_AABNUW johnson_e_Page_08.txt
821d5f246146b88dc05e13088831c27d
dfc45a6a5229fb329c6feec8edc8edee857cf9f7
20748 F20110115_AABOBV johnson_e_Page_51.QC.jpg
8f82feb4edd115f6f2e85105159179fa
69af6ecee958cf7361c9dea4f9dc9eceda978838
3761 F20110115_AABNZV johnson_e_Page_06thm.jpg
4f3cef39829fd926cecaf1ffaa1d232f
4765db57a11570bd9df87295087ec3ac66486989
111908 F20110115_AABNNC johnson_e_Page_46.jp2
6dda2c6c1da14062a44c11095895c8a1
2d64204c889f5d225e951bbe01e2e21bbc36e6d4
45404 F20110115_AABNIF johnson_e_Page_14.pro
f1464243730ba0a7e4a09de5cef3c80c
2f008779f74a26de691f5f2e1b8ed5e05adc0b9a
1736 F20110115_AABNUX johnson_e_Page_09.txt
87854a59adc6daf03c84945eba66c07a
292e1b413ce2008631d54fd35d0018950b89f2ea
5794 F20110115_AABOBW johnson_e_Page_51thm.jpg
bc86f4b643f3c07503cf8d705db460c0
c213c2ba57b5c2581ca3e116995fb84d31cce291
5503 F20110115_AABNZW johnson_e_Page_07thm.jpg
cd530fe727dfb6ca1a90925e067946b1
cad89246103cb35a4aa8ce8ce878da5400f65e3d
105661 F20110115_AABNND johnson_e_Page_47.jp2
3df1081cfca3d1cb00d341b8404307be
6f6c3a7da24ffcbac78bb6c5132bc1fdda977556
44017 F20110115_AABNSA johnson_e_Page_11.pro
305cc94f78029f3225eaa6e0ab6ea56d
188db872bc3fa2b430651962a9497f8a20713e6a
1378 F20110115_AABNUY johnson_e_Page_10.txt
74ecf0b1e088b148035a53dccf828b3c
c92f1576c8c890d713f11f0ccde666d987954983
25329 F20110115_AABOBX johnson_e_Page_52.QC.jpg
60b94284a2a83f979084d30067fb2c8a
a495c02c0db356ca96001ac1fa0b0a42abecad6c
112105 F20110115_AABNNE johnson_e_Page_48.jp2
6f29bfce1e780263e81948d698974271
1cd52031efe50a47f72ea5ccf6dc0622cb5e8896
110114 F20110115_AABNIG johnson_e_Page_65.jp2
d93b91ad6481c6e5c8a4f6c035d1bec8
0bd6a3a043c454df8f662f0d094a35150261be0d
49047 F20110115_AABNSB johnson_e_Page_12.pro
8b25b1413107814659c68eaa15fa3a9c
3f074f3c78bc6f22e76e8f2f5283caf3abf6b027
1840 F20110115_AABNUZ johnson_e_Page_11.txt
349e3194e3e890d593749d18c16e2f17
9d13404a96b23f7aa88c66cf2fd959f019243abb
6987 F20110115_AABOBY johnson_e_Page_52thm.jpg
b35a1ddefdbf755aa20152659288461a
bab53620ee485eaae98e9c8c39115bb2282f1c22
5472 F20110115_AABNZX johnson_e_Page_09thm.jpg
9240b97700320866f0571c010eb79d4c
7a0785ad9496bd4d36889aa8b59709e562289d54
69869 F20110115_AABNNF johnson_e_Page_49.jp2
356e496889b5a2df7f128ff2033899c5
ebe1cc3a50582d475b3bfb3af1bb045f02b1a620
94620 F20110115_AABNIH johnson_e_Page_11.jp2
f9549fc50fa999e45f0570beda262ec1
1f693ccb1318bd6c7e0ed84de712e03415a057a1
52991 F20110115_AABNSC johnson_e_Page_13.pro
d65b1cc6ae9b65f1304614f379fa2712
1fd4628d488cfccce05242ee2e2583a378340e86
17295 F20110115_AABOBZ johnson_e_Page_53.QC.jpg
04fc0e03a3a6404300a924ad1bc308aa
7b85400a5dbb3da7f0005792999c95a09a9aff20
16975 F20110115_AABNZY johnson_e_Page_10.QC.jpg
297ab5c6cdd3e073c651ede0d3606ba9
0faeb3a918f4f3562e48cf36b2f6fc427189436d
2043 F20110115_AABNXA johnson_e_Page_70.txt
5385e0ecb0c638d47476a61ecaba3e24
80b64099613ec1befc574f5240811ad8380c1087
2280 F20110115_AABNII johnson_e_Page_44.txt
d3be27e6630c494ebc9f152ceb1030c2
889c331ecbea5d3641587f3fd0073740b4f64ccc
29695 F20110115_AABNSD johnson_e_Page_15.pro
333b3bee52769b0d79e41ea03818cbbd
2ece9859e54af8cfe55f6057bf3e522d82a2de63
95271 F20110115_AABNNG johnson_e_Page_50.jp2
e1603cf55a07477ab7cbdc8608962270
bbd6620d08e4ea64d085906309fff64007d250e7
4947 F20110115_AABNZZ johnson_e_Page_10thm.jpg
300f30c7d157f32ea28141183179c10d
e881f96ab2f6cf89359962c96db3b63fa7d091dc
2645 F20110115_AABNXB johnson_e_Page_71.txt
772d4053b2357f7b7ca3f12abdb16de1
94b8962e82426ffe0ec72a98ef0932ba4b7aa657
2607 F20110115_AABNIJ johnson_e_Page_84.txt
2423e7cb22a0e810df1a951eb667ff04
67b5cfa9514882ea3a8a99d22897fd39cec1269a
47482 F20110115_AABNSE johnson_e_Page_16.pro
d1c43fe257a4afcc72ad72c8d6968060
c02c13a2852cfc1a2b33a6ddb90456976f820837
1036424 F20110115_AABNNH johnson_e_Page_51.jp2
3f2d19c327e0a7c71b8cec276b803a61
87942dec73c2b4cd7c761ffb1bdce7e77bcd357d
2433 F20110115_AABNXC johnson_e_Page_72.txt
56d290ef529349cf526b187f477a3e8a
b57ea72938c30a69fa35586ced5749197f22eeec
6519 F20110115_AABNIK johnson_e_Page_61thm.jpg
a5a32d32c8ff1d6e5a3e055fa947d6b6
cd98a8d3cfd6ae7928c24713b737ebd23cc8869b
52080 F20110115_AABNSF johnson_e_Page_17.pro
1c9edc91463d8a9bbd559260e7634a4d
a8cd4da9db5a7697aeab2f1a3a2d0ffcc02f5397
1051963 F20110115_AABNNI johnson_e_Page_52.jp2
dc9fd27639ad46945e5a428046b1525d
6a939f43c86b11dda5f73e6d1f8d976edb17fc4a
2509 F20110115_AABNXD johnson_e_Page_73.txt
328b67311d979fb37724a83c44047946
03ef63e93bf2673a40738a7152eede9c4e04bf3f
23044 F20110115_AABNIL johnson_e_Page_47.QC.jpg
141096f2d91288239cf4d1057bf8750f
789f53ba2c33393f1b9df3a66d733bad51089a19
50446 F20110115_AABNSG johnson_e_Page_18.pro
af1ea73998fac9a825d5a4cac7641adb
785506d33f10588ae029c6fe20d1019421cbac0d
809273 F20110115_AABNNJ johnson_e_Page_53.jp2
6c7fc383d37f053751faa6d5804507c7
8a73e34beb3cacc046f56f16a5ad86c21e8e0941
2278 F20110115_AABNXE johnson_e_Page_74.txt
c240db5243912c0edb1e95f5da4de6bd
ea1b4c07e36e4b104f3c8c51fec8bff4f440e832
101417 F20110115_AABNIM johnson_e_Page_14.jp2
0b7fa779bda2bb3d2cc9fa80652a71af
e3a5a0883226923af1ef07c3976d3982ca803f2b
49409 F20110115_AABNSH johnson_e_Page_19.pro
da42b1fce4ba07c50921dae79283ba95
0c5c6378fc374807e41c8ca5c66a1ad52419c46c
109354 F20110115_AABNNK johnson_e_Page_54.jp2
62cf763b571a740768986f85e5a4ba78
c7a8baacb4979642159b61cdfc96f89edf25804f
2561 F20110115_AABNXF johnson_e_Page_75.txt
26e4390c6f5a478ccd6e6f6d9d25f093
87dc329b8cfe787fdfcfc31c3e3dee42a07e1d28
20475 F20110115_AABNIN johnson_e_Page_43.pro
e390ebe14f740e2337e747b87b1d32db
96bc5fc8fa15dd97dc0ba062f2c4fc02c1a5949f
50950 F20110115_AABNSI johnson_e_Page_20.pro
ac8fbc11da4464f6f1ad33ed1e394c86
80aa1af281a15adaad5de302a1885ba096d04923
2548 F20110115_AABNXG johnson_e_Page_76.txt
3be878e14e65e1fcf763a9fee241940a
183acb8ecc14ed8f3be5d658c6708248f3e46454
46939 F20110115_AABNIO johnson_e_Page_34.jpg
764e88a90210f1e02c178a6ad386be70
828872491c4ce10e2cac054bb5668bd32f8a827e
39499 F20110115_AABNSJ johnson_e_Page_22.pro
49de0ae426d7b850836c4ac4a40ba38d
fa03be5df0f80d7488df3f43ad2dfbbeab35017f
1051966 F20110115_AABNNL johnson_e_Page_55.jp2
81caa6e7607ac5d887b6df42f9803cdf
1dfa1a68385c9563244399e9a12bb1fd3f10e80f
2559 F20110115_AABNXH johnson_e_Page_77.txt
ab2adddad5d2d62ab2cffcc899778bc4
15d6faf713c0abf1ee7e8d1dc0f4f72e8e680965
78022 F20110115_AABNIP johnson_e_Page_10.jp2
e2cd89167c92266de86dcc9d9e84497d
ce09a8dabf554604d7be75237d8e0708ef6752b2
52920 F20110115_AABNSK johnson_e_Page_23.pro
521e8230e3fca8237392bc2f7ee8d516
6222951834b7dac632112ece5150729a187c3768
1051983 F20110115_AABNNM johnson_e_Page_56.jp2
cb54501a5800d8f4d3dafbdb4ce09e94
c2648cb2e4378ed46c665dfa7c479ce5b8c27447
2532 F20110115_AABNXI johnson_e_Page_78.txt
fbe026008aec1521195252b2cc1244f5
846c669466342a91ca4deec132faa0b04e2b2b2e
2037 F20110115_AABNIQ johnson_e_Page_65.txt
41f45177071fb630cbdcdc3fee7508fe
f18fbe1c632916b3cb5241a82b2bf73d88113af9
7594 F20110115_AABNSL johnson_e_Page_24.pro
cc80cfd52e94f17268164f5b19254185
8cb4b4d776d1e10332e946e38f1c9c638c812718
1051981 F20110115_AABNNN johnson_e_Page_57.jp2
68e14923645f71863a8d11fb39edd9d8
049704b5792b92f11f4c88da3e8c8eaab4f0a39f
2454 F20110115_AABNXJ johnson_e_Page_80.txt
4db69d38cb12e9e3add4178fe60e410b
1d971d9aa72765bc80281b6250350c9c71c80ba9
49868 F20110115_AABNIR johnson_e_Page_31.pro
e31d9458345bd2b04a9a2cd9dcabaaa6
741810a0a9afaac94b128dabd9de234cd0a9161d
43621 F20110115_AABNSM johnson_e_Page_25.pro
7f04c4109cee3b5b38f3862f812aedbc
a7d34367915e8cdb97898f68188b86045407e849
926552 F20110115_AABNNO johnson_e_Page_58.jp2
9261908ba75b0ba04d3b2ad325629d22
e73e309bc9c0390c1a00b6d99e988e473b062999
2489 F20110115_AABNXK johnson_e_Page_81.txt
d6ba03e2a7a65cb6be14e0232c1112e9
afb12392f4723d93ce94cf95f8cee210e30fa926
25229 F20110115_AABNIS johnson_e_Page_73.QC.jpg
286ae5e31b26a9ff0e94f8950414b09c
5831709a1f759ef9dd8b97e2ba339d9ce8e29233
48165 F20110115_AABNSN johnson_e_Page_27.pro
ea664d1d273f9544ccb092317ec960a4
90d1ff881dc5f5d820e07a636eac6f219f68fa4b
912866 F20110115_AABNNP johnson_e_Page_59.jp2
358166643206bfd950ea57dd4a67c44d
ca045f25798fbff8a53f5b2d6e62626202db44c1
2596 F20110115_AABNXL johnson_e_Page_82.txt
377f3b877dfd036798a01f6e3c9d181f
4d350bf2e68deaa2994db0abd94d0f80d7df00be
6530 F20110115_AABNIT johnson_e_Page_26thm.jpg
78887e75c2ebdb96362a9284b1de3752
e3aef0d48c4543e90bcc47354218630676fb91ee
46873 F20110115_AABNSO johnson_e_Page_28.pro
76388c55103e6f8c5b78a06b5873882e
8c52a101bcf83d836c88db5f6ac98a5e1ca768b9
110504 F20110115_AABNNQ johnson_e_Page_60.jp2
0abe5b38338e7d4b1dc0aa1cdb6dfa0b
600eae40af8785f4746fabffa531bf2443b942f0
F20110115_AABNXM johnson_e_Page_83.txt
1405ed99bdf7607175e120e264aef69d
42e85d9352bd1dd68c94dadfe6c22284c7e671c1
31516 F20110115_AABNIU johnson_e_Page_49.pro
31d379d37a61cb71ad9ae2f9f121fbcf
a52b818c88f991d606727472d29ee6ff79476353
38215 F20110115_AABNSP johnson_e_Page_29.pro
85676a57013e11bdcf83a5eeb39c18d0
e518be777ce6f65b1ee2cb1d0194693509806ae9
9364 F20110115_AABNNR johnson_e_Page_62.jp2
4d1e5d994d77e2e256660f97f05d773f
999128496373f149d0e294cb3907903e38d5c8d8
2628 F20110115_AABNXN johnson_e_Page_85.txt
5d0e2db29e640cb76c403fb144533ef6
db63e484b4abb7c7baccb2588243a50f1bb2c9d4
101825 F20110115_AABNIV UFE0008337_00001.mets
35fa03212e816e1afa1da65a3b7bd59d
f6ef33716158cffbb5491433e275d160c12a245d
89047 F20110115_AABNNS johnson_e_Page_63.jp2
ad2f064414163dcf68f933768994a0a7
73df8b4c6ed66e12c4ce23091f9519d0b4217d3a
1688 F20110115_AABNXO johnson_e_Page_86.txt
1b25abdf3c15ff6c4ecee4ba26268c9d
247be5c0bf84c026b814a44c6196b09e72e33f49
44803 F20110115_AABNSQ johnson_e_Page_30.pro
378231b05b99458f45fd6bf432dcfaf6
4ecffbc32829ff03e2b1bead6ac9b72c33ef4201
106710 F20110115_AABNNT johnson_e_Page_64.jp2
25226558e86bcb7509bb4111e89fd320
203e9d75651e0ce5d7bae2fd5b417331abea76a8
1051 F20110115_AABNXP johnson_e_Page_87.txt
c08933aa60345e088c30526ddd23d375
675cc523677cf8709675fcec3cabd97aeaca37b8
46782 F20110115_AABNSR johnson_e_Page_33.pro
dad5360db31488917b56992117f7089d
3eda0f8027a65259f618154facd69ec80ee1527f
113697 F20110115_AABNNU johnson_e_Page_66.jp2
a0eea8543963896a870df59ba7b43c4d
701b63d33da58942b0c65242c1d27c729c6cd518
2908495 F20110115_AABNXQ johnson_e.pdf
d9bfc31dfb5416b7a980f75a954046ca
41d2c59e231b990a96b80dddef514e0567ccdbe4
23962 F20110115_AABNIY johnson_e_Page_01.jpg
f8953e2e979a6bdd98f0fcbd8350d830
94a1561b15cd43617fceeb1a5414b92c8dc77165
29900 F20110115_AABNSS johnson_e_Page_34.pro
2b4cfc9ff13ca62987fe129fb4e4ff69
4bd6121a321befa34992ad2f38090884c4548fff
109128 F20110115_AABNNV johnson_e_Page_68.jp2
e8f4e80a3f1fa1b8d4d73b05e69c8983
1d03a338e3c31bd391e2a7cdd3239f37e9efdacb
23787 F20110115_AABNXR johnson_e_Page_74.QC.jpg
1a126b29ff9c5fdfdd27a76ae30b0881
fcb252b023489616e7cf4a3a04daec47262acc68
10494 F20110115_AABNIZ johnson_e_Page_02.jpg
ac2cb5a816d69654160d43db3352f646
c580c595a0ff3ccec285ed6e4219082780db401c
39918 F20110115_AABNST johnson_e_Page_35.pro
9ad9e6322b44a307a862f37f1697deda
b0a241240ae97941095852b57b65c84cdeedebe6
73883 F20110115_AABNNW johnson_e_Page_69.jp2
c2a7af4d89ba231a77ea2d301891ec1b
920c756885bb3d18e6bcae79aac231b99ea3c34f
6311 F20110115_AABNXS johnson_e_Page_45thm.jpg
06f7821e82115e996336b2da96f951c9
a63111f50b7caea7f90216a712898a99733413bf
36024 F20110115_AABNSU johnson_e_Page_37.pro
b63300e7f63a453ea99f69ac52a61473
c179e9ac0f2a778b2b84df759fed736d67154254
107519 F20110115_AABNNX johnson_e_Page_70.jp2
7a5131da7a2caeaec7f7e1710d0022e1
a51294efe3f3499f4097b3f725acf52b7df550fd
24313 F20110115_AABNXT johnson_e_Page_45.QC.jpg
1fc7024597668e57b925fe8250b2c8b7
069c68a8c01e701218409553064b7215b97f1918
44329 F20110115_AABNSV johnson_e_Page_38.pro
3e0ae831e71fe2500978a3dc7f125772
46baebff4a874ba8dbebcd066b009ee964dd913c
11816 F20110115_AABNLA johnson_e_Page_62.jpg
818d8dbda84ac871df2ee4bf443a5128
b7a809d30f520f2fa9c5d8267cd3859e3c818aff
136716 F20110115_AABNNY johnson_e_Page_71.jp2
ffe3f353f068ac746bd742ce48bc120e
fb2ad7229809f414149f7de2f25a65e5905742d3
24814 F20110115_AABNXU johnson_e_Page_57.QC.jpg
d24d000be584cc0e962407a52fd06040
32c09294c7d4c5fc1beb7724722b6b9ae69bba63
24777 F20110115_AABNSW johnson_e_Page_39.pro
24339c4b188af24223fe9b71c47ada01
9834d6501fa2a6dadd076f9c3ab1a07d9dc86c8d
70774 F20110115_AABNLB johnson_e_Page_64.jpg
5bb90cf33a200758f7fa1712a1fc994b
304121964ee66078372eacca66e61263becb5093
126069 F20110115_AABNNZ johnson_e_Page_72.jp2
7945a2e25d05e11ffdb10635573fef14
f55e73ec18cb2f666dd968e57399b0afb1f217cb
34575 F20110115_AABNSX johnson_e_Page_40.pro
15caa0df7acb5c1f096a8393e35ce499
b30b046ddac43b140daab87afcb9e5cee5443c55
74050 F20110115_AABNLC johnson_e_Page_65.jpg
13ec62876221565edda86dd07649f160
cb91b145d9a5ff6c6d808846be1475b1d326a404
6455 F20110115_AABNXV johnson_e_Page_20thm.jpg
4c419c3dd9c2065a21e3123c8c3f808d
be4a1a7c1468d8307de616931da12fc45c8fcbc9
F20110115_AABNQA johnson_e_Page_43.tif
a6dd97629491345ae287a74bc808994b
140bc2727034fac7dfe4c8f96674122fd2b10916
40266 F20110115_AABNSY johnson_e_Page_41.pro
ac2fd028dc21d8b812dc94711e67d78d
a56166830931010f684b4271abc2a72dca47ed21
74325 F20110115_AABNLD johnson_e_Page_66.jpg
00987b9a9806ff249f0e9401ed115a26
2fffa5cd9215b16cc27601815ddb68dbf231dc73
23487 F20110115_AABNXW johnson_e_Page_20.QC.jpg
25f0edfa170e4005198a76cb01c73626
b42104cee5e1d1d06797dbd231f18fae1eca83d7
F20110115_AABNQB johnson_e_Page_44.tif
d04b1fc9117eaffc9aee2cf4c9c483ef
e0e39ed9544c50552995462c3daec950d4ed723a
34249 F20110115_AABNSZ johnson_e_Page_42.pro
4b1f4fb51af3c2eb9b87db126b654b81
4e45b22fde968cf990f9b17cd6ed3f5c2d1e0e60
50440 F20110115_AABNLE johnson_e_Page_69.jpg
f8e3f0ff9b92dc63ec0207c080a91479
614e8aa61652134ad79900f57228e5310a4bf399
26458 F20110115_AABNXX johnson_e_Page_55.QC.jpg
2ff3c7605199d53c23cf355f439d7dc5
b837ba7dd06d5320cf7eea044359e8389bbf4d3b
72889 F20110115_AABNLF johnson_e_Page_70.jpg
397673f6d1e508cb74a634eacb77dea6
2112f30fe182feb6023f7d844b47b050fcefa130
6470 F20110115_AABNGH johnson_e_Page_19thm.jpg
7c97caaba3b03a4c05064406436e9474
995a611614ba2084fd95844fbe3ab533552fef48
F20110115_AABNQC johnson_e_Page_45.tif
2f5604c0239bab7e511413bb3c1bd115
566e20aec718b0300464c6c4b0bd3d2cd4bfdf55
23154 F20110115_AABNXY johnson_e_Page_64.QC.jpg
31d979b317f61d0338b9b756dabd890c
ab72230ebde8e484bbcad48fdd2c4139608c15db
91898 F20110115_AABNLG johnson_e_Page_71.jpg
e89058c938699831ea175e80ffc5f09f
70259fb8f9e2106954ecf8a3d4f3c5de85fb5b50
1956 F20110115_AABNVA johnson_e_Page_12.txt
91ac585cd8953291577eb0bd632cf7d1
90c0f02130bc506528cae7618de651e4fca6110a
1137 F20110115_AABNGI johnson_e_Page_39.txt
ca4183bb6bd66b5d708ad5704be6169a
b803b845790a591483692dd1a1d94e3692b4be66
F20110115_AABNQD johnson_e_Page_46.tif
a51df0e25a3ed6d12766519c41a5c2a1
940d71d812958e6dc5516d38115002468ec0b8fc
19633 F20110115_AABNXZ johnson_e_Page_63.QC.jpg
0fdc35e3d15e4f35664ebfbd9c9ae8c3
71d21d23ae5d50b812c4812f26ef22a6f4208122
83346 F20110115_AABNLH johnson_e_Page_72.jpg
674216a7bfcc001119929a99f237ecda
c3dfa5401abc8ff25a0bb69a37271622499cc02c
2096 F20110115_AABNVB johnson_e_Page_13.txt
ee61afb87356cd78900104ca84255fb2
f1007d191b6e783d517e34d6cf8d310021810192
108601 F20110115_AABNGJ johnson_e_Page_18.jp2
0777683820727b3196270faa3082cdec
2e8980cb3e6f594f381d79d979ad550b7771e3b6
F20110115_AABNQE johnson_e_Page_48.tif
8f81f64b5ff2d03d575447afe8d9cd7f
489505b5296268437b9aabbf7b350f5daffbd7ae
92310 F20110115_AABNLI johnson_e_Page_75.jpg
e6167734810c8b442a162893593f4daf
9e8967334a81f44bd285bad1844a956743809444
1799 F20110115_AABNVC johnson_e_Page_14.txt
d844a47598fca0cb9d7da3ae65b9cadd
42dd2d0b4eaf9af44da7e6ff9a26228b13ec6c8b
F20110115_AABNGK johnson_e_Page_84.jp2
a02cd883a704c784861368b4f9fb89bc
9d7501c679d2a56b670b90124f439758b5fdb003
F20110115_AABNQF johnson_e_Page_49.tif
d5941dcfab6b103ee9c9339151a4bc09
f11d53fefad9cb8ec401c5b77d2cbed95d79df95
4798 F20110115_AABOCA johnson_e_Page_53thm.jpg
22424a0ef0d5bf3401a311a2a5530e59
5e824068477335169ff3e5fb724c7321f89a9474
1479 F20110115_AABNVD johnson_e_Page_15.txt
b2b2db5e51ad2e57dee18dad17d2f236
aecc15aa48fd4ad871a2a5fe971fe7358db2eb5e
108163 F20110115_AABNGL johnson_e_Page_67.jp2
2d1bf23b94796a5eaa5945dec7de418f
233aac330f5442b831719f880fc535ba3e3c4f51
F20110115_AABNQG johnson_e_Page_50.tif
c1e1c80f0a3fe44687d6366487c13697
a17211f0f807a42b48e53a7dd3b10ebc330ac6de
6609 F20110115_AABOCB johnson_e_Page_54thm.jpg
763b5e4d05f8aa519690fb6b8b0b3f79
917d426c8dfd82c68bc8e8237641f9539f589025
89973 F20110115_AABNLJ johnson_e_Page_76.jpg
0bf45d57aa8fce34d214ea04824cfbbd
bda2a0ca16e86ad3cc6ea624e18ab874c6c57957
2066 F20110115_AABNVE johnson_e_Page_17.txt
524724edd316b414c6cc29a94dc8a381
6bcd247a4b0200c30b4fa890d13a4c9cf632079d
2034 F20110115_AABNGM johnson_e_Page_33.txt
361b2f090f47c09cf06a6abe78a618d9
8f6a40acac3715a5cf5da21e1acf63774188c615
F20110115_AABNQH johnson_e_Page_51.tif
17cbf4347177e4f258e650a20fb47571
60d48ab7970d875c99de6c7c55ebdd16f53fa5b2
7115 F20110115_AABOCC johnson_e_Page_55thm.jpg
92cbb76fedf74f308110de8bd51ce77b
a5961c5fcff65b62b0b8cc93db806389cb56db76
90409 F20110115_AABNLK johnson_e_Page_77.jpg
5d47a214d5d45e28f36eeeff25689d99
8ea529c2b3482bee003b9af17cfc62cb54e0481a
1985 F20110115_AABNVF johnson_e_Page_18.txt
2bea49a9cc9ad0b1eeb4d292863767f6
203fe6b9c32e22fce7f539d06d7e22171f6189db
63188 F20110115_AABNGN johnson_e_Page_53.jpg
af06171b29ba89d7a770e0db9b8c476d
f24349244fd4688b4c89169d2361c339070f9473
F20110115_AABNQI johnson_e_Page_52.tif
9847f6138c15d4d1ba87842b935b42f1
5aa2670d1daa20a7cbfc1e87cdcf7ab138bdf57b
25422 F20110115_AABOCD johnson_e_Page_56.QC.jpg
034e6b2888e9f3b9e097101c608c6d30
5c1276dac3afe7491c46bd152257318f08093d3a
90238 F20110115_AABNLL johnson_e_Page_78.jpg
66f91fd39ae4e5b42e77c3849534e8af
a7a7c416eedf26fc1ec071aead7eaac2472ef3fe
2008 F20110115_AABNVG johnson_e_Page_19.txt
c947cff3e363134e4b02bbad955859b6
937c37c503b9a9b57f93a58c60195fa753e62194
75289 F20110115_AABNGO johnson_e_Page_46.jpg
27b51bce60479a892f667b4359802030
ff55222831e61354cbcfd435292739a717f11c17
F20110115_AABNQJ johnson_e_Page_53.tif
b4093b136ed7d6c60908823060636187
fc873ac2f0b8e924e1fee0c896b87e1fa99c3109
7060 F20110115_AABOCE johnson_e_Page_56thm.jpg
23eeb6827a3c12f3fb87b284cbd29960
1bc252546de07a1e32a400a7b7aad622f870e89b
89370 F20110115_AABNLM johnson_e_Page_79.jpg
15de5dccf37b2be84729e2854655a900
aca348c0044cac45cc4af62d5a1b2f253b2be353
2032 F20110115_AABNVH johnson_e_Page_20.txt
2d85fa55ae95ec2da9e46242219e0cd3
d1671e596ff42cb35136a9eb4cc000b250f908ad
25290 F20110115_AABNGP johnson_e_Page_83.QC.jpg
8ac051d3ee3b681f410d4ef7ae235d25
293e54e80c23b15609b647f6c2e8d293796ffc7b
F20110115_AABNQK johnson_e_Page_54.tif
b4d71d57483f6f52347991b9d9e4f447
f278b902a044c6632c8071e87439053f57a19a63
6822 F20110115_AABOCF johnson_e_Page_57thm.jpg
98386b3fc566807f5eb8638f23248263
c2aec613d9e8209d9bf6ada23e60858d49823fa8
84040 F20110115_AABNLN johnson_e_Page_80.jpg
92ebd057f85ab3ee30f05f16caa1a93e
73a8b9f0528bc1bd17b1550fc855ce6640a4d251
F20110115_AABNVI johnson_e_Page_21.txt
b74315f5b68703ea8acf3ec667c090bd
c9a63f3f486b41593033341e95df07034802eb34
F20110115_AABNGQ johnson_e_Page_57.tif
a17df6a46594847a889f429b390d2503
21d20c9817a3ef4782f7c2eff6445a8fcac2d078
F20110115_AABNQL johnson_e_Page_55.tif
6e785f199491dd297eb5ae07ff68702b
9578903fd0d4e01a52120a9c010ef330624091dc
21819 F20110115_AABOCG johnson_e_Page_58.QC.jpg
15dc572a081b0ab3b46947ba9429bb30
e688b77dae121882415a81758997b92cec3c987a
91925 F20110115_AABNLO johnson_e_Page_81.jpg
67d33f178349845b6370cfd82dd74305
5dc119acea59c4a0b708bb9973b9ac815ee5516b
1827 F20110115_AABNVJ johnson_e_Page_22.txt
77018f2ea0727742ab3736495151966f
8c4bb7163335eaceeb227c31883231ad3cd642d4
9600 F20110115_AABNGR johnson_e_Page_03.jp2
e67df00ec1d7d02dca98618527994f60
b4a5de8b80394a42d48d5914974900a66d2a5d94
F20110115_AABNQM johnson_e_Page_56.tif
fd2e62beaf477edb7100ad9e3d06f0e1
77ca5e9c8949dcb20f7b741b799f4dd854afe4e9



PAGE 1

SURVIVIN EXPRESSION AFTER TRAUMAT IC BRAIN INJURY: POTENTIAL ROLES IN NEUROPROTECTION By ERIK ANDREW JOHNSON 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 2004

PAGE 2

Copyright 2004 by Erik Andrew Johnson

PAGE 3

This document is dedicated to my wife, Karie, for her loving and unwavering support during this process.

PAGE 4

iv ACKNOWLEDGMENTS I would first and foremost like to tha nk my wife, Karie, for her undying support and patience during these long ye ars. No matter what the situ ation, she is always waiting there with a smile and she has he lped me more than she will ever know. I would also like to thank my parents, Arlen and Patricia J ohnson, for their love, dedication and fantastic parenting skills. Without these, I would not be the person I am today and I would never have been able to pursue this level of education. I would like to thank Dr. Ronald Hayes fo r the opportunity to pursue this novel research and for the opportunity to contribute to the scientific community. I would also like to thank past and present committee members, Dr. Douglas Anderson, Dr. William Dunn, Dr. Gerry Shaw and Dr. Brian Pike fo r their invaluable input and guidance. I would especially like to thank Dr. Stanis lav Svetlov, whose unders tanding, patience and dedication to my educational growth have not gone unappreciated or unnoticed. Additionally, I would also like to thank Dr. S. Michelle DeFord and Dr. Jose Pineda for their help and guidance in completing these studies. Lastly, I would like to thank all the memb ers of Dr. Hayes’ laboratory for their assistance and friendship throughou t the years. I w ould like to especially thank Jeremy Flint, Barbara Osteen, Dr. Rebecca Ellis, Dr. Stephen Larner, Dr. Claire Ringger, Jada Aikman, and Shannon Janssen for their effort and support.

PAGE 5

v TABLE OF CONTENTS page ACKNOWLEDGMENTS.................................................................................................iv LIST OF FIGURES..........................................................................................................vii ABSTRACT....................................................................................................................... ix CHAPTER 1 INTRODUCTION........................................................................................................1 Traumatic Brain Injury Demographics.........................................................................1 Traumatic Brain Injury Pathophysiology.....................................................................2 Traumatic Brain Injury, Apoptos is and Caspase-3 Activation.....................................3 Apoptosis Inhibition Following TBI............................................................................6 Cellular Prolifera tion Following TBI...........................................................................7 Survivin: Mitosis and Anti-apoptosis Protein...............................................................9 Survivin Protein Structure............................................................................................9 Survivin Expression and Mitosis................................................................................10 Survivin and Apoptosis Inhibition..............................................................................11 Potential Role for Survivin in TBI Pathology............................................................13 2 METHODOLOGY.....................................................................................................15 Induction of Controlled Cor tical Impact Brain Injury................................................15 Quantitative Reverse Transcriptase Polymerase Chain Reaction (Q-PCR)...............15 Rat-Specific Survivin Polyclonal Antibody Production.............................................17 Survivin Polyclonal Antibody Characterization.........................................................18 Western Blot Analyses...............................................................................................18 Preparation and Sectioning of Tissu e for Immunohistochemistry (IHC)...................20 Dual Label Fluorescent Imm unohistochemistry (IHC)..............................................21 Dual Label Fluorescent IHC fo r Same-Species Antibodies.......................................22 Experimental Group Sizes..........................................................................................22 Cell Quantification and Statistical Analysis...............................................................23 3 SURVIVIN EXPRESSION FOLLOWI NG TRAUMATIC BRAIN INJURY..........25 Induction of Survivin Expression After TBI..............................................................25 PCNA Expression After TBI......................................................................................26

PAGE 6

vi Co-Expression of Survivin and PCNA Following TBI..............................................28 Survivin and PCNA are Expressed in Astrocytes After TBI......................................30 Survivin and PCNA are Expressed in a Sub-Set of Neurons After TBI....................34 Survivin is Not Expressed in Microglia and Oligodendrocytes.................................35 Discussion of Chapter 3..............................................................................................36 4 SURVIVIN AND APOPTOSIS INHI BITION FOLLOWING TRAUMATIC BRAIN INJURY.........................................................................................................40 Caspase-3 is Activated in the Same Brai n Regions as Survivin Following TBI........40 Survivin Expression Correlates with Decreased TUNEL Labeling but not Active Caspase-3 Expression............................................................................................40 Astrocytes and Neurons Demonstrat e Cell Specific Differences in Active Caspase-3 and TUNEL Labeling...............................................................44 Discussion of Chapter 4..............................................................................................46 5 CONCLUSIONS AND FUTURE DIRECTIONS.....................................................53 Conclusions.................................................................................................................53 Future Directions........................................................................................................58 LIST OF REFERENCES...................................................................................................60 BIOGRAPHICAL SKETCH.............................................................................................77

PAGE 7

vii LIST OF FIGURES Figure page 1-1 Intrinsic and extrin sic apoptosis pathways.................................................................5 1-2 Survivin protein structure.........................................................................................12 2-1 IHC characterization of the rat-specific survivin antibody......................................19 2-2 Control section for biotin/strepta vidin same-species dual labeling IHC..................23 3-1 Survivin mRNA induction in rat brain after TBI.....................................................26 3-2 Expression of survivin protein after TBI in rats.......................................................27 3-3 Expression of PCNA after TBI in rats.....................................................................29 3-4 Immunohistochemistry of survivin and PCNA........................................................30 3-5 Co-localization of survivin a nd GFAP in brain tissue after TBI..............................31 3-6 Co-localization of PCNA and GFAP in brain tissue after TBI................................32 3-7 A sub-set of NeuN-positive neurons express survivin and PCNA after TBI...........33 3-8 No survivin expression is found in oligodendrocytes or microglia following TBI in rats.................................................................................................................35 4-1 Caspase-3 Activation in rat br ain after traumatic brain injury.................................41 4-2 Co-expression of survivin and apoptosis markers following TBI in rats.................42 4-3 Survivin expression decreases th e accumulation of TUNEL but not active caspase-3........................................................................................................43 4-4 Astrocytes express active caspase-3 and label with TUNEL following TBI in rats........................................................................................................................ 46 4-5 Neurons express active caspase-3 and label with TUNEL following TBI in rats....47

PAGE 8

viii 4-6 TUNEL labeling is cell speci fic following TBI in rats............................................48 4-7 Putative mechanism of apoptosis inhi bition by survivin following TBI.................49

PAGE 9

ix 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 SURVIVIN EXPRESSION AFTER TRAUMAT IC BRAIN INJURY: POTENTIAL ROLES IN NEUROPROTECTION By Erik Andrew Johnson December 2004 Chair: Ronald L. Hayes Major Department: Neuroscience In these studies,the expre ssion profile and cellular local ization of survivin, a novel anti-apoptotic and mitosis protein, following traumatic brain injury (TBI) in rats was examined. Specifically, survivin co-localiza tion with the cell cycle protein PCNA, the apoptosis protease active caspase-3, and the DNA fragmentation label TUNEL was determined to reveal potential role of surviv in in neuroprotection and to elucidate antiapoptotic mechanisms. Levels of survivin mRNA and protein were increased in the ipsilateral, but not contralateral, cortex and hippocampus of rats after TBI, peaking at five days post injury. Similar temporal and spatia l patterns of PCNA we re also significantly enhanced in these brain regions. Immunohist ochemistry revealed that survivin and PCNA were co-expressed in the same cells and had a focal distributi on within the injured brain. Further analysis reveal ed a frequent co-localizati on of survivin and GFAP, an astrocytic marker, in both ipsi lateral brain regions, while a much smaller subset of cells showed co-localization of su rvivin and NeuN, a mature neuronal marker. PCNA protein

PAGE 10

x expression was detected in bot h astrocytes and neurons of the ipsilateral cortex and hippocampus after TBI. Western blot analysis revealed significan t increases in the accumulation of active caspase-3 between five and f ourteen days post injury. Th e percentage of survivinpositive and negative cells labeled with active caspase-3 at five or seven days post-injury was not significantly different. However, su rvivin-negative cells exhibited a significantly greater labeling with TUNEL compared to surv ivin-positive cells, th ereby suggesting that expression of survivin may attenuate DNA cleavage and progression of apoptosis. Although a higher percentage of astrocytes a ccumulated active caspase-3 compared to neurons, these neurons showed significantly higher frequency of TUNEL labeling. These novel data demonstrate that survivin is abundantly expressed in brain cortex and hippocampus of adult rats following TBI. Survivin accumulation occurs primarily in astrocytes and a sub-set of neurons. The occasional co-expression of survivin and PCNA coupled with the low frequency of TUNEL la beling in survivin expressing cells may suggest that survivin is primarily involv ed in attenuating apoptotic cell death and secondarily may play a role in regulation of neural cell proliferativ e responses after TBI.

PAGE 11

1 CHAPTER 1 INTRODUCTION Traumatic Brain Injury Demographics Traumatic brain injury (TBI) is the leadi ng cause of death and permanent disability for children and young adults in the United St ates. Currently, there are more than 5.3 million Americans, approximately 2% of th e current U.S. population, living with TBIrelated disabilities (Thurman et al., 1999a) w ith an estimated 1.5 million additional TBIs occurring each year (Sosin et al., 1996). Approximately 230,000 cases are severe enough that the victims require transport and hospita lization (Thurman et al., 1999b). Of these, approximately 50,000 victims die from their in juries, accounting for 33% of all injuryrelated deaths (Sosin et al., 1995). Of t hose severely injured survivors, 90,000 TBI victims must live with long-term disabilitie s (Thurman et al., 1999b). The U.S. economy loses an estimated $56.3 billion a year through the direct and indir ect costs associated with TBI (Thurman et al., 1999b). Currently, most traumatic brain injuries result from motor vehicle accidents (48.9%) followed by falls (25.8%) and fir earms/ assaults (19.2%) (Thurman et al., 1999b). Young males, ages 15 to 24, are the most “at-risk” demographi c, a statistic that likely reflects lifestyle choi ces (Jennett, 1996; Thurman et al., 1999b). The magnitude of this problem led to the passing of Public Law 104-166, better known as the Traumatic Brain Injury Act of 1996, a bill designed to help prevent TBI a nd educate the public about the health consequences of this in jury (Thurman et al., 1999b). While these educational efforts have decreased TBI-rela ted deaths by an estimated 22% since 1980

PAGE 12

2 (Sosin et al., 1995), the number of people living with TBI-related disabilities has risen (Thurman et al., 1999b). To date, few pharmacological or treatment options are available to reduce these TBI-induced disabilities. Preven tion remains the only effective “cure.” True advances in clinical treatment depend on understanding the underlying pathophys iology mechanisms that regulate both cell death and cell survival following TBI. Traumatic Brain Injury Pathophysiology Traumatic brain injury is a complex inju ry that is comprised of an immediate primary injury and a progressive secondary injury cascade (Graham et al., 2000). The primary mechanical injury can be contusiv e or concussive and involves tearing and stretching of the neural tissues. Neurons and white matter tracts seem particularly vulnerable to the mechanical injury (Bal dwin et al., 1997; Maxwell et al., 1997; McCullers et al., 2002; Grady et al., 2003). The secondary injury cascade is initiated by the primary mechanical injury and is defined by unrestrained biochemical and inflammatory reactions (Gennarelli, 1993). Though the brain is remarkably adaptive, damage sustained as the result of secondary injury prevents the br ain from regaining preinjury function. While many of the biochemical proce sses seen after TB I occur under normal homeostasis, their collective dysregulation act s in a synergistic manner to contribute to the pathology associated with secondary injury. Some of the more prominent events include perturbations in blood flow (Gra ham et al., 1995; McIntosh et al., 1998; Raghupathi et al., 2000), ischemia (Lee et al., 199 9; Passineau et al., 2000), excitotoxicity (Choi and Rothman, 1990; Gennarelli, 1993), calcium deregulation (Graham et al., 1995), free radical production (Kontos, 1989; Beck man et al., 1990; Maier and Chan, 2002),

PAGE 13

3 inflammation (Povlishock and Kontos, 1985; Giulian, 1991; Morganti-Kossmann et al., 2001), edema (Choi, 1988; Bullock et al., 1991) and protease activation (Pike et al., 1998; Clark et al., 2000; Eldadah and Faden, 2000; Raghupathi et al., 2000; Knoblach et al., 2002; Larner et al., 2004). Ultimately, activation of these processes disrupts fragile homeostatic states and creates an inhospita ble environment for neural cell survival. Traumatic Brain Injury, Apopto sis and Caspase-3 Activation Cell death following TBI is distinguished by necrotic and apoptotic processes (Conti et al., 1998; Clark et al., 2000; Yakovlev and Faden, 2001). Necrosis and apoptosis lie on a continuum (N icotera et al., 1999) wherein the mode of cell death is dictated by several factors in cluding ATP availability (G reen and Reed, 1998), calpain activity (Wang, 2000), intracellu lar calcium levels (Gwag et al., 1999; Zipfel et al., 2000), presence of anti-apoptotic factors (Raghup athi et al., 2000) and the presence of activated caspases (Denecker et al., 2001). W ithin hours of a TBI, neural cells around the contusion area exhibit classic signs of necr osis including cytotoxi c edema, mitochondrial swelling, nuclear pyknosis, ruptured plasma membranes, organelle breakdown and vacuolated cytoplasm (Sutton et al., 1993; Dietrich, 1994; Denecker et al., 2001). However, as time progresses, many cells including neurons, astrocytes and oligodendrocytes begin to e xhibit characteristic s of apoptosis including chromatin condensation, cell shrinkage, apoptotic body formation and DNA laddering (Conti et al., 1998; Newcomb et al., 1999). It is well docum ented that apoptotic cell death continues for many months following injury, thereby ma king it a chronic cont ributor to post-TBI pathology (Cervos-Navarro and Lafuente, 1991). Apoptosis has been well characterized fo llowing traumatic brain injury (Pike et al., 1998; Beer et al., 2000; Clar k et al., 2000) and utilizes both the intrinsic and extrinsic

PAGE 14

4 apoptotic pathways (Fig. 1-1). Each path way involves a unique set of upstream and downstream cysteine specific proteases cal led caspases that cleave a variety of intracellular substrates and drive apoptosis. Synthesized as inactive zymogens, caspases require the cleavage of a pro-domain to become active. Caspase activation is achieved in multiple manners including proximity-induced autoproteolysis or cleavage by another caspase (Stennicke and Salvesen, 1999; Van de Craen et al., 1999). Both the intrinsic and extrinsic pathways lead to cleavage a nd activation of caspase -3, the most abundant executioner caspase in the brain (Cha n and Mattson, 1999; Slee et al., 2001). Among the numerous structural and regulat ory protein targets of active caspase-3 are stress response proteins ( e.g ., PARP, Rb and p21), signal transduction proteins ( e.g ., phospholipase A2, NF B and PKC), structural proteins ( e.g ., -II-spectrin, actin and vimentin), nuclear matrix proteins ( e.g ., lamins A, B1 and C) and mitochondrial proteins ( e.g ., Bcl-2, Bcl-xl and Bid) (Cohen, 1997; Chan and Mattson, 1999; Earnshaw et al., 1999; Wang, 2000). Cleavage of proteins su ch as iCAD/DFF45 (Ena ri et al., 1998; Liu et al., 1998b; Sakahira et al., 1998), poly ( ADP-ribose) polymerase (PARP) (Ferrer and Planas, 2003), DNA-dependent protein kinase (DNA-PK) (L azebnik et al., 1994) and acinus (Sahara et al., 1999) prevents DNA repair and pr omotes DNA condensation and fragmentation (Woo et al., 1998). When unr egulated, even moderate activation of caspase-3 can rapidly lead to cell death. Therefore, nature has developed various mechanisms to temper the deleterious effect s of caspase-3 over-ac tivity and counter the progression of apoptosis.

PAGE 15

5 Figure 1-1: Intrinsic and extrinsic apoptosis pathways. Apoptosis progresses primarily through the extrinsic and intrinsic apoptosis pathwa ys. The extrinsic pathway is mediated by ligand binding to membrane bound death receptors and caspase-8 activation. Activ ation of this pathway can promote cell death by intrinsic pathway activation or apop tosis prevention by up-regulation of apoptosis inhibitors. The intrinsic pathway is mediated by mitochondrial stress. Caspase-9 is activated in th e apoptosome complex. Both pathways promote caspase-3 activation. Acti ve caspase-3 can cleave several intracellular proteins. Cleavage of pr oteins such as iCAD (DFF45), PARP and acinus can lead to DNA fr agmentation and cell death.

PAGE 16

6 Apoptosis Inhibition Following TBI While many pro-apoptotic proteins are expressed following TBI, there is a concomitant increase in pro-survival factor s (Nowak and Jacewicz, 1994; Iwata et al., 1997; Buytaert et al., 2001; Hermann et al ., 2001; Sanz et al., 2001; Alzheimer and Werner, 2002; Maroni et al., 2003). Of these pr o-survival factors, a family of proteins known as inhibitor of apoptosis proteins (I APs) can attenuate apoptotic cell death by directly binding to the active site of activat ed caspases such as caspase-3 (Tamm et al., 1998; Conway et al., 2000; Shin et al., 2001) The IAP family contains eight known members including survivin (Li, 2003). The pr oteins are highly conserved across species (LaCasse et al., 1998) and the expression of each IAP appears to be cell type specific. Each IAP has one to three baculovirus IAP re peat (BIR) domains that possess the ability to bind and directly inhibit active caspase-3 (Tamm et al ., 1998; Conway et al., 2000; Shin et al., 2001), caspase-7 (Tamm et al ., 1998; Shin et al., 2001) and caspase-9 (LaCasse et al., 1998; Deveraux and Reed, 1999) Mutation studies have demonstrated that the BIR domain is responsible for caspase interaction and is th erefore necessary for anti-apoptotic action of the IAPs (Roy et al., 1997; Takahashi et al., 1998; Vucic et al., 1998; Muchmore et al., 2000).Although few IAPs have been extensively characterized in the context of TBI pathophysiol ogy, increases in XIAP (Keane et al., 2001; Lotocki et al., 2003), NAIP (Xu et al., 1997; Hu tchison et al., 2001; Thom pson et al., 2004), cIAP-1 (Keane et al., 2001; Belluardo et al., 2002) and cIAP-2 (Kean e et al., 2001) have been reported in neurons following brain injury. Th e potential role of survivin following TBI has not been investigated.

PAGE 17

7 Cellular Proliferation Following TBI In addition to pro-survival factors, new cell production plays a pivotal role in the brain following injury. Large pools of neur al progenitor cells have recently been identified in the germinal centers of th e dentate gyrus subgranular zone (SGZ) and subventricular zones (SVG) of the adult brai n (Gage et al., 1998; Magavi et al., 2000; Gage, 2002; Sanai et al., 2004). Followi ng both ischemia and TBI, these neural progenitor cells proliferate (Gould and Ta napat, 1997; Yagita et al., 2001) and differentiate into mature neurons (Gage et al., 1998; Doetsch et al ., 1999; Magavi et al., 2000; Cameron and McKay, 2001; Dash et al., 2001; Kernie et al., 200 1; Yagita et al., 2001; Peterson, 2002), astrocytes (Dash et al., 2001; Gould et al., 2001; Chirumamilla et al., 2002; Chen et al., 2003) and oligodendrocyt es (Gould et al., 2001). Consistent with these findings, many cell cycle proteins ( e.g ., cyclins A, B and D1, cdk4 and PCNA) are also up-regulated after brain injury (Miyake et al., 1992; Kaya et al., 1999a; Chen et al., 2003; McPherson et al., 2003). Cellular proliferation following TBI can have both beneficial and detrimental consequences to the recovery of the damage d brain. These consequences can also vary by cell type. Neuronal progenitor cells have been shown to prolif erate following brain injury (Parent, 1997; Hill-Fel berg et al., 1999; Dash et al ., 2001; Kernie et al., 2001; Yagita et al., 2001; Chirumamilla et al., 2002; Rice et al., 2003) but the functional viability and therefore significance of these newly formed neurons is not clear. New neurons appear to migrate away from the ge rminal centers of the subventricular zone (SVZ) and subgranular zones of the dentate gyr us (SGZ) but not towa rds areas of injury (Rice et al., 2003). Additionally, as many as 80% of all newly formed neurons undergo apoptosis within two weeks of their forma tion in normal conditions (Morshead and van

PAGE 18

8 der Kooy, 1992; Morshead et al., 1994). The proliferation of neurons following TBI may be advantageous but their in ability to survive and cont ribute to recovery requires additional clarification. Glial cell proliferation, specifically astroc ytes and oligodendrocytes, can serve to both support and inhibit natura l recovery processes. A dult oligodendrocyte precursor cells (OPC) can develop into both astrocyt es and oligodendrocyte s following injury. Furthermore, their distribution in the adult brain is not as restri cted as the neuronal precursor populations (Dawson et al., 2000). An increase in both the astrocyte and oligodendrocyte population ma y contribute positively to the post-injury milieu. Astrocytes metabolize extracellular glutamat e, neutralize free radicals, modulate the immunological response by production of cytoki nes and modulating nitric oxide activity (Gabryel and Trzeciak, 2001; Bambrick et al ., 2004; Heales et al., 2004). Similarly, oligodendrocytes can help re-myelinate damaged axons. Furthermore, glial cell proliferation may contribute to formation of th e glial scar. This barrier can act to protect non-damaged brain regions from advancing se condary injury proce sses (Ridet et al., 1997; Bush et al., 1999; Smith et al., 2001). However, the glial scar also produces chondroitin sulfate proteoglycans which ma y then act to form an impermissible environment for axonal growth (Fawcett and Asher, 1999; Chen et al., 2002). With developmental origins as hematopoiet ic cells, microglia are one of the few mature neural cell types that retain the ability to divide (Simard and Rivest, 2004). After various types of brain injury, microglia proliferate rapidly (L iu et al., 1998a; Csuka et al., 2000; Liu et al., 2000; Grady et al., 2003) to remove cellular debris, protect injured neurons and promote functional recovery (Giulian, 1991). Howe ver, microglia have been

PAGE 19

9 documented as a major source of proteases a nd inflammatory cytokines following various CNS injuries (Nakajima and Kohsaka, 1993; Streit and Kincaid-Colton, 1995; Streit, 1996; Aldskogius et al., 1999; Fawcett and Asher, 1999; Gong et al., 2000). Functional replacement of injured and dying cel ls may contribute to more complete recovery following TBI. Because the neural environment becomes hostile for new cells to survive during the injury state, the identifi cation of proteins that promote both cellular proliferation and survival in compromised cellular environments may prove useful in treating the injured brain. A very delicate balance between proliferation and cell death inhibition is desired. Survivin is a protein that has recently been identified as having roles in both mitosis regula tion and apoptosis inhibition in other non-central nervous system (CNS) pathological conditions and may contribute to this balance following TBI. Survivin: Mitosis and An ti-apoptosis Protein Survivin was discovered in 1997 as a prot ein expressed only by rapidly dividing cells during development (Ambrosini et al., 1997). As its expression is prominent in apoptosis-resistant tumor cells, survivin becam e an intensely studied protein in cancer research. These studies demonstrated that survivin functioned to inhibit apoptosis and was essential for the proper co mpletion of mitosis. Because it has an integral role in cellular proliferation and a poptotic cell death, both of which contribute to the pathophysiology of TBI, survivin may have an important role in the secondary injury cascade. Survivin Protein Structure The survivin protein is composed of 142 am ino acids with a molecular weight of 17 kDa per monomer (Ambrosini et al., 1997). Cellular surviv in exists as a homodimer bound together by an intermolecular Zn+ atom giving the complex a “bow-tie “

PAGE 20

10 appearance and is the only IAP known to homodimerize in so lution (Chantalat et al., 2000; Muchmore et al., 2000; Verdecia et al., 20 00) (Fig.1-2). Survivin is the smallest IAP to have anti-apoptotic properties, containing only a single BIR domain and microtubule-binding coiled coil dom ain (Ambrosini et al., 1997). A distinct subcellular pool of survivin ex ists in the cytoplas m and nucleus of the cell (Conway et al., 2000; Li, 2003; Badran et al., 2004) with a ratio of 6:1, respectively (Fortugno et al., 2002). Recent evidence suggest s that the subcellular localization of survivin may designate its role. The nuclear pool appears to be asso ciated with cellular proliferation while cytoplasmic survivin appears to be more predictive of caspase inhibition (Moon and Tarnawski, 2003). Survivin is a relatively shor t-lived protein with a half-life of approximately 30 minutes (Zhao et al., 2000), th ough phosphorylation may enhance its stability (O'Connor et al., 2000a; O' Connor et al., 2002). Survivin is removed from the cell by polyubiquitination and prot easomal destruction (Zhao et al., 2000). Survivin Expression and Mitosis As a protein found almost exclusively in apoptosis-regulated embryonic and fetal tissue (Adida et al., 1998; K obayashi et al., 1999), surviv in is not normally found in differentiated adult tissues. Howe ver, it is present at very low levels in adult cells with a high mitotic index (Ambrosini et al., 1997). The function of survivin during mitosis is intimately related to its ability to bind microtubules. Survivin is required for the assembly of a bipolar mitotic apparatus by controlling microtubule stability (Altieri, 2001, 2003b). Homozygous deletion of the surviv in gene causes def ects in microtubule assembly, mitotic spindle formation and cel l division resulting in multi-nucleation and total lethality of the orga nism by E3.5-4.5 in knockout mice (Uren et al., 2000).

PAGE 21

11 Beyond development, survivin is prominen tly expressed in many cancers and is linked to poor survival prognosis, higher ra tes of cancer reoccurrence and elevated mortality rates (Altieri, 2003a). Many neur al derived cancer cell lines have been shown to over-express survivin incl uding astrocytes (glioma), neurons (neuroblastoma) and oligodendrocytes (oligodendrogl ioma) (Shankar et al., 2001; Bo rriello et al., 2002; Sasaki et al., 2002; Kajiwara et al., 2003; Kleinschmidt-DeMasters et al., 2003; Jiao et al., 2004), indicating that mature, albeit abnormal, ne ural cells retain the ability to express survivin beyond differentiation. Additionally, proliferating neural stem cells express mitosis proteins after brai n injury (Cameron and McKay, 1998; Doetsch et al., 1999; Cameron and McKay, 2001; Song et al., 2002) indicating that non-transformed neural cells may also express survivin following TBI. Survivin and Apoptosis Inhibition The ability of survivin to inhibit apopto sis is known to occu r in conjunction with the cell cycle but also has been shown to be independent of mitosis. For example, many tumor cells express survivin when not actively dividing and can inhibit apoptosis caused by chemotherapeutic agents (Li et al., 1998). Beyond cancer, survivin expression has been reported in non-prolifer ating, non-tumor cells after is chemic brain injury without activating mitosis and with the ability to inhibit ce ll death (Blanc-Brude et al., 2002; Tran et al., 2002; Conway et al., 2003). Therefor e, survivin expression may occur without activation of the cell cycle. It has been demonstrated repeatedly th at survivin over-exp ression can inhibit apoptosis in cancer cells (Amb rosini et al., 1998; Grossman et al., 1999; Muchmore et al., 2000; Shin et al., 2001; Kim et al., 2004). Survivin expressing gastric and esophageal squamous cell cancers exhibit significantly lower rates of apoptosis compared to

PAGE 22

12 Figure 1-2: Survivin protein st ructure. Survivin is a 142 am ino acid (17 kDa) protein that contains a single baculovirus IAP repeat (BIR) domain (red) and a C-terminus -helical coiled coil domain (orange). In solution, survivin homodimerizes and is held together by a zinc ion inte raction. The survivin BIR domain has been shown to bind activated caspas e-3 and inhibit apoptosis induced by many factors. The coiled coil domai n can bind and stabilize microtubules during assembly of the bipolar mitotic apparatus and keep it in close proximity to caspase activity as mitosi s progresses. 3-D survivin structure adapted from Verdecia et al 2000. survivin-negative cancer cells (Lu et al., 1998). Molecula r antagonists of survivin ( e.g., siRNA, antisense, dominant negative mutants) cause caspase-dependent cell death and magnify the effects of ot her pro-apoptotic signals in vitro and in vivo (Li et al., 1999; Kanwar et al., 2001; Kasof and Gomes, 2001; Shankar et al., 2001; Xia et al., 2002a; Zhou et al., 2002; Choi et al., 2003 ). In addition, survivin has been shown to protect cells from a variety of apoptotic stimuli including IL-3 withdrawal (Amb rosini et al., 1997), Fas stimulation (Tamm et al., 1998; Jiang et al., 2001), anoikis (Pap apetropoulos et al., 2000), cytochrome c administration (Takah ashi et al., 1998; Ta mm et al., 1998), Bax

PAGE 23

13 over-expression (Deveraux et al., 1997; Tamm et al., 1998), active caspase-3 (Tamm et al., 1998), active caspase-7 (Tamm et al., 1998; Jiang et al., 2001), Taxol (Li et al., 1998), and etoposide (Tamm et al., 1998; Jiang et al., 2001). In these models, survivin appears to ex ert its anti-apoptotic effects by directly binding to active caspase-3 (Tamm et al., 1998; Kobayashi et al., 1999). It is possible, however, that survivin may also act at other, less clearly defined points in the apoptotic cascade (Suzuki et al., 2000; Grossman and Altieri, 2001; Grossman et al., 2001a; Fortugno et al., 2003) or outside of apoptotic caspase activat ion to prevent cell death (Shankar et al., 2001; Chakravarti et al., 2004). Potential Role for Survivin in TBI Pathology There are currently no comprehensive studies of survivin in neural cells following CNS injury. Moreover, the pot ential involvement of surviv in in TBI pathophysiology is unknown. The role of survivin in apoptosis inhibition and cellular proliferation in various in vitro and in vivo models supports the hypothesi s that survivin may also contribute to the pathophysiology of TBI. Bo th apoptosis and cellular proliferation occur following traumatic brain injury and create an environment where survivin expression may be important in balancing two contrasting yet related pro cesses. From the literature, it is clear that survivin is ubiquitously e xpressed by all cells early in development and that this expression may be restored in certain mature cells following CNS injury. Therefore, based on the existing data de scribed above from the areas of cancer, mitosis and apoptosis, a thorough investigation of survivin following TBI was warranted. Thus, the main goal of this work is to reveal and characterize potential roles for survivin in neural cell responses following traumatic brain injury. The gene ral hypothesis of the study is that survivin is up-regulated followi ng TBI and plays a role in anti-apoptotic and

PAGE 24

14 cell cycle activation mechanisms to oppose TB I pathogenesis. Specifically, I propose that (i) survivin up-regulat ion inhibits caspase-3 mediat ed DNA fragmentation in a cellspecific manner following TBI, and (ii) survivin plays a role in cell cycle progression following TBI.

PAGE 25

15 CHAPTER 2 METHODOLOGY Induction of Controlled Cortical Impact Brain Injury The surgical and cortical impact injury procedures were conducted as previously described (Dixon et al., 1991; Pi ke et al., 1998). Briefly, ad ult male Sprague-Dawley rats (250-300 g) were anesthetized with 4% isoflurane (Halocar bon Laboratories; River Edge, NJ) in 1:1 O2/ N2O for 4 minutes and maintained dur ing surgery with 2.5% isoflurane. Core body temperature was continuously monito red using a rectal thermistor probe and maintained at 36.5-37.5o C using an adjustable heating pad. A unilateral craniotomy (ipsilateral to injury) was performed over th e right cortex between the sagittal suture, bregma and lambda while leaving the dura intact. Traumatic insult was generated by impacting the exposed cortex with a 5 mm di ameter aluminum tip at a velocity of 4 m/sec, a 150ms dwell time and 1.6 mm compression. Craniotomy control animals received the craniotomy but not the impact injury. All procedur es were performed according to guidelines established by the Univ ersity of Florida Institutional Animal Care and Use Committee (IACUC) and the National Institutes of Health (NIH). In the following studies, “ipsilateral” refers to the same side as the impact injury whereas “contralateral” refers to the opposite side of the injury. “C raniotomy control” refers to animals that received the craniotomy but did not receive the impact injury. Quantitative Reverse Transcriptase Polymerase Chain Reaction (Q-PCR) Survivin primers were generated using GeneBank locus AF 276775: forward primer 5’ TAAGC CACTT GTCCC AGCTT 3’ and reverse primer 5’ AGGAT GGTAC

PAGE 26

16 CCCAT TACCT 3’. GAPDH: forward prim er 5’ GGCTG CCTTC TCTTG TGAC 3’ and the reverse primer 5’ CACCA CTTCG TCCGC CGG 3’. Cortical and hippocampal tissues from the ipsilateral and contralateral hemispheres were rapidl y excised at either 1 day, 2 days, 3 days, 5 days, 7 days or 14 da ys and ‘snap-frozen’ with liquid nitrogen. Total RNA was isolated from the samples us ing TRIzol reagent (In vitrogen, Carlsbad, CA, USA) according to the manufacturer’s inst ructions. Final RNA concentrations were determined via spectrophotometry and were st ored at -20 C in diethyl pyrocarbonate (DEPC) water for future cDNA preparation. cDNA synthesis was performed using 1 g of total RNA with the SuperScript First-Strand Synthesis System for RT-PCR k it (Invitrogen/Life Technologies, Carlsbad, CA) according to the manufacturer ’s instructions. Any DNA contamination was detected in the RNA samples by “no reverse transcript ase” reactions that were performed in conjunction with the cDNA synthesis reaction. Q-PCR was performed as previously desc ribed (Tolentino et al., 2002) using the LightCycler-FastStart DNA Master SYBR Green I reacti on mix (Roche Diagnostics, Indianapolis, IN) in combination with 0.5 M primers, 2.5 mM MgCl2 in the Light Cycler rapid thermal cycler system (Roche Diagnostics, Indianapolis, IN). Briefly, the products were amplified then continuously quantified by online monitoring. Each PCR reaction has its kinetics represented by an am plification curve. Each amplification curve (fluorescence vs. cycle number) is assigned a crossing point value (CPV), which is the exact time point at which the logarithmic lin ear phase could be distinguished from the background. A lower CPV indicates a more ra pid increase in the level of fluorescence indicative of a higher concentra tion of specific message present in the sample. Therefore,

PAGE 27

17 those samples with a lower CPV have more am plified message than those with a higher CPV. The survivin primer sets were subjecte d to serial dilution and linear regression analysis of the logarithm of the dilution factor vs. the CPV generated a standard curve for each transcript-specific template. The sp ecificity of the amplified products were confirmed using melting curve analysis and ge l electrophoresis. The relative amounts of RNA from the unknown samples were extrapolated from its calculated CVP in relation to the generated standard curve. Results are pres ented as percentage of craniotomy control. Data were analyzed by ANOVA with a post-hoc Bonferroni-test and are given as mean SEM. Differences were consider ed significant at the level of p 0.05. Rat-Specific Survivin Polyclon al Antibody Production Commercially available survivin antibodies were not adequate to label survivin in tissue sections. Therefore, a new rat-specific antibody was developed for use in fluorescent immunohistochemistry. Two rat-spec ific survivin sequence peptides were synthesized using the protein sequen ce from GeneBank, accession number AF276775 (Swissprot Q9JHY7), for antibody production. The two pep tides corresponded to regions in the conserved BIR domain (CPTENEPDLAQC) and from the C-terminus coiled coil domain (CFKELEGWEPDDNPIEE). The peptides used to develop the survivin antibody (R51) are specific to survivin and do not r ecognize other IAP family proteins according to SDSC Biology Workbench BLASTP (2.2.2) (Altschul et al., 1997) and CLUSTAL W (1.81) analysis (Higgins et al., 1992; Thomps on et al., 1994) resulting in the survivin antibody’s specificity. Alignment scores for CLUSTALW (1.81) were computed with the following multiple alignment parameters: Matrix : Gonnet, Gap Open Penalty : 10.00,

PAGE 28

18 % Identity for Delay : 30, Penalize End Gaps : on, Gap Separation Distance : 0, Negative Matrix? : no, Gap Extension Penalty : 0.20, Residue-Specific Gap Penalties : on, Hydrophilic Gap Penalties : on, Hydrophilic Residues : GPSNDQEKR. Rabbits were immunized with these peptides allowed to produce antibodies to the peptides and finally serum was extracted fr om the immunized rabbits. The rat specific survivin antibodies were removed and affinity purified using a SulfoLink kit (Pierce Inc; Rockford, IL) as per the manufacturers instructions. Survivin Polyclonal Antibody Characterization The specificity of the survivin antibody (R 51; Dr. G. Shaw) was compared to other commercially available survivin antibodies (Chemicon; Temecula, CA and Novus Biologicals; Littleton, CO) on we stern blots and in cell cultur e. On western blots using dividing cell culture lysates (H eLa and SY5Y) and injured ti ssue lysates, R51 and the Novus survivin antibody show a similar labeling pattern and recognized the 17 kDa monomer of survivin. For IHC, R51 show ed characteristic staining of the cleavage furrow between dividing HeLa and SY5Y cells consistent with other reports (Li et al., 1998; Li et al., 1999; Uren et al., 2000) (Figure 2-1). In addition, dual-labeling in dividing cell cultures of both HeLa and SY5Y cells with R51 and the Chemicon survivin antibody showed co-localizati on at the cleavage furrow. Western Blot Analyses The cortex and hippocampus from each set of brain tissues was excised, rinsed with cold PBS, snap frozen in liquid nitrogen a nd homogenized in ice-cold triple detergent lysis buffer containing a CompleteTM protease inhibitor cockta il (Roche Biochemicals, Indianapolis, IN). Protein concentration was determined by bicinchoninic acid (BCA)

PAGE 29

19 Figure 2-1: IHC characterization of the ratspecific survivin an tibody. The survivin antibody (R51) reveals a characteristic and previously described labeling pattern on western blot (A) and in IHC (B ). Western blot analysis revealed a classic 17 kDa band in cell culture lysates (lanes 1-3) and in injured rat tissue lysates (lanes 6-7) but not in un-injure d rat tissue lysates (lanes 4-5). IHC using the survivin antibody revealed a well-characterized survivin (green) staining pattern around the nuclei (DAPI, bl ue) of proliferating SY5Y cells in various stages of mitosis including G2/M interphase (I), pro-metaphase (PM), anaphase (A) and telophase (T). micro protein assays (Pierce, Inc., Rockfor d, IL). Forty micrograms of protein per well was loaded and separated by SDS-PAGE, tran sferred to PVDF membranes and probed with either goat-anti-rabbit survivin antibody (Novus Biolog icals; Littleton, CO; 1:1000) or goat-anti-rabbit active caspase-3 (Ce ll Signaling; 9661L; 1:100) After incubation with goat anti-rabbit HRP-la beled secondary antibody (Bio rad, Hercules, CA), the membranes were developed using Enhanced Chemiluminescence Plus reagents (ECL Plus; Amersham, Arlington Heights, IL). Fo r further PCNA analysis, developed PVDF membranes were incubated in stripping buffer, rinsed twice in TBST and incubated with PCNA (Santa Cruz Biotech; Santa Cruz, CA; 1:1000) antibody with goat-anti-mouse

PAGE 30

20 HRP conjugated secondary antibody. Semi-qua ntitative, densitome tric analysis was performed using the AlphaImager 2000 Digital Imaging System (San Leandro, CA). The blots were not labeled with an antibody, such as actin or GAPDH, to act as an internal standard because our previous studi es found that many “stable” proteins are the targets of proteolytic cleavage and thus c ould not act as a proper internal control. Transformed data (experimental densitometr y value/ craniotomy control densitometry value x 100) was evaluated by ANOVA and a post-hoc Dunnet-test. Values were expressed as percentage of craniotomy controls and are reported as mean SEM. Differences were considered si gnificant at the level of p 0.05. Preparation and Sectioning of Tiss ue for Immunohistochemistry (IHC) Tissue was prepared and sectioned for vi bratome and cryostat sectioning. For vibratome sectioning, animals were transcardi ally perfused with 2% Heparin (ElkinsSinn, Inc.; Cherry Hill, NJ) in 0.9% saline solution (pH 7.4) followed by 4% paraformaldehyde in 0.1M phosphate buffer (pH 7.4) The brains were post-fixed in 4% paraformaldehyde and stored in 0.1M PBS or cryobuffer. Sections were cut on a Series 1000 vibratome (Ted Pella; Redding, CA) at forty microns. For cryostat sectioning, animals were anesthetized w ith 4% isoflurane (Halocarbon Laboratories; River Edge, NJ) in 1:1 O2/ N2O for 4 minutes, then the head was re moved. The brains were blocked in O.C.T. (Ted Pella; Redding, CA), snap frozen in liquid nitrogen and cut on a Leica CM3050 cryostat. Five micron sections were attached to Fropen (Ted Pella; Redding, CA)-treated coverslips, fixed in cold methanol for 20 minutes at –20 C.

PAGE 31

21 Dual Label Fluorescent Immunohistochemistry (IHC) Sections were fluorescent immunolabeled with two primary antibodies in the following experiments: survivin (1:500)/GFAP for astrocytes (Sternberger; Lutherville, MD; 1:1000), survivin/NeuN for mature ne urons (Chemicon; Temecula, CA; 1:1000), survivin/PCNA (Santa Cruz Biotech; Santa Cruz, CA; 1:200), PCNA/GFAP, PCNA/NeuN, active caspase-3 (1:100)/GFA P, active caspase-3 /NeuN and active caspase-3 /survivin (G. Shaw; 1:250). In addition, sections were labeled with the Apoptag Cell Death Labeling kit (termina l deoxynucleotidyl transferase-mediated biotinylated dUTP nick-end labeling or T UNEL) to mark double stranded DNA breaks as per the manufacturer’s instructions. This k it was used in conjunction with the following antibodies: TUNEL/GFAP, TU NEL/NeuN and TUNEL/survivin. The nuclear dye DAPI (in Vectashield; H-1200; Vector Laboratories ; Burlingame, CA) was used to label the nuclei in all sections. The first primary antibody was incubated at 4 C for 2448 h in a 2% goat serum/ 2% horse serum/ 0.2% Triton-X 100 in 0.1 M PBS (block) solution followed by the second primary antibody at 4 C for 1 h in block solution. Fluorescenttagged secondary antibody (Molecular Probes; Eugene, OR) was used for visualization. Sections were viewed and digitally captur ed with a Zeiss Axioplan 2 microscope equipped with a SPOT Real Time Slider high-resolution color CCD digital camera (Diagnostic Instruments, Inc., Ster ling Heights, MI). A Bio-Rad 1024 ES confocal microscope was used to confirm single cell localization of the label pairings. The settings for these images were as follows: power = 100%; for the red field: iris = 2.7 – 5.2, gain = 1400, Blev = -3; for the green fiel d: iris = 3.0 – 5.7, gain = 1400, Blev = -3. The number of animals used for each label pa iring for dual-labeling IHC was four (n=4).

PAGE 32

22 Dual Label Fluorescent IHC for Same-Species Antibodies Two systems were used for dual-labeli ng using same species antibodies; the tyramide signal amplification (TSA) kit (P erkinElmer Life Sciences, Boston, MA) and a biotin/streptavidin antibody protocol. Both t echniques rely on steric hindrance to block same-species binding sites. Control sections showed the secondary/tertiary complex was sufficient for steric hindrance of same speci es sites for both protocols (Figure 2-2). Tyramide signal amplification (TSA) was accomplished using the TSA kit (PerkinElmer Life Sciences, Boston, MA) acco rding to the manufacturer’s instructions and as previously described (Stone et al., 2002 ). Biotin/streptavidin same species duallabeling begins with an endogenous biotin blocking step (Vector Laboratories; Burlingame, CA) followed by incubation of the first primary antibody as described above followed sequentially by a biotin-conjugate d secondary antibody a nd fluorescent-labeled streptavidin (Molecular Probes; Eugene, OR), both steps at room temperature for 1 h in block solution. The second antigen was then labeled as described above. Experimental Group Sizes The number of animals used for western blot an alysis is as follows (per time point): survivin = 6, PCNA = 6, activ e caspase-3 = 6. The number of animals used for duallabeling IHC is as follows (presented as 5 days post injury or both 5 / 7 days post injury): survivin x PCNA = 4, survivin x GFAP = 6, survivin x NeuN = 4, PCNA x GFAP = 4, PCNA x NeuN = 4, active caspase-3 x survivin = 4/4, active caspase-3 x GFAP = 4/4, active caspase-3 x NeuN = 4/4, TUNEL x GF AP = 4/4, TUNEL x NeuN = 4/4 & TUNEL x survivin = 4/4.

PAGE 33

23 Figure 2-2: Control section for biotin/strep tavidin same-species dual labeling IHC. Biotin/streptavidin same-species dual labeling IHC is a technique based on steric hindrance of sa me species binding sites to prevent the second fluorescent-labeled secondary from bindi ng to the first primary antibody. To ensure that this process was suffici ent, mouse antibodies for astrocytes (GFAP, green) and neurons (NeuN, red) we re used to label a section of brain. These two protein targets were chosen b ecause of their abundance and distinct labeling patterns in adult rat brain. On the left (3 Control), neurons were labeled with only the primary and bi otin secondary with no fluorescent streptavidin tertiary anti body while astrocytes were labeled with a primary and fluorescent-tagged secondary antibody. On the right (Complete), neurons were labeled with a primary, the biotin conjugated secondary and a fluorescent-tagged streptavidin tertiary while astrocytes were labeled as described previously. The absence of red fluorescent labeling in the picture on the left indicates that the biotin se condary antibody was sufficiently large to prevent the green fluorescent-labeled secondary antibody to bind to it thus confirming steric hindran ce of NeuN binding sites. Cell Quantification and Statistical Analysis Cell counts were obtained by comparing the num ber of dual-labeled cells to total singlelabeled cells in the following groups: survivin/NeuN positive cells to total NeuN positive cells, survivin/PCNA positive cells to tota l PCNA positive cells, PCNA/NeuN positive cells to total NeuN positive cells, surviv in/GFAP positive cells to total GFAP positive cells, active caspase-3/NeuN positive cells to total NeuN positive cells, active caspase-

PAGE 34

24 3/survivin positive cells to total survivin positive cells, active caspase-3/GFAP positive cells to total GFAP positive cells, TUNEL/Ne uN positive cells to total NeuN positive cells, TUNEL/survivin positive cells to tota l survivin positive cells and TUNEL/GFAP positive cells to total GFAP positive cells. Pe rcentages were calculated by dividing the number of dual-labeled cells with the to tal number of single-labeled cells. For each group, representative photomicrographs were se lected and counted. Cells were counted in a total area of 188,000 m2 for each label pairing in both cortical and hippocampal regions. These numbers were then transformed into percentage of either total cells or total cell type. Individual comparisons be tween groups were made using an unpaired Student t-test. Results were considered significant at p<0.05. Two additional blinded observers were used to count representative sa mples. Inter-rater reliability was calculated using the inter-rater re liability formula created by R.L. Eb el (Ebel, 1951). The intra-class correlation (ICC) value achieved was 0.97 indi cating little variation between raters.

PAGE 35

25 CHAPTER 3 SURVIVIN EXPRESSION FOLLOWI NG TRAUMATIC BRAIN INJURY Induction of Survivin Expression After TBI Q-PCR analysis revealed an initial increase in survivin mRNA at 2 days post injury in the ipsilateral cortex and hippocampus. These transcripts remained elevated in both regions, reached maximum levels at day 5 post-in jury and declined at 7 days in the cortex and at 14 days in the hippocampus. All e xperimental animals remained alive and exhibited slightly impaired motor and cogniti ve impairments (data not shown). Cortical mRNA levels reached a maximum of 448 10.0%, whereas hippocampal mRNAs attained 606 10.0% compared to craniotomy control values (Figure 3-1). To determine if the induction of survivin mRNA resulted in corresponding increases in survivin protein, western blot analysis was performed. Survivin ( 17 kDa protein) was readily detectable in the ipsilateral cortex and hi ppocampus of TBI rats, wh ile it was negligible in contralateral cortex and hi ppocampus (Figure 3-2A). Surviv in was expressed in a timedependant manner with a maximum increase at 5 days after injury followed by a gradual decline by 14 days. Specifically, the levels of survivin in cortical tissue were at 616 257% at 3 days and at 839 339% at 5 days compared to craniotomy controls (Figure 32B). Similar increases of survivin protein in the ipsilateral hippocampus were detected at 3 days and 5 days post injury: 464 196% and 545 102% compared to craniotomy control, respectivel y (Figure 3-2C).

PAGE 36

26 Figure 3-1: Survivin mRNA induction in rat br ain after TBI. Rats were subjected to craniotomy followed by controlled corti cal impact brain injury. Total RNA was isolated from injured (ipsilateral ) cortex (ic) and hippocampus (ih) at indicated post-injury tim es. cDNA was synthesized, and quantitative PCR using survivin primers was performed as described in detail under Materials and Methods. Data are given as percent of survivin expression over craniotomy controls; each tim e point represents mean + SEM of 4 independent measurements in cranio tomy control or TBI group. ** p<0.01 versus craniotomy control (one-way ANOVA test with post hoc Bonferroni analysis). PCNA Expression After TBI. For detection of proliferating cell nu clear antigen (PCNA), PVDF membranes immunostained for survivin were stripped and re-probed using a PCNA-specific antibody. PCNA (36 kDa protein) wa s significantly detectable in the ipsila teral cortex and hippocampus of TBI rats, but only ne gligible amounts were observed in the contralateral cortex and hippo campus (Figure 3-3A). The te mporal patterns exhibited by PCNA protein were similar to that of surv ivin protein. Namely, PCNA expressed in a time-dependant fashion with a maximum increas e at 5 days after injury followed by a

PAGE 37

27 Figure 3-2: Expression of surv ivin protein after TBI in ra ts. Brain tissue homogenate proteins (40 g) were separated us ing SDS-PAGE, immunoblotted with survivin antibody and visualized as de scribed in detail under Materials and Methods. A-Representative western blot of surviv in (17 kDa protein) in ipsilateral cortex (ic) and hippocampus (ih), contralateral cortex (cc) and hippocampus (ch) obtained from injure d rats, and from craniotomy control rats without cortical imp act (craniotomy control.). Densitometry analysis representation of survivin-positive bands in ipsilateral (ic) and contralateral (cc) cortex (B) and ipsi lateral (ih) and contrala teral (ch) hippocampus (C) after TBI is shown as per cent of craniotomy control values. Each data point represents the mean + SEM of 4 to 6 independe nt experiments. *p<0.05, ** p< 0.001 versus craniotomy cont rol (one-way ANOVA test with post hoc Bonferroni analysis).

PAGE 38

28 gradual decline by 14 days. The levels of PCNA in ipsilateral cortical tissue were raised over craniotomy control by 919 459% at 3 days, 2263 333% at 5 days, and 1035 356% at 7 days post injury (Figure 3-3B). Similar increases of PCNA protein in ipsilateral hippocampus were detected at 5 days post injury with a maximum of 1006 229% compared to craniotomy controls (Fig ure 3-3C). No significant increase was found in the contralateral regions when compared to craniotomy controls (Figure 3-3A). Co-Expression of Survivin and PCNA Following TBI To examine spatial co-localization of survivin and PCNA, dual-label immunohistochemistry was performed on five-day post injury brain ti ssue sections, when peak expression of these proteins was observed. Survivin and PCNA immunoreactivity was found in the ipsilateral cortex (Figure 34A) and ipsilateral hippocampus (Figure 3-4B ) consistent with data obtained using Western blot analyses. Within both regions, focal co-expressi on patterns of survivin and PCNA in single cells were detected, wh ich was demonstrated by both separate fluorescent visualization of i ndividual proteins and by mergin g the images of dual-stained slides (Figure 3-4C-E). However, the dua l expression of survivin and PCNA occurred infrequently as survivin and PCNA immunor eactivity could readily be found separately (Figure 3-4C-E). Approximately 12% of the total number of PCNA-positive cells also labeled with survivin. The nuclear morphology of dual survivin and PCNA-positive cells was ambiguous as indicated by DAPI staining (Figure 3-4F). Therefore, DAPI staining was simply used for cell identificatio n in all subsequent experiments.

PAGE 39

29 Figure 3-3: Expression of PC NA after TBI in rats. PVDF membranes visualized for survivin were stripped and re-probe d with PCNA antibody as described in Materials and Methods. Representati ve western blots showing PCNA (36 kDa) (A) and densitometry analysis of PCNA-positive bands (B, C) are presented. Experimental conditions, sample size and abbreviations are identical to those in Fig. 3-2. *p<0.0 5, ** p< 0.01 versus craniotomy control (one-way ANOVA test with post hoc Bonferroni analysis). Values are mean SEM with n=6.

PAGE 40

30 Figure 3-4: Immunohistochemistry of surviv in and PCNA. Dual-label fluorescent immunostaining for survivin (red) a nd PCNA (green) was performed in the ipsilateral cortex (A) and hippocampus (B) at 5 day postinjury as described in detail under Materials and Methods. Surv ivin is expressed in the cytoplasm (C, red) while PCNA is expressed in th e nucleus (D, green). The white arrow indicates the typical focal co-expressi on of survivin and PCNA as shown in merged survivin and PCNA images (E). PCNA expression was co-incident with DAPI staining (F, blue, white ar row). Magnification: 200x, scale bar 50 m (A and B); 400x, scal e bar 20 m (C-F). Survivin and PCNA are Expressed in Astrocytes After TBI To determine the cell types expres sing survivin and PCNA, dual-label immunohistochemistry for these proteins and GFAP, a marker of astrocytes, was performed in five-day post injury tissue. In accordance with western blot data, survivinpositive immunoreactivity was observed in the ipsilateral cortex and hippocampus proximal to the injury cavity (Figure 3-5A & G, green) but not in the contralateral areas (Figure 3-5B & H). Survivin was co-localized with GFAP in the cells of injured cortex and hippocampus, which strongly suggested prim ary accumulation of survivin in cells of

PAGE 41

31 Figure 3-5: Co-localization of survivin and GFAP in brain tissue af ter TBI. Fluorescent immunohistochemistry for survivin (gr een) and GFAP (red) was performed in the ipsilateral and contralateral cortex (A, B) and in the CA1 and dentate gyrus regions of the hippocampus (G, H) at 5 day post-injury as described in Materials and Methods. The injury has co mpletely destroyed the cortex in G leaving only the hippocampus in this pict ure. Survivin was expressed in the cytoplasm (D, J, green) of GFAP-positiv e astrocytes (C, I, red) of the ipsilateral cortex and hippocampus and wa s found to co-localiz e to these cells as shown in merged C/D and I/J imag es (E, K, respectively, yellow). White arrows indicate typical survivin-positiv e astrocytes. Nuclei are shown using DAPI (F, L, blue). Magnification: 100x, scale bar 50 m (A, B, G, H); 400x, scale bar 20 m (C – F, and I – L). astrocytic lineage (Figure 3-5C-E, I-L). It was further observed that survivin was uniformly distributed in the cytoplasm and pr ocesses of astrocytes in both cortex and hippocampus (Figure 3-5D & J). DAPI staini ng is shown in Figures 3-5F & L. Approximately 88% of the total number of GFAP-positive cells also labeled with survivin. PCNA-positive immunoreactivity staining was observed in the ipsilateral cortex (Figure 3-6A, green) and hippocampus (Figur e 3-6G, green) of injured brain, while contralateral cortex and hippocampus exhi bited negligible PCNA immunoreactivity

PAGE 42

32 Figure 3-6: Co-localization of PCNA and GFAP in brain tissue after TBI. Dual-label immunostaining for PCNA (green) and GFAP (red) was performed in the ipsilateral and contralateral cortex (A, B) and the CA1 and dentate gyrus regions of the hippocampus (G, H) at 5 day post-injury. PCNA is present in GFAP positive cells of ipsilateral cort ex (C, D) and, to a lesser extent hippocampus (I, J). E and K depict me rged C/D and I/J, respectively. White arrows indicate typical PCNA-positive astrocytes. PCNA expression was coincident with DAPI staining (F, L, blue ). Magnification: 100x, scale bar 50 m (A, B, G, H); 400x, scale ba r 20 m (C – F, and I – L). (Figure 3-6B & H). PCNA (Figures 3-6C & I) was partially co-localized with GFAP (Figures 3-6D & J, red) in both regions, a nd was characteristically distributed in the nucleus of the cells in both cortex and hi ppocampus (Figures 3-6E & K). DAPI staining is shown in Figures 3-6F & L. Taken together, dual-label immunohistochemist ry data provides evidence that both survivin and PCNA can be detected in GFAP-positive astrocytes following traumatic insult. Since survivin and PCNA immunoreac tivity was not exclusively localized in GFAP-positive cells, other cell type s must also express survivin.

PAGE 43

33 Figure 3-7: A sub-set of NeuN -positive neurons express surv ivin and PCNA after TBI. Dual-label fluorescent immunohistochemist ry for survivin (green) and NeuN (red) in the ipsilateral cortex (A & B) and the CA1 pyramidal layer of the contralateral hippocampus (E & F) was performed as described in Materials and Methods. Survivin is expressed in the cytoplasm and, to a limited extent, in the processes of NeuN-positive ne urons (merged images C & G). Dual staining for PCNA (green) and NeuN (red) is shown in the ipsilateral cortex (I & J) and the CA1 pyramidal layer of the ipsilateral hippocampus (M & N).

PAGE 44

34 The nuclei are shown using DAPI staining (D & H, blue). PCNA is expressed in the nucleus of NeuN-positive neurons (merged images K & O). PCNA expression was co-incident with DAPI st aining in these examples (L & P, blue). White arrows indicate focal co-localization of survivin/NeuN and PCNA/NeuN. Survivin/NeuN co-localizat ion of survivin (green) and NeuN (red) was seen only in TBI rats as opposed to either hemisphere of craniotomy control (Q & R). (Magnificati on of A-P = 400x, scale bar = 20 m; magnification of Q & R 50x, scale bar = 100,000 m). Survivin and PCNA are Expressed in a Sub-Set of Neurons After TBI As can be seen in Figure 3-7, survivin a nd PCNA were each co-expressed with NeuN, a marker of mature neurons. NeuN-positive cel ls were found to express survivin in the ipsilateral cortex distal to the injury cav ity (Figure 3-7A-D) and in the contralateral hippocampus (Figure 3-7E-H). It should be noted, however, that NeuN-positive cells that also expressed survivin occurred infre quently. For example, the number of dual survivin/NeuN positive cells was estimated at 0.1% to 1.5% of the total number of NeuNpositive cells in these regions. Survivin im munoreactivity was negligible in either hemisphere of craniotomy control brains (F igures 3-7Q & R). No co-localization of survivin and NeuN was observed in ipsilatera l hippocampus (data not shown). As can be seen in Figures 3-7B & F, survivin was pr edominantly localized to the cytoplasm and axons of NeuN-positive neurons. DAPI stai ning is shown in Figures 3-7D & H. PCNA-positive neurons were found in the ips ilateral cortex (Figures 3-7I-L) and hippocampus after TBI (Figure 3-7M-P), wher eas craniotomy control tissue exhibited only trace amounts of PCNA (data not shown) Similar to the survivin/NeuN colocalization data, dual PCNA/ NeuN immunos taining was a rare event accounting for approximately 4% of the total number of NeuN positive cells. PCNA was distributed in the nuclei of these neurons (Figures 3-7K & O) although the nuclear morphology of these cells was not clearly resolved by DAP I staining (Figures 3-7L & P).

PAGE 45

35 Figure 3-8: Survivin expressi on is absent in oligodendroc ytes and microglia following TBI in rats. Dual-label fluorescen t immunohistochemistry for survivin (green), CNPase (red, A) and OX42 (red, B) in the ipsilateral cortex and hippocampus was performed as descri bed in Materials and Methods. Negligible co-localization was seen w ith survivin, CNPase and OX42 in the ipsilateral cortex (A, B respectively) and hippocampus (data not shown). Higher magnification photomicrographs (inset, A and B) show survivinpositive cells (white arrowheads) su rrounded by oligodendrocytes (white arrows, A) and microglia (w hite arrows, B) that do not show co-localization. (Magnification of A and B = 400x, scale bar = 20 m). Survivin is Not Expressed in Mi croglia and Oligodendrocytes To further determine the neural cell types expressing survivin, dual-label immunohistochemistry for survivin, OX42, a ma rker of microglia, and CNPase, a marker for oligodendrocytes, was performed in five-d ay post injury tissue sections. No colocalization with survivin and either CNPa se (Figure 3-8A) or OX-42 (Figure 3-8B) is observed following traumatic brain injury. In addition, attempts were made to localize survivin with the neuronal progen itor cell markers nestin, doublecortin, -internexin and -III-tubulin. However, these antibodies did no t prove to be of accep table quality to use in western blot and IHC analyses in this model leaving prolif erating progenitors undetected.

PAGE 46

36 Discussion of Chapter 3 Traumatic brain injury (TBI) initiates va rious biochemical cascades that induce neural tissue injury and cell death. To c ounteract these cascades, several proteins expressed in neural cells afte r TBI are directed to resist cell death and promote recovery in the injured CNS (Ridet et al., 1997; Ch en and Swanson, 2003). Survivin is a multifunctional protein that inhibits apoptosis and is also required for the proper completion of mitosis. Anti-apoptotic and pro-mitogenic ro les for survivin have been documented in proliferating cells of neural origin in vitr o, such as in neuroblastoma and glioma cells (LaCasse et al., 1998; Tamm et al., 1998; Deveraux and Reed, 1999; Conway et al., 2000; Shin et al., 2001; Sasaki et al., 2002). However, no studies have investigated the potential role of survivin in the adult brain after TBI, wh en a sub-population of CNS cells may initiate a cell cycle-related process in response to injury. These data demonstrate the induction of su rvivin expression in rat brain subjected to TBI. The expression of survivin was timedependent, cell-specific and was present in astrocytes and, to a much lesser extent, in neurons in ipsilateral cortex and hippocampus. Induction of survivin in these cells was accompanied by occasional expression of PCNA, a cell cycle protein involved in mitotic G1/S progression. These data are the first to show that survivin mRNA and protein are significantly up-re gulated after TBI in rats. PCNA expression after TBI has been described prev iously (Miyake et al., 1992; Chen et al., 2003), suggesting its role in mechanisms of br ain recovery after inju ry. The concurrent up-regulation of survivin with a similar temporal profile as PCNA shown herein further suggests that survivin may play a role in cellular prolif eration after TBI. Brain injury evoked the expression of survivin and PCNA in a time-dependent manner (Figures 3-2 & 3-3). Western blot an alysis revealed maximal co-expression of

PAGE 47

37 both survivin and PCNA at five days post injury. Immunohistochemistry demonstrated co-localization of these proteins (Figure 3-4) although most cells were labeled separately with PCNA and survivin. In fact, only 12 % of the total number of PCNA-positive cells were also survivin positive. It has been reported that PCNA is expressed predominantly in G1/S (Bravo et al., 1987), while survivin is found at the G2/M phase of the cell cycle (Bravo et al., 1987; Otaki et al., 2000). Hence, a lack of strict co-loc alization of survivin and PCNA in this study may be explained by th eir expression at di fferent points in the cell cycle. To determine if the differing expression patterns of survivin and PCNA contributed to lower incidence of co-local ization, other cell cy cle proteins were investigated including cdk4 (G 1), cyclin B (G2), cyclin D (G1) and AIM-1 (M). Only PCNA provided clear results in both western blot and IHC analyses. Survivin-positive and PCNA-positive astrocytes were observed in the proximal area of the injury and in the ipsilateral hippocampus. Prolifer ation of astrocytes is well documented after TBI as shown by cell labeli ng with BrdU as well as expression of PCNA (Latov et al., 1979; Dunn-Meynell a nd Levin, 1997; Carbonell and Grady, 1999; Norton, 1999; Csuka et al., 2000; Kernie et al., 2001; Chen et al., 2003). Because survivin and PCNA were expresse d in astrocytes following TBI (Figures 3-5 & 3-6), it is possible that survivin plays an important role linking astrocyte survival and proliferation after traumatic insult. Astrocyte proliferation has been implicated in the formation of the glial scar observed after injury (Latov et al., 197 9) and creates a non-permissive environment for repair (Sykova et al., 1999). However, glial proliferation may also enhance neuronal survival (Smith et al., 2001; We i et al., 2001).

PAGE 48

38 Of particular interest is a sub-set of NeuN-positive neurons found to express survivin only after TBI (Figure 3-7). These cells were much less abundant than survivinpositive astrocytes and their functional si gnificance is currently unknown. However, both neurons and astrocytes have been documented previously to expre ss cell cycle proteins after various insults such as exposure to -amyloid activated microglia (Wu et al., 2000), TBI (Kaya et al., 1999a; Kaya et al., 1999b), chlorin e6 toxicity, (Magavi et al., 2000) or as a consequence of Alzheimer’s Disease (Ya ng et al., 2001). The ramifications of cell cycle protein expression in mature neurons is still controversial and may be a marker of cell death rather than cell pr oliferation (Herrup an d Busser, 1995; Li et al., 1997; Kaya et al., 1999a; Kaya et al., 1999b; Wu et al., 2000; Yang et al., 2001). These papers underscore the significant contr oversy that exists regardi ng the function of cell cycle proteins such as PCNA in neurons after different types of injury. It should be noted that dual staining of survivin and PCNA could not be directly attributed to a specific cell type due to th e technical difficulties of triple labeling antibody-based IHC. Therefore, other cell types, such as endo thelial (Conway et al 2003), inflammatory cells (Hill-Felberg et al., 1999) or neural progenitor cell s (Ignatova et al., 2002), may also contribute to survivin and PCNA expression after TBI. The appearance of survivin and PCNA separately in neur ons (NeuN-positive) and astrocytes (GFAPpositive) along with co-localization of surv ivin with PCNA in the same cells provide correlative data to suggest an activation of cell cycle-like program in astrocytes and possibly in a small subtype of neurons after TBI. In these experiments, survivin colocalization with PCNA does sugge st that survivin may be a ssociated with a pro-mitotic process. In an attempt to clarify these pr otein’s roles after TBI, the nuclear morphology

PAGE 49

39 of survivin-positive cells was analyzed to defi ne the apoptotic or mitotic architecture of nuclei. DAPI staining proved too ambiguous in identifying apoptotic versus mitotic phenotypes likely due to the thic kness of the brain sections (4 0 m). Further studies using direct markers of mitosis such as BrdU in corporation as well as simultaneous labeling with cell death related proteins is required to delineate anti-apoptotic and pro-mitotic activities of survivin and PCNA in these cells. To summarize, an induction of surviv in was found in rat brain cortex and hippocampus after TBI in a time-dependent fa shion. Expression of survivin occurred predominantly in astrocytes and a sub-set of neurons but not in oligodendrocytes or microglia, and was occasionally accompanied by expression of PCNA. However, survivin expression is only found in 12% of PCNA positive cells, which suggests that the primary role of survivin after traumatic brai n injury is not related to the cell cycle but rather to apoptosis inhibition. Thus, the next specific aim of this study was to examine the link between survivin, active caspase-3 a nd downstream DNA fragmentation following traumatic brain injury in rats.

PAGE 50

40 CHAPTER 4 SURVIVIN AND APOPTOSIS INHIBITI ON FOLLOWING TRAUMATIC BRAIN INJURY Caspase-3 is Activated in the Same Br ain Regions as Survivin Following TBI To determine the temporal and regional pr ofile of caspase-3 ac tivation, western blot analysis was performed on cortical and hippoc ampal TBI samples. Active caspase-3 (19 kDa protein) was readily dete ctable in the ipsilateral co rtex and hippocampus of rats subjected to TBI (Figure 4-1A). Caspase3 activation occurred in a time-dependant manner in the ipsilateral cortex and hippo campus with prominent activation occurring between five and fourteen days post-injury, with peak accumulation occurring at seven days post-injury. In the ipsi lateral cortex, significant increases in active caspase-3 levels reached 3468 1088% at five days, 4019 1291% at seven days and 2984 1058% fourteen days post-injury, compared with cr aniotomy controls. Similar increases in caspase-3 activation were detected in the ip silateral hippocampus with increases of 671 257% at five days, 2662 738% at seven da ys and 1487 405% at fourteen days postinjury, compared with cranioto my controls (Figure 4-1B). Survivin Expression Correlates with D ecreased TUNEL Labeling but not Active Caspase-3 Expression. Immunohistochemistry (IHC) was performed on brain sections at five days post injury to investigate the expression of ac tive caspase-3 and TUNEL labeling at peak survivin expression (Johnson et al., 2004). IHC revealed moderate co-localization of survivin with active caspase-3 and TUNEL in the ipsilateral cortex (Figure 4-2A,F) and

PAGE 51

41 Figure 4-1: Caspase-3 Activati on in rat brain after traumatic brain injury. Rats were subjected to craniotomy followed by cont rolled cortical impa ct brain injury. Brain tissue homogenate pr oteins (40 g) were separated using SDS-PAGE, immunoblotted with antibody specific for active caspase-3 an d visualized as described in detail under Materials an d Methods. Representative western blots of active caspase-3 (19 kDa) in ipsilateral cortex and hippocampus obtained from injured rats and from cran iotomy control rats without cortical impact revealed the accumulation of active caspase-3 after TBI (A). Densitometry analysis of the active caspase-3 bands in ipsilateral cortex and hippocampus after TBI show significant increases in a time dependent manner (B). Data are given as percent of the active fragment of caspase-3 related to craniotomy controls; each tim e point represents mean + SEM of 6 independent measurements in cranioto my control or TBI group. p<0.05, ** p<0.01 versus craniotomy contro l (one-way ANOVA test with post hoc Dunnet analysis). ipsilateral hippocampus (Figure 4-2B,G) of injured animals. These data are summarized in Table 1 (pg 45). Survivin was found primar ily in the cytoplasm a nd to a lesser extent in the nucleus of the ipsilateral cortex (Figure 4-2C,H). A similar pattern was seen in the

PAGE 52

42 Figure 4-2: Co-expression of survivin and a poptosis markers following TBI in rats. Dual-label fluorescent immunostaining for survivin (red) and active caspase-3 (green, A-E) or TUNEL (green, F-J) wa s performed in the ipsilateral cortex (A, F) and hippocampus (B, G) at 5 day post-injury as described in detail under Materials and Methods. Survivin is expressed in both the cytoplasm and in the nucleus (C & H, red) whil e active caspase-3 (D, green) and TUNEL (I, green) label predominantly the nu cleus. The white arrow indicates the typical focal co-localizat ion of survivin and active caspase-3 (E) and TUNEL (J) as shown in the merged images. Magnification: 200x, scale bar 50 m (A, B, F, G); 200x, scale bar 10 m (C-E, H-J). ipsilateral hippocampus (data not shown). Active caspase-3 (Figure 4-2D) and TUNEL (Figure 4-2I) labeling were both found in th e nucleus in both regions. Survivin colocalization with active caspas e-3 and TUNEL is shown at high magnification in Figure 4-2E and Figure 4-2J, respectiv ely. Confocal microscopy was used to assure single cell co-localization of survivin, active cas pase-3 and TUNEL (data not shown). Quantitative analysis revealed no signif icant difference in the accumulation of active caspase-3 in survivin-positive cells co mpared to survivin-negative cells at five days post injury in either the cortex or hippocampus (Figure 4-3A,B). However, significantly higher percentage of TUNEL labeling was observed in survivin-negative

PAGE 53

43 Figure 4-3: Survivin expre ssion decreases the accumulati on of TUNEL but not active caspase-3. Cells were quantified and vi sualized as described in detail under Materials and Methods. The percentage of cells expressing active caspase-3 and labeling with TUNEL was compared in two cell populations, survivinpositive and survivin-negative, in the ipsilateral cortex (A) and hippocampus (B). No differences were found in th e percentage of survivin-positive and survivin-negative cells e xpressing active caspase-3 in either the cortex (A, black bars) or hippocampus (B, black ba rs). Significantly more survivinnegative cells were TUNEL-positive comp ared to survivin-positive cells in both the cortex (p<0.01, A, white ba rs) and hippocampus (p<0.001, B, white bars). No differences were found in the percentage of TUNEL-positive (C, white bars) or TUNEL-negative cells (C, gray bars) that also labeled with active caspase-3. Each bar represents mean + SEM of 4 (A, cortex) or 3 (B, hippocampus) independent measurements using an unpaired Student t-test for statistical analysis.

PAGE 54

44 cells as compared to surviv in-positive cells in both re gions (p<0.01) (Figure 4-3A,B, Table 1A). At seven days post injury, there was no significant difference in the accumulation of both active caspase-3 and TUNEL labeling in survivin-positive cells compared to survivin-negative cells in eith er the cortex or hippocampus (Table 1B). To verify that caspase-3 activation does not necessarily result in irreversible cell death, possibly due to survivin inhibition, quan tification data was gathered on cells that accumulate active caspase-3 and are TUNEL label-positive and compared to active caspase-3-positive cells that do not label with TUNEL. Indeed, we f ound that the number of dual-labeled active caspase-3 and TUNEL cells was not st atistically different from those cells labeled with activ e caspase-3 only (Figure 4-3C ). This finding further supports the notion that severa l counteractive factors, including survivin, may inhibit active caspase-3 and diminish cell death following TBI. Astrocytes and Neurons Demonstrate Cell Spe cific Differences in Active Caspase-3 and TUNEL Labeling To determine cell types that express activ e caspase-3 or labeled with TUNEL, dual immunohistochemistry of these apoptosis ma rkers was performed with GFAP, a marker of astrocytes, and NeuN, a ma rker of mature neurons. Co -localization of GFAP was observed with both active caspase-3 and TUNEL labeling in the ipsilateral cortex (Figure 4-4A,F respectively) and ipsilateral hippocampus (Figur e 4-4B,G respectively). Higher magnification photomicrographs show a typi cal astrocyte expre ssing active caspase-3 (Figure 4-4C-E) or labeling w ith TUNEL (Figure 4-4H-J). NeuN-positive cells exhibited modest accumulation of active caspase-3 and considerable labeling with TUNEL in the ipsilateral cortex (Figure 4-5A,F) and ipsilateral hippocampus (Figure 4-5B,G). These data are summarized in Table 1.

PAGE 55

45 Table 1 Cell Quantification Data for Imm unohistochemistry Labeling Pairs This table summarizes the results from all cell count experiment s investigating survivin, cell type and the apoptosis markers. The numbers given are percentages calculated as described in the Materi als and Methods. Each percentage represents the mean + SEM of 4 (cortex) or 3 (hippocampus) independent measurements at either five days postinjury (A) or seven days post-injury (B). ** p<0.01 versus survivin-negative cells, ## p<0.01, ### p<0.001 versus NeuN-positive cells, $$ p<0.01 versus TUNEL-negative cells (unpaired Student t-test). Specifically, both active caspase-3 (Figure 45D) and TUNEL (Figure 4-5I) were present in neuronal nuclei (Figure 4-5C,H), which wa s indicated by co-local ization with NeuN (Figure 4-5E, J). Confocal microscopy was us ed to assure single ce ll co-localization of NeuN and GFAP with active caspase-3 and TUNEL (data not shown). Quantitative analysis revealed a substantia lly different expression profile of active caspase-3 and TUNEL in neurons and astroc ytes. A significantly higher number of astrocytes accumulated active caspase-3 as comp ared to neurons in the cortex, but not the hippocampus (p<0.001) (Table 1). Conversely, a significantly greater number of neurons were labeled with TUNEL compared to astr ocytes in both the cortex and hippocampus (p<0.001) (Figure 4-6, Table 1A). A similar expression profile of active caspase-3 and

PAGE 56

46 Figure 4-4: Astrocytes expre ss active caspase-3 and label with TUNEL following TBI in rats. Dual-label fluorescent immuno staining for GFAP (red) and active caspase-3 (green, A-E) or TUNEL (g reen, F-J) was performed in the ipsilateral cortex (A, F) and hippocam pus (B, G) at 5 day post-injury as described in detail under Materials and Methods. GF AP is expressed in the cytoplasm (C & H, red) while activ e caspase-3 (D, green) and TUNEL (I, green) are expressed in the nucleus. Th e white arrow indicates the typical focal co-expression of GFAP and active caspase-3 (E) and TUNEL (J) as shown in the merged images. Magnificatio n: 200x, scale bar 50 m (A, B, F, G); 200x, scale bar 10 m (C-E, H-J). TUNEL was observed in astrocyt es and neurons seven days post injury (Table 1B). Discussion of Chapter 4 Caspase-3 activation is a prominent feat ure of apoptosis and its role in DNA fragmentation after TBI has b een well documented (Nicholson et al., 1995; Tewari et al., 1995; Pike et al., 1998; Tang and Kidd, 1998; Wolf et al., 1999; Beer et al., 2000; Buki et al., 2000; Clark et al., 2000). Survivin is an inhibitor of apoptosis protein (IAP), which can inhibit active caspase-3 and thereby mode rate cell death in various tissues, including CNS (Shankar et al., 2001; Sasaki et al., 2002; Van Hare n et al., 2004). However, no

PAGE 57

47 Figure 4-5: Neurons express active caspase3 and label with TUNEL following TBI in rats. Dual fluorescent immunostaining for NeuN (red) and active caspase-3 (green, A-E) or TUNEL (green, F-J) wa s performed in the ipsilateral cortex (A, F) and hippocampus (B, G) at 5 day post-injury as described in detail under Materials and Methods. NeuN is e xpressed primarily in the nucleus but also in the cytoplasm (C & H, red) while active caspase-3 (D, green) and TUNEL (I, green) are found in the nucl eus. The white arrow indicates the typical focal co-localization of NeuN and active caspase-3 (E) and TUNEL (J) as shown in the merged images. Magnif ication: 200x, scale bar 50 m (A, B, F, G); 200x, scale bar 10 m (C-E, H-J). studies have investigated the potential anti-apoptotic role of survivin in the adult brain after TBI. These data characterize the relationshi p between survivin expression and two apoptosis events: the accumulation of active caspase-3 and downstream DNA fragmentation (as shown by TUNEL labeling) in rat brain subjected to TBI. The use of TUNEL labeling in conjunction wi th active caspase-3 is considered a reliable tool to assess apoptosis progression (L ei et al., 2004; Marciano et al ., 2004; Nakase et al., 2004). The appearance of active caspase-3 was time-de pendent and region specific (Figure 4-1)

PAGE 58

48 Figure 4-6: TUNEL labeling is cell specific foll owing TBI in rats. Cells were quantified and visualized as described in deta il under Materials and Methods. The percentage of cells labeling with TUN EL was compared in astrocytes (black bars) and neurons (gray bars) in the ipsilateral cortex and hippocampus. Significantly more TUNEL labeling was seen in neurons than in astrocytes in both the cortex and hippocampus (p<0.001). Each bar represents mean + SEM of 4 (cortex) or 3 (hippocampus) independent measurements using an unpaired Student t-test for statistical analysis. with a pattern similar to survivin expressi on after TBI which suggests that survivin may inhibit caspase-3 activity to di minish the deleterious conse quences of proteolysis after TBI (Tamm et al., 1998; Kobayashi et al., 1999; O'Connor et al., 2000b; Shin et al., 2001). Other IAPs are up-regulated in concer t with activation of caspases after brain injury (Keane et al., 2001). In addition, survivin expression is up-regulated by the prosurvival PI3-kinase/Akt path way which is activated after TBI (Kitagawa et al., 1999; Xia et al., 2002b; Kim et al., 2004). Survivin-positive cells expressed active caspase-3 and were labeled with TUNEL (Figure 4-2) though survivin-positive cells showed no significant difference in accumulation of active caspase-3 compared to survivin-negative cells (Figure 4-3).

PAGE 59

49 Figure 4-7 : Putative mechanism of apoptosis inhibi tion by survivin following TBI. Traumatic brain injury induces activa tion of upstream caspases-8 and 9 that can process pro-caspase-3 to its active form. Once activated, caspase-3 can cleave several intracellular substrates and activate restrictases that may lead to DNA fragmentation. Concomitant surviv in expression is up-regulated in response to the same TBI signals. Surv ivin has the ability to inhibit the activity of active caspase-3, which results in the attenuation of DNA fragmentation. These data are in accordance with the ability of survivin (Tamm et al., 1998) and other IAPs (Shankar et al., 2001; Maie r et al., 2002) to inhibit the activity but not the activation of caspase-3. In contrast, fewer survivin -positive cells exhibi ted DNA fragmentation (TUNEL labeling) compared to survivin-negativ e cells at five days post injury (Figure 43). These data suggest that survivin expr ession may attenuate the apoptotic cascade by inhibiting the cleavage of caspase-3 specific substrates that result in DNA fragmentation. Furthermore, the finding that active casp ase-3 accumulation led to positive TUNEL

PAGE 60

50 labeling in 51% of all active caspase-3-positiv e cells (Figure 4-3) is consistent with the observation that endogenous fact ors, including survivin, may inhibit active caspase-3 and attenuate DNA fragmentation following TBI. In an attempt to more directly investigate whether survivin could inhibit active caspase-3 activity, survivin was co-localized with markers of caspase-3 activity including the cleaved speci es of PARP, DFF45/iCAD and -II-spectrin (120 kDa). Unfo rtunately, these markers proved to be of unacceptable quality to use in western blot and IHC analys es in this model. Therefore, it remains unknown whether survivin correlates with a decrease in active cas pase-3 activity as measured by caspase-3-specific breakdown products The ability of survivin to inhibit DNA fragmentation has been shown in gastric ca ncer cells (Lu et al ., 1998). In addition, survivin antisense treatment increases DNA fragmentation in neuroblastoma and oligodendroglioma (S hankar et al., 2001). Taken together, these findings suggest that survivin likely inhibits the proteolytic activity of caspas e-3 after TBI to attenuate DNA cleavage. The labeling patterns of ac tive caspase-3 and TUNEL in astrocytes and neurons were investigated. Data descri bed in Chapter 3 showed that a large majority of astrocytes but few neurons express survivin after TBI. These data show that both astrocytes and neurons expressed active caspa se-3 and label with TUNEL post-injury but the prevalence of this labeling was drastically different (F igures 4-4, 4-5). A higher percentage of astrocytes accumulate active caspase-3 but fe wer astrocytes label with TUNEL compared to neurons (Figure 4-6). It is unclear at present why these experiments revealed few neurons labeling with active caspase-3 (T able 1). Some active caspase-3-positive neurons may not have been detected due to caspase-3 mediated loss of NeuN antigenicity

PAGE 61

51 (Unal-Cevik et al., 2004). Other groups ha ve found prominent caspase-3 activation in apoptotic neurons after TBI (B eer et al., 2000). In addition, caspase-independent necrosis may also be a major contributor of neuronal cell death after TBI (Newcomb et al., 1999; Wennersten et al., 2003). Cell specific survivin expression may cont ribute to the lower incidence of TUNEL labeling in astrocytes as compared to neurons (Table 1). Cell type specific expression of IAPs after TBI has been previ ously shown. For example, XIAP is expressed primarily by neurons (Lotocki et al., 2003) and a subset of oligodendrocytes (Keane et al., 2001) following brain injury. NAIP is expressed in neurons after ischemia (Xu et al., 1997) and TBI (Hutchison et al., 2001). Lastly, RIAP-2 is abundantly expressed in neurons as opposed to astrocytes after kainic acid treatm ent in rats (Belluardo et al., 2002). Figure 4-7 shows a putative mechanism for apoptos is inhibition by survivin. Following upstream caspase activation and cleavage of procaspase-3 to activ e caspase-3, survivin acts to attenuate the apoptotic cascade a nd finally DNA cleavage and cell death by inhibiting caspase-3 activity. Taken together, data from Chapter 4 demons trate that the activation of caspase-3 in the rat brain after TBI follows the same ti meframe, regions and cell type expression patterns as survivin. Quantitative correlative analysis reveals that significantly fewer cells expressing survivin undergo final stag e of apoptosis and cell death. These data suggest that DNA cleavage may be attenuate d via inhibition of active caspase-3 by survivin after traumatic brain injury in a cel l-specific fashion. Name ly, astrocytes have significantly lower TUNEL labeling than neur ons suggesting a more robust anti-apoptotic role for survivin in astrocytes. Collectively, these results suggest that survivin plays a

PAGE 62

52 role in diminishing apoptosis and DNA fragme ntation following traumatic brain injury in rats.

PAGE 63

53 CHAPTER 5 CONCLUSIONS AND FUTURE DIRECTIONS Conclusions Traumatic brain injury (TBI) remains a majo r health care and economic issue in the United States to this date. Despite con tinued education and improved first responder care, the Centers for Disease Control estim ate that there are more than 5.3 million Americans living with disabilities from TB I with another 1.5 million new TBIs sustained in the U.S each year. Cognitive and memory deficits resulting from TBI are especially difficult to cure and no effective treatment options are available. TBI is a complex injury that induces bot h apoptotic cell death and neural cell proliferation. Apoptosis is opposed by up-re gulation of inhibitor of apoptosis proteins (IAPs) that attenuate apoptosis through direct inhibition of active caspases. Neural cell proliferation may contribute to neural ti ssue healing by repopulating damaged regions with new, functional cells but may also preven t normal recovery of the injured brain by forming impermissible barriers for axonal growth. The recently discovered protein, survivin, may play a significant role in these processes following TBI. Survivin is a protein that is both integral for mitosis and is an IAP with anti-apoptotic properties. Though not normally found in quiescent adult tissues, survivin can be induced in certain mature non-neural cells afte r CNS insult or cancer transformation to resist apoptosis. Furthe rmore, brain injury can induce stem cell proliferation, a process that likely require s the survivin protein for proper completion.

PAGE 64

54 However, no study to date had investigated the temporal, regional and neural cell expression patterns of this unique protein following TBI. Chapter 3 of this dissertation critically examined the transcriptional and translational expression of survivin following TBI in rats. This dissertation is the first to demonstrate that survivin mR NA and protein are expressed in neural cells following TBI and that this expression is cell type specific. QPCR analysis confirme d elevated levels of survivin following TBI that peaked at five da ys post-injury in the ipsilateral cortex and hippocampus. Immunoblot analys is confirmed survivin transl ation with peak expression at five days post-injury in both regions. Survivin localiz ation at this time point was observed in approximately 88% of the astr ocytes in the ipsilateral cortex and hippocampus. Survivin expression was also observed in a much smaller sub-set of neurons, where no more than 1.5% of neurons expressed survivin. Survivin expression was not observed in microglia and oligodendroc ytes. Like other IAP proteins, expression of survivin appears to be cell type specific following TBI. In contrast to the IAPs XIAP, NAIP, cIAP-1 and cIAP-2 which are predomin antly expressed in neurons following brain insult, survivin is expressed primarily in astrocytes. Attempts were made to localize survivin with the neuronal progen itor cell markers nestin, doublecortin, -internexin and -III-tubulin. However, these antibodies did no t prove to be of accep table quality to use in western blot and IHC analyses in this model leaving prolif erating progenitors undetected. Survivin has two biochemically distinct f unctions, that of apoptosis inhibition and to properly separate DNA during mitosis. To reveal the relations hip between survivin and cellular proliferation, se veral cell cycle proteins we re investigated including cdk4,

PAGE 65

55 cyclin B, cyclin D, AIM-1 and PCNA. Only PCNA provided clear results in both western blot and IHC analyses. Therefore, the regional, tempor al and cell specific protein expression of the prev iously characterized cell cy cle protein, PCNA and its colocalization with survivin wa s investigated. PCNA protein accumulated in the ipsilateral cortex and hippocampus peaking at five days post-injury. In addition, astrocytes and a small subset of neurons expressed PCNA in a pattern similar to survivin. However, only 12% of PCNA-positive cells also expressed su rvivin. The cellular identity of these duallabeled cells remains unknown due to difficulties with triple-label IHC. Therefore, the ramifications of cell cycle protein expressi on in individual cell types remains unclear. However, these data seem to indicate that the primary function of survivin following TBI is not cellular proliferation. A more complete picture of survivin’s role in cellular proliferation following brain insult would re quire co-localization with other cell cycle related proteins. Thus far, additional invest igations with proteins such as AIM-1, cdk4, cyclin D and cyclin B1 have been inconclu sive. Further studies will be required to determine the extent to which survivin acts as an indicator of cellular proliferation and the cell types that may be actively proliferating following TBI. Chapter 4 revealed the relationship betw een survivin and apoptosis inhibition by investigating the accumulation of active ca spase-3, the main executioner caspase in apoptosis, and the appearance of DNA frag mentation (TUNEL) following TBI. Immunoblot studies revealed that active cas pase-3 did accumulate following TBI with significant accumulation at five, seven and four teen days post-injury in the ipsilateral cortex and at seven and fourteen days pos t-injury in the ipsilateral hippocampus. Because survivin and active caspase-3 levels peaked on different post-injury days, IHC

PAGE 66

56 was performed at the peak expression times of five days (survivin) and seven days (active caspase-3) post-injury. IHC anal ysis revealed that survivin and both active caspase-3 and TUNEL did co-localize to the same cells at th ese time points. Attempts were made to colocalize survivin with markers of caspase-3 ac tivity to correlate the presence of survivin to an absence in caspase-3 specific brea kdown products. Antibodies for the caspase-3specific cleaved species of PARP, DFF45/iCAD and -II-spectrin (120 kDa) proved to be of unacceptable quality to use in western blot and IHC analyses in this model leaving markers of caspase-3 activity undetected. Quantitative analysis revealed no signif icant difference in the accumulation of active caspase-3 in survivin-positive cells co mpared to survivin-negative cells at five days post injury in either the cortex (37 2% v. 26 6%) or hippoc ampus (21 5% v. 22 3%). Conversely, a significantly higher percentage of TUNEL labeling was observed in survivin-negative cells as compared to surv ivin-positive cells in ipsilateral cortex (49 5% v. 25 2%) and hippocampus (55 3% v. 30 4%). These data are consistent with my hypothesis that survivin expression may a ttenuate the apoptotic cascade by inhibiting the cleavage of caspase-3 substrates. Howe ver, by seven days post injury, there was no significant difference in the accumulation of either active caspase-3 or TUNEL labeling in survivin-positive cells compared to surviv in-negative cells. It is unclear why this pattern was observed. In particular, the sh arp decrease in survivin-negative, TUNELpositive cells from five days to seven days post-injury was unexpected. This decrease in DNA fragmentation may be e xplained by death and remova l of TUNEL-positive cells between five and seven days as evidenced by progressive increases in cavity size seen after brain injury (data not shown). The tu rnover from healthy to TUNEL-positive in the

PAGE 67

57 survivin-negative cel l population may not be steady, as indicated by the biphasic fluctuations of cell death i ndicators after brain injury (Holmin and Mathiesen, 1995; Domanska-Janik, 1996; Kampfl et al., 1996; Baskaya et al., 1997). Again because of the difficulty of triple-label IHC, the cell type of survivin dual-labeled cells could not be identified. Because survivin is expressed in astrocyt es and neurons, the accumulation of active caspase-3 and TUNEL-labeling in these cel ls was investigated. Both cell types accumulated active caspase-3 and labeled w ith TUNEL following TBI. Quantification studies revealed a significan tly greater percentage of neurons labeled with TUNEL compared to astrocytes in both the cortex and hippocampus at both five and seven days post injury. Taken with the observation that a majority of astrocytes express survivin after TBI, it is possible that survivin expression contributes to the low DNA fragmentation observed in these cells desp ite prominent caspase-3 activation. The opposite appears to be the case with neurons. Few neurons express survivin following TBI and many of these cells show prominent DNA fragmentation. Neurons are particularly vulnerable to apopt osis signals followi ng brain injury and absence of survivin expression may contribute to this vulnerabil ity. Therefore the hypothesis does not seem to support an anti-apoptosis role for survivin in the majority of neurons following TBI. Collectively, these data i ndicate that survivin, a developmental protein normally absent in adult tissues, is up-regulated at bot h the transcriptional a nd translational level following traumatic brain injury. Survivin is expressed predominantly by astrocytes and a small sub-set of neurons. In addition, this is the first study to pr ovide indirect evidence that survivin functions as both an apoptosis inhibitor a nd as a cell cycle protein following

PAGE 68

58 brain trauma. These data suggest that furt her studies are necessary to show the overall ramifications of survivin expression on th e secondary injury cascade following TBI. Future Directions Additional studies of survivin expression and function must be completed before survivin may be considered a potential therapeu tic agent/target for brain injury treatment. First, survivin activities following TBI in the intact animal must be inhibited to observe the histological and behavioral changes associated with its expression. Of interest are heterozygous survivin knockout mi ce strains that appear to be particularly vulnerable to even mild apoptotic stimuli (Conway et al ., 2002). TBI experiments comparing the histological and behavioral differences between hetero zygous survivin knockouts and their wild type littermates w ould give more direct evidence of survivin function following brain insults. Additionally, a host of new techniques to inhi bit survivin expression in the whole animal are currently being developed for cancer tr eatment and can be readily applied to a TBI model. Of practical inte rest to this model is “molecular antagonism” and pharmacological inhibition. “Molecular antagonism” using siRNA, antisense and dominant-negative survivin mutants are effective in vivo at interfering with survivin expression and function (Grossman et al., 2001b ; Kanwar et al., 2001; Yamamoto et al., 2002; Williams et al., 2003). Pharmacological inhibition by flavopi ridol (Zhai et al., 2002) or Purv.A (Gray et al., 1998) can in crease survivin turnover and reduce its effectiveness as an apoptosis inhibitor by re ducing the phosphorylati on state of survivin (O'Connor et al., 2000a; Grossman et al., 2001b). Based on the findings in this dissertation, inhibition of survivin following TBI will likely increase apoptotic cell death, cavity size and have a negative im pact on behavioral recovery.

PAGE 69

59 Second, the activities of surviv in must be enhanced to see if increased survivin has a beneficial role to the overall recovery of the organism. Both pharmacological and gene transfection can be used to accomplish this ta sk. Gene transfection has been successfully used to temporarily up-regulate proteins of interest in the adult brain (Yenari and Sapolsky, 2004) and may be used to increase survivin expression following TBI. A more flexible approach may include pharmacological agents because they can be used in established wild-typ e animal models. Compounds that enhance Cdk phosphorylation activity such as PD0166285 (Li et al., 2002) may inhibit surv ivin turnover and promote cell survival by increasing th e phosphorylation state of surv ivin. Based on the findings from this dissertation and work by other groups enhancing survivin activity in the brain following TBI will likely decrease cell deat h, reduce cavity size and enhance behavioral recovery compared to untreated animals. This set of proposed experiments will reveal the functiona l consequences of endogenous survivin expression in injured brai n tissues. With this knowledge, it can be determined whether survivin is indeed a viable therapeutic agent for brain injury treatment.

PAGE 70

60 LIST OF REFERENCES Adida C, Crotty PL, McGrath J, Berrebi D, Diebold J, Altieri DC (1998) Developmentally regulated expression of the novel cancer anti-apoptosis gene survivin in human and mouse diffe rentiation. Am J Pathol 152:43-49. Aldskogius H, Liu L, Svensson M (1999) G lial responses to synaptic damage and plasticity. J Neurosci Res 58:33-41. Altieri DC (2001) Cytokinesis, apoptosis a nd survivin: three for tango? Cell Death Differ 8:4-5. Altieri DC (2003a) Blocking survivin to ki ll cancer cells. Methods Mol Biol 223:533542. Altieri DC (2003b) Survivin in apoptosis control and cell cy cle regulation in cancer. Prog Cell Cycle Res 5:447-452. Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new gene ration of protein database search programs. Nucleic Acids Res 25:3389-3402. Alzheimer C, Werner S (2002) Fibroblast gr owth factors and neuroprotection. Adv Exp Med Biol 513:335-351. Ambrosini G, Adida C, Altieri DC (1997) A novel anti-apoptosis gene, survivin, expressed in cancer and ly mphoma. Nat Med 3:917-921. Ambrosini G, Adida C, Sirugo G, Altieri DC (1998) Induction of apoptosis and inhibition of cell proliferation by survivin gene targeting. J Biol Chem 273:11177-11182. Badran A, Yoshida A, Ishikawa K, Goi T, Yamaguchi A, Ueda T, Inuzuka M (2004) Identification of a novel sp lice variant of the human anti-apoptopsis gene survivin. Biochem Biophys Res Commun 314:902-907. Baldwin SA, Gibson T, Callihan CT, Sullivan PG, Palmer E, Scheff SW (1997) Neuronal cell loss in the CA3 subfield of the hippocampus following cortical contusion utilizing the optical disector method for cell counting. J Neurotrauma 14:385-398. Bambrick L, Kristian T, Fiskum G (2004) Astrocyte mitochondrial mechanisms of ischemic brain injury and neur oprotection. Neurochem Res 29:601-608.

PAGE 71

61 Baskaya MK, Rao AM, Dogan A, Donalds on D, Dempsey RJ (1997) The biphasic opening of the blood-brain barrier in the cortex and hippocampus after traumatic brain injury in rats. Neurosci Lett 226:33-36. Beckman JS, Beckman TW, Chen J, Mars hall PA, Freeman BA (1990) Apparent hydroxyl radical production by peroxynitrite: imp lications for endothelial injury from nitric oxide and superoxide. Proc Natl Acad Sci U S A 87:1620-1624. Beer R, Franz G, Srinivasan A, Hayes RL, Pike BR, Newcomb JK, Zhao X, Schmutzhard E, Poewe W, Kampfl A (2000) Temporal profile and cell subt ype distribution of activated caspase-3 following experiment al traumatic brain injury. J Neurochem 75:1264-1273. Belluardo N, Korhonen L, Mudo G, Li ndholm D (2002) Neuronal expression and regulation of rat inhibitor of apoptosis protein-2 by ka inic acid in the rat brain. Eur J Neurosci 15:87-100. Blanc-Brude OP, Yu J, Simosa H, Conte MS, Sessa WC, Altieri DC (2002) Inhibitor of apoptosis protein survivin regulates vascular injury. Nat Med 8:987-994. Borriello A, Roberto R, Della Ragione F, Io lascon A (2002) Proliferate and survive: cell division cycle and apoptosis in hum an neuroblastoma. Haematologica 87:196214. Bravo R, Frank R, Blundell PA, Macdonald-Bravo H (1987) Cyclin/PCNA is the auxiliary protein of DNA polymer ase-delta. Nature 326:515-517. Buki A, Okonkwo DO, Wang KK, Povlishock JT (2000) Cytochrome c release and caspase activation in traumatic axon al injury. J Neurosci 20:2825-2834. Bullock R, Maxwell WL, Graham DI, Teas dale GM, Adams JH (1991) Glial swelling following human cerebral contusion: an ul trastructural study. J Neurol Neurosurg Psychiatry 54:427-434. Bush TG, Puvanachandra N, Horner CH, Polito A, Ostenfeld T, Svendsen CN, Mucke L, Johnson MH, Sofroniew MV (1999) Leukocyte infiltration, neuronal degeneration, and neurite outgrowth af ter ablation of scar-forming, reactive astrocytes in adult transg enic mice. Neuron 23:297-308. Buytaert KA, Kline AE, Montanez S, Likler E, Millar CJ, Hernandez TD (2001) The temporal patterns of c-Fos and basic fi broblast growth factor expression following a unilateral anteromedial cort ex lesion. Brain Res 894:121-130. Cameron HA, McKay R (1998) Stem cells and ne urogenesis in the adult brain. Curr Opin Neurobiol 8:677-680. Cameron HA, McKay RD (2001) Adult neurog enesis produces a large pool of new granule cells in the dentate gyrus. J Comp Neurol 435:406-417.

PAGE 72

62 Carbonell WS, Grady MS (1999) Regional and temporal char acterization of neuronal, glial, and axonal response after trauma tic brain injury in the mouse. Acta Neuropathol (Berl) 98:396-406. Cervos-Navarro J, Lafuente JV (1991) Trau matic brain injuries: st ructural changes. J Neurol Sci 103 Suppl:S3-14. Chakravarti A, Zhai G, Zhang M, Malhotra R, Latham D, Delaney M, Robe P, Nestler U, Song Q, Loeffler J (2004) Survivin enha nces radiation resistance in primary human glioblastoma cells via caspase -independant mechanisms. Oncogene 23:7494-7506. Chan SL, Mattson MP (1999) Caspase and calpain substrates: roles in synaptic plasticity and cell death. J Neurosci Res 58:167-190. Chantalat L, Skoufias DA, Kleman JP, Jung B, Dideberg O, Margolis RL (2000) Crystal structure of human survivin reveals a bow tie-shaped dimer with two unusual alpha-helical extensions. Mol Cell 6:183-189. Chen XH, Iwata A, Nonaka M, Browne KD, Smith DH (2003) Neurogenesis and glial proliferation persist for at least one year in the subventricular zone following brain trauma in rats. J Neurotrauma 20:623-631. Chen Y, Swanson RA (2003) Astrocytes a nd brain injury. J Cereb Blood Flow Metab 23:137-149. Chen ZJ, Negra M, Levine A, Ughrin Y, Levine JM (2002) Oli godendrocyte precursor cells: reactive cells that inhibit axon growth and re generation. J Neurocytol 31:481-495. Chirumamilla S, Sun D, Bullock M, Colello R (2002) Traumatic Brain Injury Induced Cell Proliferation in the Adult Mamm alian Nervous System. Journal of Neurotrauma 19:693-703. Choi DW (1988) Calcium-mediated neurotoxic ity: relationship to specific channel types and role in ischemic damage. Trends Neurosci 11:465-469. Choi DW, Rothman SM (1990) The role of gl utamate neurotoxicity in hypoxic-ischemic neuronal death. Annu Re v Neurosci 13:171-182. Choi KS, Lee TH, Jung MH (2003) Ribozyme-me diated cleavage of the human survivin mRNA and inhibition of antiapoptotic f unction of survivin in MCF-7 cells. Cancer Gene Ther 10:87-95. Clark RS, Kochanek PM, Watkins SC, Chen M, Dixon CE, Seidberg NA, Melick J, Loeffert JE, Nathaniel PD, Jin KL, Gr aham SH (2000) Caspase-3 mediated neuronal death after traumatic brain injury in rats. J Neurochem 74:740-753.

PAGE 73

63 Cohen GM (1997) Caspases: the executi oners of apoptosis. Biochem J 326:1-16. Conti AC, Raghupathi R, Trojanowski JQ, Mc Intosh TK (1998) Experimental brain injury induces regionally distinct ap optosis during the acu te and delayed posttraumatic period. J Neurosci 18:5663-5672. Conway EM, Zwerts F, Van Eygen V, DeVrie se A, Nagai N, Luo W, Collen D (2003) Survivin-dependent angiogenesis in isch emic brain: molecular mechanisms of hypoxia-induced up-regulation. Am J Pathol 163:935-946. Conway EM, Pollefeyt S, Cornelissen J, De Baere I, Steiner-Mosonyi M, Ong K, Baens M, Collen D, Schuh AC (2000) Three di fferentially expressed survivin cDNA variants encode proteins with distin ct antiapoptotic f unctions. Blood 95:14351442. Conway EM, Pollefeyt S, Steiner-Mosonyi M, Luo W, Devriese A, Lupu F, Bono F, Leducq N, Dol F, Schaeffer P, Collen D, Herbert JM (2002) Deficiency of survivin in transgenic mice exacerbate s Fas-induced apoptosis via mitochondrial pathways. Gastroenterology 123:619-631. Csuka E, Hans VH, Ammann E, Trentz O, Kossmann T, Morganti-Kossmann MC (2000) Cell activation and inflammatory response following traumatic axonal injury in the rat. Neuroreport 11:2587-2590. Dash PK, Mach SA, Moore AN (2001) E nhanced neurogenesis in the rodent hippocampus following traumatic brain injury. J Neurosci Res 63:313-319. Dawson MR, Levine JM, Reynolds R (2000) NG2 -expressing cells in the central nervous system: are they oligodendroglial pr ogenitors? J Neuros ci Res 61:471-479. Denecker G, Vercammen D, Steemans M, Vanden Berghe T, Brouckaert G, Van Loo G, Zhivotovsky B, Fiers W, Grooten J, D eclercq W, Vandenabeele P (2001) Death receptor-induced apoptotic and necrotic ce ll death: differential role of caspases and mitochondria. Cell Death Differ 8:829-840. Deveraux QL, Reed JC (1999) IAP family pr oteins--suppressors of apoptosis. Genes Dev 13:239-252. Deveraux QL, Takahashi R, Salvesen GS, R eed JC (1997) X-linked IAP is a direct inhibitor of cell-death pr oteases. Nature 388:300-304. Dietrich WD (1994) Morphological manifestations of reperfus ion injury in brain. Ann N Y Acad Sci 723:15-24. Dixon CE, Clifton GL, Lighthall JW, Yagh mai AA, Hayes RL (1991) A controlled cortical impact model of traumatic brain injury in the rat. J Neurosci Methods 39:253-262.

PAGE 74

64 Doetsch F, Caille I, Lim DA, Garcia -Verdugo JM, Alvarez-Buylla A (1999) Subventricular zone astrocytes are neural stem cells in the adult mammalian brain. Cell 97:703-716. Domanska-Janik K (1996) Protein serine /threonine kinases (PKA, PKC and CaMKII) involved in ischemic brain pathology. Acta Neurobiol Exp (Wars) 56:579-585. Dunn-Meynell AA, Levin BE (1997) Histologi cal markers of neuronal, axonal and astrocytic changes after lateral rigid impact traumatic brain injury. Brain Res 761:25-41. Earnshaw WC, Martins LM, Kaufmann SH (1999) Mammalian caspases: structure, activation, substrates, and functions during apoptosi s. Annu Rev Biochem 68:383424. Ebel RL (1951) Estimation of the Reliab ility of Ratings. Psycometrika 16:407-424. Eldadah BA, Faden AI (2000) Caspase pathwa ys, neuronal apoptosis, and CNS injury. J Neurotrauma 17:811-829. Enari M, Sakahira H, Yokoyama H, Okawa K, Iwamatsu A, Nagata S (1998) A caspaseactivated DNase that degrades DNA duri ng apoptosis, and its inhibitor ICAD. Nature 391:43-50. Fawcett JW, Asher RA (1999) The glial scar and central nervous sy stem repair. Brain Res Bull 49:377-391. Ferrer I, Planas AM (2003) Signaling of cell death and cell survival following focal cerebral ischemia: life and death struggl e in the penumbra. J Neuropathol Exp Neurol 62:329-339. Fortugno P, Beltrami E, Plescia J, Fontana J, Pradhan D, Marchisio PC, Sessa WC, Altieri DC (2003) Regulation of surviv in function by Hsp90. Proc Natl Acad Sci U S A 100:13791-13796. Fortugno P, Wall NR, Giodini A, O'Connor DS, Plescia J, Padgett KM, Tognin S, Marchisio PC, Altieri DC (2002) Survivin exists in immunochemically distinct subcellular pools and is involved in sp indle microtubule function. J Cell Sci 115:575-585. Gabryel B, Trzeciak HI (2001) Role of astr ocytes in pathogenesis of ischemic brain injury. Neurotox Res 3:205-221. Gage FH (2002) Neurogenesis in th e adult brain. J Neurosci 22:612-613. Gage FH, Kempermann G, Palmer TD, Peterson DA, Ray J (1998) Multipotent progenitor cells in the adult dent ate gyrus. J Neurobiol 36:249-266.

PAGE 75

65 Gennarelli TA (1993) Mechanisms of br ain injury. J Emerg Med 11 Suppl 1:5-11. Giulian D (1991) Microglia-Neu ron Interactions After Injury to the Central Nervous System. In: Peripheral Signaling of the Brai n: role in neural-immune interactions, learning and memory (Frederickson R, McGaugh J, Felton D, eds), pp 73-82. Lewiston, NY: Hogrefe and Huber. Gong C, Hoff JT, Keep RF (2000) Acute infl ammatory reaction following experimental intracerebral hemorrhage in rat. Brain Res 871:57-65. Gould E, Tanapat P (1997) Lesion-induced pr oliferation of neurona l progenitors in the dentate gyrus of the adult rat. Neuroscience 80:427-436. Gould E, Vail N, Wagers M, Gross CG (2001) Adult-generated hippocampal and neocortical neurons in macaques have a tr ansient existence. Proc Natl Acad Sci U S A 98:10910-10917. Grady MS, Charleston JS, Maris D, Witgen BM Lifshitz J (2003) Ne uronal and glial cell number in the hippocampus after experiment al traumatic brain injury: analysis by stereological estimati on. J Neurotrauma 20:929-941. Graham DI, McIntosh TK, Maxwell WL, Nicoll JA (2000) Recent advances in neurotrauma. J Neuropathol Exp Neurol 59:641-651. Graham DI, Adams JH, Nicoll JA, Maxwe ll WL, Gennarelli TA (1995) The nature, distribution and causes of traumatic brain injury. Brain Pathol 5:397-406. Gray NS, Wodicka L, Thunnissen AM, Norman TC, Kwon S, Espinoza FH, Morgan DO, Barnes G, LeClerc S, Meijer L, Kim SH, Lockhart DJ, Schultz PG (1998) Exploiting chemical libraries structure, and genomics in the search for kinase inhibitors. Science 281:533-538. Green DR, Reed JC (1998) Mitochondr ia and apoptosis. Science 281:1309-1312. Grossman D, Altieri DC (2001) Drug resist ance in melanoma: mechanisms, apoptosis, and new potential therapeutic target s. Cancer Metastasis Rev 20:3-11. Grossman D, McNiff JM, Li F, Altieri DC (1999) Expression and targeting of the apoptosis inhibitor, surv ivin, in human melanoma. J Invest Dermatol 113:10761081. Grossman D, Kim PJ, Schechner JS, Altieri DC (2001a) Inhibition of melanoma tumor growth in vivo by survivin targeti ng. Proc Natl Acad Sci U S A 98:635-640. Grossman D, Kim PJ, Blanc-Brude OP, Bras h DE, Tognin S, Marchisio PC, Altieri DC (2001b) Transgenic expression of surviv in in keratinocytes counteracts UVBinduced apoptosis and cooperates with loss of p53. J Clin Invest 108:991-999.

PAGE 76

66 Gwag BJ, Canzoniero LM, Sensi SL, Demaro JA, Koh JY, Goldberg MP, Jacquin M, Choi DW (1999) Calcium ionophores can indu ce either apoptosis or necrosis in cultured cortical neurons. Neuroscience 90:1339-1348. Heales SJ, Lam AA, Duncan AJ, Land JM ( 2004) Neurodegeneration or neuroprotection: the pivotal role of astroc ytes. Neurochem Res 29:513-519. Hermann DM, Kilic E, Kugler S, Isenmann S, Bahr M (2001) Adenovirus-mediated GDNF and CNTF pretreatment protects agai nst striatal injury following transient middle cerebral artery occlusion in mice. Neurobiol Dis 8:655-666. Herrup K, Busser JC (1995) The induction of multiple cell cycle events precedes targetrelated neuronal deat h. Development 121:2385-2395. Higgins DG, Bleasby AJ, Fuchs R (1992) CLUS TAL V: improved software for multiple sequence alignment. Comput Appl Biosci 8:189-191. Hill-Felberg SJ, McIntosh TK, Oliver DL, Ra ghupathi R, Barbarese E (1999) Concurrent loss and proliferation of astrocytes follo wing lateral fluid percussion brain injury in the adult rat. J Neurosci Res 57:271-279. Holmin S, Mathiesen T (1995) Biphasic ed ema development after experimental brain contusion in rat. Ne urosci Lett 194:97-100. Hutchison JS, Derrane RE, Johnston DL, Ge ndron N, Barnes D, Fliss H, King WJ, Rasquinha I, MacManus J, Robertso n GS, MacKenzie AE (2001) Neuronal apoptosis inhibitory protein expression after traumatic brai n injury in the mouse. J Neurotrauma 18:1333-1347. Ignatova T, Kukekov V, Layw ell E, Suslov O, Vrionis F, Steindler D (2002) Human Cortical Glial Tumors Contain Neural Stem-Like Cells Expressing Astroglial and Neuronal Markers in vitro. Glia 39:online. Iwata E, Asanuma M, Nishibayashi S, K ondo Y, Ogawa N (1997) Di fferent effects of oxidative stress on activation of transcri ption factors in primary cultured rat neuronal and glial cells. Brai n Res Mol Brain Res 50:213-220. Jennett B (1996) Epidemiology of head inju ry. J Neurol Neurosur g Psychiatry 60:362369. Jiang X, Wilford C, Duensing S, Munger K, Jones G, Jones D (2001) Participation of Survivin in mitotic and apoptotic activi ties of normal and tumor-derived cells. J Cell Biochem 83:342-354. Jiao BH, Yao ZG, Geng SM, Zuo SH (2004) E xpression of survivin, a novel apoptosis inhibitor and cell cycle regul atory protein, in human gliomas. Chin Med J (Engl) 117:612-614.

PAGE 77

67 Johnson EA, Svetlov SI, Pike BR, Tolentino PJ Shaw G, Wang KKW, Hayes RL, Pineda JA (2004) Cell-specific Upregulation of Survivin After Experimental Traumatic Brain Injury in Rats. Journal of Neurotrauma 21:1183-1195. Kajiwara Y, Yamasaki F, Hama S, Yahara K, Yoshioka H, Sugiyama K, Arita K, Kurisu K (2003) Expression of survivin in astroc ytic tumors: correla tion with malignant grade and prognosis. Cancer 97:1077-1083. Kampfl A, Posmantur R, Nixon R, Grynspan F, Zhao X, Liu SJ, Newcomb JK, Clifton GL, Hayes RL (1996) mu-calpain activa tion and calpain-mediated cytoskeletal proteolysis following traumatic br ain injury. J Neurochem 67:1575-1583. Kanwar JR, Shen WP, Kanwar RK, Berg RW, Krissansen GW (2001) Effects of survivin antagonists on growth of established tu mors and B7-1 immunogene therapy. J Natl Cancer Inst 93:1541-1552. Kasof GM, Gomes BC (2001) Livin, a novel in hibitor of apoptosis protein family member. J Biol Chem 276:3238-3246. Kaya SS, Mahmood A, Li Y, Yavuz E, Chopp M (1999a) Expression of cell cycle proteins (cyclin D1 and cdk4) after contro lled cortical impact in rat brain. J Neurotrauma 16:1187-1196. Kaya SS, Mahmood A, Li Y, Yavuz E, Goksel M, Chopp M (1999b) Apoptosis and expression of p53 response proteins and cyc lin D1 after cortical impact in rat brain. Brain Res 818:23-33. Keane RW, Kraydieh S, Lotocki G, Alonso OF Aldana P, Dietrich WD (2001) Apoptotic and antiapoptotic mechanisms after trau matic brain injury. J Cereb Blood Flow Metab 21:1189-1198. Kernie SG, Erwin TM, Parada LF (2001) Brain remodeling due to neuronal and astrocytic proliferation afte r controlled cortical injury in mice. J Neurosci Res 66:317-326. Kim S, Kang J, Qiao J, Thomas RP, Ev ers BM, Chung DH (2004) Phosphatidylinositol 3-kinase inhibition down-regulates surv ivin and facilitates TRAIL-mediated apoptosis in neuroblastomas. J Pediatr Surg 39:516-521. Kitagawa H, Warita H, Sasaki C, Zhang WR Sakai K, Shiro Y, Mitsumoto Y, Mori T, Abe K (1999) Immunoreactive Akt, PI3-K a nd ERK protein kinase expression in ischemic rat brain. Neurosci Lett 274:45-48. Kleinschmidt-DeMasters BK, Heinz D, McCa rthy PJ, Bobak JB, Lillehei KO, Shroyer AL, Shroyer KR (2003) Survivin in g lioblastomas. Protein and messenger RNA expression and comparison with telome rase levels. Arch Pathol Lab Med 127:826-833.

PAGE 78

68 Knoblach SM, Nikolaeva M, Huang X, Fan L, Krajewski S, Reed JC, Faden AI (2002) Multiple caspases are activated after traumatic brain injury: evidence for involvement in functional outcome. J Neurotrauma 19:1155-1170. Kobayashi K, Hatano M, Otaki M, Ogasaw ara T, Tokuhisa T ( 1999) Expression of a murine homologue of the inhibitor of apoptosis protein is related to cell proliferation. Proc Natl Acad Sci U S A 96:1457-1462. Kontos HA (1989) Oxygen radicals in CNS damage. Chem Biol Interact 72:229-255. LaCasse EC, Baird S, Korneluk RG, MacKenzi e AE (1998) The inhib itors of apoptosis (IAPs) and their emerging role in cancer. Oncogene 17:3247-3259. Larner SF, Hayes RL, McKins ey DM, Pike BR, Wang KK (2004) Increased expression and processing of caspase-12 after trauma tic brain injury in rats. J Neurochem 88:78-90. Latov N, Nilaver G, Zimmerman EA, Johnson WG, Silverman AJ, Defendini R, Cote L (1979) Fibrillary astrocytes proliferat e in response to brain injury: a study combining immunoperoxidase technique for glial fibrillary acidic protein and radioautography of tritiated t hymidine. Dev Biol 72:381-384. Lazebnik YA, Kaufmann SH, Desnoyers S, Poirier GG, Earnshaw WC (1994) Cleavage of poly(ADP-ribose) polymerase by a protei nase with properties like ICE. Nature 371:346-347. Lee SM, Wong MD, Samii A, Hovda DA (1999) Evidence for energy failure following irreversible traumatic brain in jury. Ann N Y Acad Sci 893:337-340. Lei B, Popp S, Capuano-Waters C, Cottrell JE, Kass IS (2004) Lidocaine attenuates apoptosis in the ischemic penumbra and re duces infarct size after transient focal cerebral ischemia in ra ts. Neuroscience 125:691-701. Li F (2003) Survivin study: what is the next wave? J Cell Physiol 197:8-29. Li F, Ambrosini G, Chu EY, Plescia J, Tognin S, Marchisio PC, Altieri DC (1998) Control of apoptosis and mitotic spindl e checkpoint by survivin. Nature 396:580584. Li F, Ackermann EJ, Bennett CF, Rothermel AL Plescia J, Tognin S, Villa A, Marchisio PC, Altieri DC (1999) Pleiotropic cell-di vision defects and apoptosis induced by interference with survivin f unction. Nat Cell Biol 1:461-466. Li J, Wang Y, Sun Y, Lawrence TS (2002) Wild-type TP53 inhibits G(2)-phase checkpoint abrogation and radiosen sitization induced by PD0166285, a WEE1 kinase inhibitor. Radiat Res 157:322-330.

PAGE 79

69 Li Y, Chopp M, Powers C, Jiang N ( 1997) Immunoreactivity of cyclin D1/cdk4 in neurons and oligodendrocytes after focal cerebral ischem ia in rat. J Cereb Blood Flow Metab 17:846-856. Liu L, Rudin M, Kozlova EN (2000) Glial cell pr oliferation in the spin al cord after dorsal rhizotomy or sciatic nerve transection in the adult rat. Exp Brain Res 131:64-73. Liu L, Persson JK, Svensson M, Aldskogius H (1998a) Glial cell re sponses, complement, and clusterin in the central nervous syst em following dorsal root transection. Glia 23:221-238. Liu X, Li P, Widlak P, Zou H, Luo X, Garrard WT, Wang X (1998b) The 40-kDa subunit of DNA fragmentation factor indu ces DNA fragmentation and chromatin condensation during apoptosis. Pr oc Natl Acad Sci U S A 95:8461-8466. Lotocki G, Alonso OF, Frydel B, Dietrich WD, Keane RW (2003) Monoubiquitination and cellular distribution of XIAP in neurons after trau matic brain injury. J Cereb Blood Flow Metab 23:1129-1136. Lu CD, Altieri DC, Tanigawa N (1998) E xpression of a novel antiapoptosis gene, survivin, correlated with tumor cell apoptosis and p53 accumulation in gastric carcinomas. Cancer Res 58:1808-1812. Magavi SS, Leavitt BR, Macklis JD (2000) Indu ction of neurogenesis in the neocortex of adult mice. Nature 405:951-955. Maier CM, Chan PH (2002) Role of superoxi de dismutases in oxi dative damage and neurodegenerative disorders. Neuroscientist 8:323-334. Maier JK, Lahoua Z, Gendron NH, Fetni R, J ohnston A, Davoodi J, Rasper D, Roy S, Slack RS, Nicholson DW, MacKenzie AE (2002) The neuronal apoptosis inhibitory protein is a direct inhibito r of caspases 3 and 7. J Neurosci 22:20352043. Marciano PG, Brettschneider J, Manduchi E, Davis JE, Eastman S, Raghupathi R, Saatman KE, Speed TP, Stoeckert CJ, Jr ., Eberwine JH, McIntosh TK (2004) Neuron-specific mRNA complexity respons es during hippocampal apoptosis after traumatic brain injury. J Neurosci 24:2866-2876. Maroni P, Bendinelli P, Tiberio L, Rovetta F, Piccoletti R, Schiaffonati L (2003) In vivo heat-shock response in the brain: sign alling pathway and transcription factor activation. Brain Res Mol Brain Res 119:90-99. Maxwell WL, Povlishock JT, Graham DL (1997) A mechanistic analysis of nondisruptive axonal injury: a re view. J Neurotrauma 14:419-440.

PAGE 80

70 McCullers DL, Sullivan PG, Scheff SW, Herm an JP (2002) Mifepristone protects CA1 hippocampal neurons following traumatic brain injury in rat. Neuroscience 109:219-230. McIntosh TK, Saatman KE, Raghupathi R, Gr aham DI, Smith DH, Lee VM, Trojanowski JQ (1998) The Dorothy Russell Memorial Lecture. The molecular and cellular sequelae of experimental traumatic br ain injury: pathogenetic mechanisms. Neuropathol Appl Neurobiol 24:251-267. McPherson CA, Kubik J, Wine RN, D'Hellenc ourt CL, Harry GJ ( 2003) Alterations in cyclin A, B, and D1 in mouse dentat e gyrus following TMT-induced hippocampal damage. Neurotox Res 5:339-354. Miyake T, Okada M, Kitamura T (1992) Reac tive proliferation of astrocytes studied by immunohistochemistry for proliferating cell nuclear antigen. Brain Res 590:300302. Moon WS, Tarnawski AS (2003) Nuclear transl ocation of survivin in hepatocellular carcinoma: a key to cancer cell growth? Hum Pathol 34:1119-1126. Morganti-Kossmann MC, Rancan M, Otto VI Stahel PF, Kossmann T (2001) Role of cerebral inflammation after traumatic brai n injury: a revisite d concept. Shock 16:165-177. Morshead CM, van der Kooy D (1992) Postmito tic death is the fate of constitutively proliferating cells in the subependymal la yer of the adult mouse brain. J Neurosci 12:249-256. Morshead CM, Reynolds BA, Craig CG, Mc Burney MW, Staines WA, Morassutti D, Weiss S, van der Kooy D (1994) Neural stem cells in the adult mammalian forebrain: a relatively quiescent subp opulation of subependymal cells. Neuron 13:1071-1082. Muchmore SW, Chen J, Jakob C, Zakula D, Ma tayoshi ED, Wu W, Zhang H, Li F, Ng SC, Altieri DC (2000) Crystal structure and mutagenic analysis of the inhibitorof-apoptosis protein surv ivin. Mol Cell 6:173-182. Nakajima K, Kohsaka S (1993) Functional roles of microglia in the brain. Neurosci Res 17:187-203. Nakase T, Sohl G, Theis M, Willecke K, Naus CC (2004) Increased apoptosis and inflammation after focal brain ischemia in mice lacking connexin43 in astrocytes. Am J Pathol 164:2067-2075. Newcomb JK, Zhao X, Pike BR, Hayes RL (1999) Temporal profile of apoptotic-like changes in neurons and astrocytes followi ng controlled cortical impact injury in the rat. Exp Neurol 158:76-88.

PAGE 81

71 Nicholson DW, Ali A, Thornberry NA, Vaillanc ourt JP, Ding CK, Gallant M, Gareau Y, Griffin PR, Labelle M, Laze bnik YA, et al. (1995) Iden tification and inhibition of the ICE/CED-3 protease necessary for mammalian apoptosis. Nature 376:37-43. Nicotera P, Leist M, Single B, Volbracht C (1999) Execution of apoptosis: converging or diverging pathways? Biol Chem 380:1035-1040. Norton WT (1999) Cell reactions following acute brain inju ry: a review. Neurochem Res 24:213-218. Nowak TS, Jr., Jacewicz M (1994) The heat shock/stress response in focal cerebral ischemia. Brain Pathol 4:67-76. O'Connor DS, Wall NR, Porter AC, Altieri DC (2002) A p34(cdc2) survival checkpoint in cancer. Cancer Cell 2:43-54. O'Connor DS, Grossman D, Plescia J, Li F, Zhang H, Villa A, Tognin S, Marchisio PC, Altieri DC (2000a) Regulation of a poptosis at cell division by p34cdc2 phosphorylation of survivin. Proc Natl Acad Sci U S A 97:13103-13107. O'Connor DS, Schechner JS, Adida C, Mesri M, Rothermel AL, Li F, Nath AK, Pober JS, Altieri DC (2000b) Control of apoptosis during angiogenesis by survivin expression in endothelial cells. Am J Pathol 156:393-398. Otaki M, Hatano M, Kobayashi K, Ogasawar a T, Kuriyama T, T okuhisa T (2000) Cell cycle-dependent regulation of TIAP/m -survivin expression. Biochim Biophys Acta 1493:188-194. Papapetropoulos A, Fulton D, Mahboubi K, Ka lb RG, O'Connor DS, Li F, Altieri DC, Sessa WC (2000) Angiopoietin-1 inhib its endothelial cell apoptosis via the Akt/survivin pathway. J Biol Chem 275:9102-9105. Parent A (1997) The brain in evolution and involution. Bioche m Cell Biol 75:651-667. Passineau MJ, Zhao W, Busto R, Dietri ch WD, Alonso O, Loor JY, Bramlett HM, Ginsberg MD (2000) Chronic metabolic sequelae of traumatic brain injury: prolonged suppression of somatosensory activation. Am J Physiol Heart Circ Physiol 279:H924-931. Peterson DA (2002) Stem cells in brain plas ticity and repair. Curr Opin Pharmacol 2:3442. Pike BR, Zhao X, Newcomb JK, Posmantu r RM, Wang KK, Hayes RL (1998) Regional calpain and caspase-3 proteolysis of alpha -spectrin after trau matic brain injury. Neuroreport 9:2437-2442. Povlishock JT, Kontos HA (1985) Continuing axonal and vascular change following experimental brain trauma. Ce nt Nerv Syst Trauma 2:285-298.

PAGE 82

72 Raghupathi R, Graham DI, McIntosh TK (2000) Apoptosis after trauma tic brain injury. J Neurotrauma 17:927-938. Rice AC, Khaldi A, Harvey HB, Salman NJ White F, Fillmore H, Bullock MR (2003) Proliferation and neuronal differentiati on of mitotically active cells following traumatic brain injury. Expe rimental Neurology 183:406-417. Ridet JL, Malhotra SK, Privat A, Gage FH (1997) Reactive astrocytes: cellular and molecular cues to biological f unction. Trends Neurosci 20:570-577. Roy N, Deveraux QL, Takahashi R, Salvesen GS, Reed JC (1997) The c-IAP-1 and cIAP-2 proteins are direct inhibitors of specific caspases. Embo J 16:6914-6925. Sahara S, Aoto M, Eguchi Y, Imamoto N, Yoneda Y, Tsujimoto Y (1999) Acinus is a caspase-3-activated protein required for apoptotic chromatin condensation. Nature 401:168-173. Sakahira H, Enari M, Nagata S (1998) Cleav age of CAD inhibitor in CAD activation and DNA degradation during a poptosis. Nature 391:96-99. Sanai N, Tramontin AD, Quinones-Hinojosa A, Barbaro NM, Gupta N, Kunwar S, Lawton MT, McDermott MW, Parsa AT, Manuel-Garcia Verdugo J, Berger MS, Alvarez-Buylla A (2004) Unique astrocyt e ribbon in adult human brain contains neural stem cells but lacks ch ain migration. Nature 427:740-744. Sanz O, Acarin L, Gonzalez B, Castellano B (2001) Expression of 27 kDa heat shock protein (Hsp27) in immature rat brain after a cortical as piration lesion. Glia 36:259-270. Sasaki T, Lopes MB, Hankins GR, Helm GA (2002) Expression of survivin, an inhibitor of apoptosis protein, in tumors of the nervous system. Acta Neuropathol (Berl) 104:105-109. Shankar SL, Mani S, O'Guin KN, Kandimalla ER, Agrawal S, Shafit-Zagardo B (2001) Survivin inhibition induces human ne ural tumor cell death through caspaseindependent and -dependent pathways. J Neurochem 79:426-436. Shin S, Sung BJ, Cho YS, Kim HJ, Ha NC, Hwang JI, Chung CW, Jung YK, Oh BH (2001) An anti-apoptotic protein human surviv in is a direct inhi bitor of caspase-3 and -7. Biochemistry 40:1117-1123. Simard AR, Rivest S (2004) Bone marrow st em cells have the ab ility to populate the entire central nervous system into fu lly differentiated parenchymal microglia. Faseb J 18:998-1000. Slee EA, Adrain C, Martin SJ (2001) Executi oner caspase-3, -6, and -7 perform distinct, non-redundant roles during the demolition phase of apoptosis. J Biol Chem 276:7320-7326.

PAGE 83

73 Smith C, Berry M, Clarke WE, Logan A ( 2001) Differential expr ession of fibroblast growth factor-2 and fibroblast growth factor receptor 1 in a scarring and nonscarring model of CNS injury in the rat. Eur J Neurosci 13:443-456. Song H, Stevens CF, Gage FH (2002) Astroglia induce neurogenesis from adult neural stem cells. Nature 417:39-44. Sosin DM, Sniezek JE, Waxweiler RJ (1995) Tr ends in death associated with traumatic brain injury, 1979 through 1992. Success and failure. Jama 273:1778-1780. Sosin DM, Sniezek JE, Thurman DJ (1996) Inci dence of mild and moderate brain injury in the United States, 1991. Brain Inj 10:47-54. Stennicke HR, Salvesen GS (1999) Catalytic pr operties of the caspases. Cell Death Differ 6:1054-1059. Stone JR, Okonkwo DO, Singleton RH, Mutlu LK, Helm GA, Povlishock JT (2002) Caspase-3-mediated cleavage of amyloi d precursor protein and formation of amyloid Beta peptide in traumatic axonal injury. J Neurotrauma 19:601-614. Streit WJ (1996) The role of microglia in brain injury. Neurotoxicology 17:671-678. Streit WJ, Kincaid-Colton CA (1995) The br ain's immune system. Sci Am 273:54-55, 5861. Sutton RL, Lescaudron L, Stein DG (1993) Unilateral cortical c ontusion injury in the rat: vascular disruption and tem poral development of corti cal necrosis. J Neurotrauma 10:135-149. Suzuki A, Ito T, Kawano H, Hayashida M, Hayasaki Y, Tsutomi Y, Akahane K, Nakano T, Miura M, Shiraki K (2000) Survivin initiates procaspase 3/p21 complex formation as a result of interaction with Cdk4 to resist Fasmediated cell death. Oncogene 19:1346-1353. Sykova E, Vargova L, Prokopova S, Simonova Z (1999) Glial swelli ng and astrogliosis produce diffusion barriers in the rat spinal cor d. Glia 25:56-70. Takahashi R, Deveraux Q, Tamm I, Welsh K, Assa-Munt N, Salvesen GS, Reed JC (1998) A single BIR domain of XIAP suffi cient for inhibiting caspases. J Biol Chem 273:7787-7790. Tamm I, Wang Y, Sausville E, Scudiero DA, Vigna N, Oltersdorf T, Reed JC (1998) IAP-family protein survivin inhibits cas pase activity and apoptosis induced by Fas (CD95), Bax, caspases, and anticancer drugs. Cancer Res 58:5315-5320. Tang D, Kidd VJ (1998) Cleavage of DFF-45/ ICAD by multiple caspases is essential for its function during apoptosi s. J Biol Chem 273:28549-28552.

PAGE 84

74 Tewari M, Quan LT, O'Rourke K, Desnoye rs S, Zeng Z, Beidler DR, Poirier GG, Salvesen GS, Dixit VM (1995) Yama/CPP32 beta, a mammalian homolog of CED-3, is a CrmA-inhibitable protease that cleaves the death substrate poly(ADPribose) polymerase. Cell 81:801-809. Thompson C, Gary D, Mattson M, Mackenzi e A, Robertson GS (2004) Kainic acidinduced naip expression in the hippocam pus is blocked in mice lacking TNF receptors. Brain Res Mol Brain Res 123:126-131. Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence al ignment through sequence weighting, position-specific gap penalties and wei ght matrix choice. Nucleic Acids Res 22:4673-4680. Thurman DJ, Alverson C, Dunn KA, Guerrero J, Sniezek JE (1999a) Traumatic brain injury in the United States: A public hea lth perspective. J He ad Trauma Rehabil 14:602-615. Thurman DJ, Alverson C, Browne D, Dunn KA, Guerrero J, Johnson R, Johnson V, Langlois J, Pilkey D, Sniezek JE, Toal S (1999b) Traumatic Brain Injury in the United States: A Report to Congress. In, p http://www.cdc.gov/doc.do/id/0900f0903ec8001011c: National Center for Injury Prevention and Control (NCIPC) date retrieved 08/23/2004. Tolentino PJ, DeFord SM, Notterpek L, Glenn CC, Pike BR, Wang KK, Hayes RL (2002) Up-regulation of tissue-type transg lutaminase after trau matic brain injury. J Neurochem 80:579-588. Tran J, Master Z, Yu JL, Rak J, Dumont DJ Kerbel RS (2002) A role for survivin in chemoresistance of endothelial cells me diated by VEGF. Proc Natl Acad Sci U S A 99:4349-4354. Unal-Cevik I, Kilinc M, Gursoy-Ozdemir Y, Gurer G, Dalkara T (2004) Loss of NeuN immunoreactivity after cerebral ischemia does not indicate neuronal cell loss: a cautionary note. Brain Res 1015:169-174. Uren AG, Wong L, Pakusch M, Fowler KJ, Burrows FJ, Vaux DL, Choo KH (2000) Survivin and the inner centromere prot ein INCENP show similar cellcycle localization and gene knockout phenotype. Curr Biol 10:1319-1328. Van de Craen M, Declercq W, Van den bra nde I, Fiers W, Vandenabeele P (1999) The proteolytic procaspase activ ation network: an in vitr o analysis. Cell Death Differ 6:1117-1124. Van Haren K, van der Voorn JP, Peterson DR, van der Knaap MS, Powers JM (2004) The life and death of oligodendrocytes in vanishing white matter disease. J Neuropathol Exp Neurol 63:618-630.

PAGE 85

75 Verdecia MA, Huang H, Dutil E, Kaiser DA, H unter T, Noel JP (2000) Structure of the human anti-apoptotic protein survivin reveals a dimeric arrangement. Nat Struct Biol 7:602-608. Vucic D, Kaiser WJ, Miller LK (1998) Inhib itor of apoptosis protei ns physically interact with and block apoptosis induced by Dr osophila proteins HID and GRIM. Mol Cell Biol 18:3300-3309. Wang KK (2000) Calpain and caspase: can you tell the difference? Trends Neurosci 23:20-26. Wei LH, Huang CY, Cheng SP, Chen CA, Hs ieh CY (2001) Carcinosarcoma of ovary associated with previous radiothe rapy. Int J Gynecol Cancer 11:81-84. Wennersten A, Holmin S, Mathiesen T (2003) Characterization of Bax and Bcl-2 in apoptosis after experimental traumatic brai n injury in the rat. Acta Neuropathol (Berl) 105:281-288. Williams NS, Gaynor RB, Scoggin S, Verma U, Gokaslan T, Simmang C, Fleming J, Tavana D, Frenkel E, Becerra C (2003) Identification and validation of genes involved in the pathogenesis of color ectal cancer using c DNA microarrays and RNA interference. Clin Cancer Res 9:931-946. Wolf BB, Schuler M, Echeverri F, Green DR (1999) Caspase-3 is the primary activator of apoptotic DNA fragmentat ion via DNA fragmentation factor-45/inhibitor of caspase-activated DNase inactivation. J Biol Chem 274:30651-30656. Woo M, Hakem R, Soengas MS, Duncan GS, Shahinian A, Kagi D, Hakem A, McCurrach M, Khoo W, Kaufman SA, Se naldi G, Howard T, Lowe SW, Mak TW (1998) Essential contribution of cas pase 3/CPP32 to apoptosis and its associated nuclear changes. Genes Dev 12:806-819. Wu Q, Combs C, Cannady SB, Geldmacher DS Herrup K (2000) Beta -amyloid activated microglia induce cell cycling and cell death in cultured cortical neurons. Neurobiol Aging 21:797-806. Xia C, Xu Z, Yuan X, Uematsu K, You L, Li K, Li L, McCormick F, Jablons DM (2002a) Induction of apoptosis in mesothelioma cells by antisurvivin oligonucleotides. Mol Cancer Ther 1:687-694. Xia XG, Hofmann HD, Deller T, Kirsch M (2002b) Induction of STAT3 signaling in activated astrocytes and sprouting sept al neurons following entorhinal cortex lesion in adult rats. Mol Cell Neurosci 21:379-392. Xu DG, Crocker SJ, Doucet JP, St-Jean M, Tamai K, Hakim AM, Ikeda JE, Liston P, Thompson CS, Korneluk RG, MacKenzie A, Robertson GS (1997) Elevation of neuronal expression of NAIP reduces isch emic damage in the rat hippocampus. Nat Med 3:997-1004.

PAGE 86

76 Yagita Y, Kitagawa K, Ohtsuki T, Takasawa K, Miyata T, Okano H, Hori M, Matsumoto M (2001) Neurogenesis by progenitor cells in the ischemic adult rat hippocampus. Stroke 32:1890-1896. Yakovlev AG, Faden AI (2001) Caspase-depend ent apoptotic pathways in CNS injury. Mol Neurobiol 24:131-144. Yamamoto T, Manome Y, Nakamura M, Tani gawa N (2002) Downregulation of survivin expression by induction of th e effector cell protease receptor-1 reduces tumor growth potential and results in an increas ed sensitivity to anticancer agents in human colon cancer. Eur J Cancer 38:2316-2324. Yang Y, Geldmacher DS, Herrup K (2001) DNA replication precedes neuronal cell death in Alzheimer's disease. J Neurosci 21:2661-2668. Yenari MA, Sapolsky RM (2004) Gene thera py in neurological disease. Methods Mol Med 104:75-88. Zhai S, Senderowicz AM, Sausville EA, Fi gg WD (2002) Flavopiri dol, a novel cyclindependent kinase inhibitor, in clinic al development. Ann Pharmacother 36:905911. Zhao J, Tenev T, Martins LM, Downward J, Lemoine NR (2000) The ubiquitinproteasome pathway regulates survivin degradation in a cell cycle-dependent manner. J Cell Sci 113 Pt 23:4363-4371. Zhou M, Gu L, Li F, Zhu Y, Woods WG Findley HW (2002) DNA damage induces a novel p53-survivin signaling pathway regula ting cell cycle and apoptosis in acute lymphoblastic leukemia cells. J Pharmacol Exp Ther 303:124-131. Zipfel GJ, Babcock DJ, Lee JM, Choi DW (2000) Neuronal apoptosis after CNS injury: the roles of glutamate and calcium. J Neurotrauma 17:857-869.

PAGE 87

77 BIOGRAPHICAL SKETCH Erik Andrew Johnson was born in Louisville KY, and raised in Kansas City, MO. He graduated high school from Lincoln College Preparatory Academy (Kansas City, MO) in 1994. He attended M acalester College (St. Paul, MN) where he received a Bachelor of Arts degree in 1998 with majors in biology, psychology and neuroscience. After a year of graduate study at the University of Texas-H ouston, he transferred to the Interdisciplinary Program in Biological Sc iences at the University of Florida (Gainesville, FL) to complete his doctorate in the laboratory of Dr. Ronald Hayes. Erik has been awarded top honors at the Nationa l and International Neurotrauma Society Student Poster Competition in 2001. In additi on, Erik has twice been awarded the B.W. Robinson Research Endowment Grant-in-Aid Achievement Award in 2003 and 2004. Erik finished his doctoral work with two peer reviewed first author papers and five total papers to his credit.


Permanent Link: http://ufdc.ufl.edu/UFE0008337/00001

Material Information

Title: Survivin Expression after Traumatic Brain Injury: Potential Roles in Neuroprotection
Physical Description: Mixed Material
Copyright Date: 2008

Record Information

Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
System ID: UFE0008337:00001

Permanent Link: http://ufdc.ufl.edu/UFE0008337/00001

Material Information

Title: Survivin Expression after Traumatic Brain Injury: Potential Roles in Neuroprotection
Physical Description: Mixed Material
Copyright Date: 2008

Record Information

Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
System ID: UFE0008337:00001


This item has the following downloads:


Full Text












SURVIVIN EXPRESSION AFTER TRAUMATIC BRAIN INJURY: POTENTIAL
ROLES IN NEUROPROTECTION















By

ERIK ANDREW JOHNSON


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


2004

































Copyright 2004

by

Erik Andrew Johnson

































This document is dedicated to my wife, Karie, for her loving and unwavering support
during this process.















ACKNOWLEDGMENTS

I would first and foremost like to thank my wife, Karie, for her undying support

and patience during these long years. No matter what the situation, she is always waiting

there with a smile and she has helped me more than she will ever know. I would also like

to thank my parents, Arlen and Patricia Johnson, for their love, dedication and fantastic

parenting skills. Without these, I would not be the person I am today and I would never

have been able to pursue this level of education.

I would like to thank Dr. Ronald Hayes for the opportunity to pursue this novel

research and for the opportunity to contribute to the scientific community. I would also

like to thank past and present committee members, Dr. Douglas Anderson, Dr. William

Dunn, Dr. Gerry Shaw and Dr. Brian Pike for their invaluable input and guidance. I

would especially like to thank Dr. Stanislav Svetlov, whose understanding, patience and

dedication to my educational growth have not gone unappreciated or unnoticed.

Additionally, I would also like to thank Dr. S. Michelle DeFord and Dr. Jose Pineda for

their help and guidance in completing these studies.

Lastly, I would like to thank all the members of Dr. Hayes' laboratory for their

assistance and friendship throughout the years. I would like to especially thank Jeremy

Flint, Barbara Osteen, Dr. Rebecca Ellis, Dr. Stephen Lamer, Dr. Claire Ringger, Jada

Aikman, and Shannon Janssen for their effort and support.
















TABLE OF CONTENTS

page

A C K N O W L E D G M E N T S ................................................................................................. iv

LIST OF FIGURE S ......... ..................................... ........... vii

ABSTRACT .............. .......................................... ix

CHAPTER

1 IN TR OD U CTION ............................................... .. ......................... ..

Traum atic Brain Injury Dem ographics..................................................... ..............1
Traumatic Brain Injury Pathophysiology ........................ ........... .... .........2
Traumatic Brain Injury, Apoptosis and Caspase-3 Activation.................................
A poptosis Inhibition Follow ing TBI ........................................ ........................ 6
Cellular Proliferation Follow ing TB I ........................................ ........ ............... 7
Survivin: M itosis and A nti-apoptosis Protein.................................... .....................9
Survivin Protein Structure ................................................. ............................... 9
Survivin Expression and M itosis .......................... ............ ................. 10
Survivin and Apoptosis Inhibition................................... ........... ..... .......... 11
Potential Role for Survivin in TBI Pathology .................................... ............... 13

2 M E T H O D O L O G Y ........................................................................ .......................15

Induction of Controlled Cortical Impact Brain Injury ...............................................15
Quantitative Reverse Transcriptase Polymerase Chain Reaction (Q-PCR) ..............15
Rat-Specific Survivin Polyclonal Antibody Production...........................................17
Survivin Polyclonal Antibody Characterization .. ............... .. ............... ................... 18
W western B lot A nalyses ... ......... ... .. .......... .... ........................ ........... ....... .... 18
Preparation and Sectioning of Tissue for Immunohistochemistry (IHC) .................20
Dual Label Fluorescent Immunohistochemistry (IHC)...........................................21
Dual Label Fluorescent IHC for Same-Species Antibodies ....................................22
Experim ental Group Sizes ..................................... .......................................22
Cell Quantification and Statistical Analysis.................................... ............... 23

3 SURVIVIN EXPRESSION FOLLOWING TRAUMATIC BRAIN INJURY..........25

Induction of Survivin Expression After TBI................................... .................25
PCN A Expression After TBI. ............................................. ............................. 26









Co-Expression of Survivin and PCNA Following TBI.................. ..................28
Survivin and PCNA are Expressed in Astrocytes After TBI........................... 30
Survivin and PCNA are Expressed in a Sub-Set of Neurons After TBI ....................34
Survivin is Not Expressed in Microglia and Oligodendrocytes ..............................35
D discussion of Chapter 3 .................. ........................... .... .... .. ........ .... 36

4 SURVIVING AND APOPTOSIS INHIBITION FOLLOWING TRAUMATIC
B R A IN IN JU R Y ............ ..................................................................... ........ .. ....... .. 40

Caspase-3 is Activated in the Same Brain Regions as Survivin Following TBI........40
Survivin Expression Correlates with Decreased TUNEL Labeling but not Active
Caspase-3 Expression. ..... ............. .................... .... .. ......... ..... ................ 40
Astrocytes and Neurons Demonstrate Cell Specific Differences in
Active Caspase-3 and TUNEL Labeling .................................... ............... 44
D iscu ssion of C chapter 4 ...................................................................... .................. 46

5 CONCLUSIONS AND FUTURE DIRECTIONS ............................................. 53

C o n c lu sio n s.................................................... .................. 5 3
F future D directions .......................................................................58

LIST OF REFEREN CES ..................................................................... ............... 60

BIO GRAPH ICAL SK ETCH .................................................. ............................... 77
















LIST OF FIGURES


Figure pge

1-1 Intrinsic and extrinsic apoptosis pathways..... .......... ....................................... 5

1-2 Survivin protein structure ........................................................................... ... .... 12

2-1 IHC characterization of the rat-specific survivin antibody .............. ...................19

2-2 Control section for biotin/streptavidin same-species dual labeling IHC..................23

3-1 Survivin mRNA induction in rat brain after TBI. .................................................26

3-2 Expression of survivin protein after TBI in rats.....................................................27

3-3 Expression of PCNA after TBI in rats ........................................... ...............29

3-4 Immunohistochemistry of survivin and PCNA.....................................................30

3-5 Co-localization of survivin and GFAP in brain tissue after TBI.............................31

3-6 Co-localization of PCNA and GFAP in brain tissue after TBI.............................32

3-7 A sub-set of NeuN-positive neurons express survivin and PCNA after TBI........... 33

3-8 No survivin expression is found in oligodendrocytes or microglia following
T B I in rats. ............................................................................35

4-1 Caspase-3 Activation in rat brain after traumatic brain injury..............................41

4-2 Co-expression of survivin and apoptosis markers following TBI in rats ...............42

4-3 Survivin expression decreases the accumulation of TUNEL but not
active caspase-3 ................................................. .... ........ ........... 43

4-4 Astrocytes express active caspase-3 and label with TUNEL following TBI
in rats ................... ...................................... ............................46

4-5 Neurons express active caspase-3 and label with TUNEL following TBI in rats....47









4-6 TUNEL labeling is cell specific following TBI in rats. ................ ............... 48

4-7 Putative mechanism of apoptosis inhibition by survivin following TBI .................49














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

SURVIVIN EXPRESSION AFTER TRAUMATIC BRAIN INJURY: POTENTIAL
ROLES IN NEUROPROTECTION

By

Erik Andrew Johnson

December 2004

Chair: Ronald L. Hayes
Major Department: Neuroscience

In these studies,the expression profile and cellular localization of survivin, a novel

anti-apoptotic and mitosis protein, following traumatic brain injury (TBI) in rats was

examined. Specifically, survivin co-localization with the cell cycle protein PCNA, the

apoptosis protease active caspase-3, and the DNA fragmentation label TUNEL was

determined to reveal potential role of survivin in neuroprotection and to elucidate anti-

apoptotic mechanisms. Levels of survivin mRNA and protein were increased in the

ipsilateral, but not contralateral, cortex and hippocampus of rats after TBI, peaking at five

days post injury. Similar temporal and spatial patterns of PCNA were also significantly

enhanced in these brain regions. Immunohistochemistry revealed that survivin and

PCNA were co-expressed in the same cells and had a focal distribution within the injured

brain. Further analysis revealed a frequent co-localization of survivin and GFAP, an

astrocytic marker, in both ipsilateral brain regions, while a much smaller subset of cells

showed co-localization of survivin and NeuN, a mature neuronal marker. PCNA protein









expression was detected in both astrocytes and neurons of the ipsilateral cortex and

hippocampus after TBI.

Western blot analysis revealed significant increases in the accumulation of active

caspase-3 between five and fourteen days post injury. The percentage of survivin-

positive and negative cells labeled with active caspase-3 at five or seven days post-injury

was not significantly different. However, survivin-negative cells exhibited a significantly

greater labeling with TUNEL compared to survivin-positive cells, thereby suggesting that

expression of survivin may attenuate DNA cleavage and progression of apoptosis.

Although a higher percentage of astrocytes accumulated active caspase-3 compared to

neurons, these neurons showed significantly higher frequency of TUNEL labeling.

These novel data demonstrate that survivin is abundantly expressed in brain cortex

and hippocampus of adult rats following TBI. Survivin accumulation occurs primarily in

astrocytes and a sub-set of neurons. The occasional co-expression of survivin and PCNA

coupled with the low frequency of TUNEL labeling in survivin expressing cells may

suggest that survivin is primarily involved in attenuating apoptotic cell death and

secondarily may play a role in regulation of neural cell proliferative responses after TBI.














CHAPTER 1
INTRODUCTION

Traumatic Brain Injury Demographics

Traumatic brain injury (TBI) is the leading cause of death and permanent disability

for children and young adults in the United States. Currently, there are more than 5.3

million Americans, approximately 2% of the current U.S. population, living with TBI-

related disabilities (Thurman et al., 1999a) with an estimated 1.5 million additional TBIs

occurring each year (Sosin et al., 1996). Approximately 230,000 cases are severe enough

that the victims require transport and hospitalization (Thurman et al., 1999b). Of these,

approximately 50,000 victims die from their injuries, accounting for 33% of all injury-

related deaths (Sosin et al., 1995). Of those severely injured survivors, 90,000 TBI

victims must live with long-term disabilities (Thurman et al., 1999b). The U.S. economy

loses an estimated $56.3 billion a year through the direct and indirect costs associated

with TBI (Thurman et al., 1999b).

Currently, most traumatic brain injuries result from motor vehicle accidents

(48.9%) followed by falls (25.8%) and firearms/ assaults (19.2%) (Thurman et al.,

1999b). Young males, ages 15 to 24, are the most "at-risk" demographic, a statistic that

likely reflects lifestyle choices (Jennett, 1996; Thurman et al., 1999b). The magnitude of

this problem led to the passing of Public Law 104-166, better known as the Traumatic

Brain Injury Act of 1996, a bill designed to help prevent TBI and educate the public

about the health consequences of this injury (Thurman et al., 1999b). While these

educational efforts have decreased TBI-related deaths by an estimated 22% since 1980









(Sosin et al., 1995), the number of people living with TBI-related disabilities has risen

(Thurman et al., 1999b).

To date, few pharmacological or treatment options are available to reduce these

TBI-induced disabilities. Prevention remains the only effective "cure." True advances in

clinical treatment depend on understanding the underlying pathophysiology mechanisms

that regulate both cell death and cell survival following TBI.

Traumatic Brain Injury Pathophysiology

Traumatic brain injury is a complex injury that is comprised of an immediate

primary injury and a progressive secondary injury cascade (Graham et al., 2000). The

primary mechanical injury can be contusive or concussive and involves tearing and

stretching of the neural tissues. Neurons and white matter tracts seem particularly

vulnerable to the mechanical injury (Baldwin et al., 1997; Maxwell et al., 1997;

McCullers et al., 2002; Grady et al., 2003). The secondary injury cascade is initiated by

the primary mechanical injury and is defined by unrestrained biochemical and

inflammatory reactions (Gennarelli, 1993). Though the brain is remarkably adaptive,

damage sustained as the result of secondary injury prevents the brain from regaining pre-

injury function.

While many of the biochemical processes seen after TBI occur under normal

homeostasis, their collective dysregulation acts in a synergistic manner to contribute to

the pathology associated with secondary injury. Some of the more prominent events

include perturbations in blood flow (Graham et al., 1995; McIntosh et al., 1998;

Raghupathi et al., 2000), ischemia (Lee et al., 1999; Passineau et al., 2000), excitotoxicity

(Choi and Rothman, 1990; Gennarelli, 1993), calcium deregulation (Graham et al., 1995),

free radical production (Kontos, 1989; Beckman et al., 1990; Maier and Chan, 2002),









inflammation (Povlishock and Kontos, 1985; Giulian, 1991; Morganti-Kossmann et al.,

2001), edema (Choi, 1988; Bullock et al., 1991) and protease activation (Pike et al., 1998;

Clark et al., 2000; Eldadah and Faden, 2000; Raghupathi et al., 2000; Knoblach et al.,

2002; Larner et al., 2004). Ultimately, activation of these processes disrupts fragile

homeostatic states and creates an inhospitable environment for neural cell survival.

Traumatic Brain Injury, Apoptosis and Caspase-3 Activation

Cell death following TBI is distinguished by necrotic and apoptotic processes

(Conti et al., 1998; Clark et al., 2000; Yakovlev and Faden, 2001). Necrosis and

apoptosis lie on a continuum (Nicotera et al., 1999) wherein the mode of cell death is

dictated by several factors including ATP availability (Green and Reed, 1998), calpain

activity (Wang, 2000), intracellular calcium levels (Gwag et al., 1999; Zipfel et al.,

2000), presence of anti-apoptotic factors (Raghupathi et al., 2000) and the presence of

activated caspases (Denecker et al., 2001). Within hours of a TBI, neural cells around the

contusion area exhibit classic signs of necrosis including cytotoxic edema, mitochondrial

swelling, nuclear pyknosis, ruptured plasma membranes, organelle breakdown and

vacuolated cytoplasm (Sutton et al., 1993; Dietrich, 1994; Denecker et al., 2001).

However, as time progresses, many cells including neurons, astrocytes and

oligodendrocytes begin to exhibit characteristics of apoptosis including chromatin

condensation, cell shrinkage, apoptotic body formation and DNA laddering (Conti et al.,

1998; Newcomb et al., 1999). It is well documented that apoptotic cell death continues

for many months following injury, thereby making it a chronic contributor to post-TBI

pathology (Cervos-Navarro and Lafuente, 1991).

Apoptosis has been well characterized following traumatic brain injury (Pike et

al., 1998; Beer et al., 2000; Clark et al., 2000) and utilizes both the intrinsic and extrinsic









apoptotic pathways (Fig. 1-1). Each pathway involves a unique set of upstream and

downstream cysteine specific proteases called caspases that cleave a variety of

intracellular substrates and drive apoptosis. Synthesized as inactive zymogens, caspases

require the cleavage of a pro-domain to become active. Caspase activation is achieved in

multiple manners including proximity-induced autoproteolysis or cleavage by another

caspase (Stennicke and Salvesen, 1999; Van de Craen et al., 1999). Both the intrinsic

and extrinsic pathways lead to cleavage and activation of caspase-3, the most abundant

executioner caspase in the brain (Chan and Mattson, 1999; Slee et al., 2001).

Among the numerous structural and regulatory protein targets of active caspase-3

are stress response proteins (e.g., PARP, Rb and p21), signal transduction proteins (e.g.,

phospholipase A2, NFKB and PKC), structural proteins (e.g., a-II-spectrin, actin and

vimentin), nuclear matrix proteins (e.g., lamins A, B and C) and mitochondrial proteins

(e.g., Bcl-2, Bcl-xl and Bid) (Cohen, 1997; Chan and Mattson, 1999; Earnshaw et al.,

1999; Wang, 2000). Cleavage of proteins such as iCAD/DFF45 (Enari et al., 1998; Liu

et al., 1998b; Sakahira et al., 1998), poly (ADP-ribose) polymerase (PARP) (Ferrer and

Planas, 2003), DNA-dependent protein kinase (DNA-PK) (Lazebnik et al., 1994) and

acinus (Sahara et al., 1999) prevents DNA repair and promotes DNA condensation and

fragmentation (Woo et al., 1998). When unregulated, even moderate activation of

caspase-3 can rapidly lead to cell death. Therefore, nature has developed various

mechanisms to temper the deleterious effects of caspase-3 over-activity and counter the

progression of apoptosis.










Extrinsic


Intrinsic


pa-!X. \;e-s 0 1

\Apoptosome


Caspase-3
Substrates
Stress Response
Rb T 5 & 'a a ^ ^
Signal Transduction
PKC
Phospholipase A2
NFkB
ua llspectrin
Actin DNA Fragmentation
Vimentin
Nuclear Matrix
Lamins A, B1 & C
Mitochondrial

i Cell Death


Figure 1-1: Intrinsic and extrinsic apoptosis pathways. Apoptosis progresses primarily
through the extrinsic and intrinsic apoptosis pathways. The extrinsic pathway
is mediated by ligand binding to membrane bound death receptors and
caspase-8 activation. Activation of this pathway can promote cell death by
intrinsic pathway activation or apoptosis prevention by up-regulation of
apoptosis inhibitors. The intrinsic pathway is mediated by mitochondrial
stress. Caspase-9 is activated in the apoptosome complex. Both pathways
promote caspase-3 activation. Active caspase-3 can cleave several
intracellular proteins. Cleavage of proteins such as iCAD (DFF45), PARP
and acinus can lead to DNA fragmentation and cell death.


Death i eeptol llsaind


I ~II~

)~









Apoptosis Inhibition Following TBI

While many pro-apoptotic proteins are expressed following TBI, there is a

concomitant increase in pro-survival factors (Nowak and Jacewicz, 1994; Iwata et al.,

1997; Buytaert et al., 2001; Hermann et al., 2001; Sanz et al., 2001; Alzheimer and

Werner, 2002; Maroni et al., 2003). Of these pro-survival factors, a family of proteins

known as inhibitor of apoptosis proteins (IAPs) can attenuate apoptotic cell death by

directly binding to the active site of activated caspases such as caspase-3 (Tamm et al.,

1998; Conway et al., 2000; Shin et al., 2001). The IAP family contains eight known

members including survivin (Li, 2003). The proteins are highly conserved across species

(LaCasse et al., 1998) and the expression of each IAP appears to be cell type specific.

Each IAP has one to three baculovirus IAP repeat (BIR) domains that possess the ability

to bind and directly inhibit active caspase-3 (Tamm et al., 1998; Conway et al., 2000;

Shin et al., 2001), caspase-7 (Tamm et al., 1998; Shin et al., 2001) and caspase-9

(LaCasse et al., 1998; Deveraux and Reed, 1999). Mutation studies have demonstrated

that the BIR domain is responsible for caspase interaction and is therefore necessary for

anti-apoptotic action of the IAPs (Roy et al., 1997; Takahashi et al., 1998; Vucic et al.,

1998; Muchmore et al., 2000).Although few IAPs have been extensively characterized in

the context of TBI pathophysiology, increases in XIAP (Keane et al., 2001; Lotocki et al.,

2003), NAIP (Xu et al., 1997; Hutchison et al., 2001; Thompson et al., 2004), clAP-1

(Keane et al., 2001; Belluardo et al., 2002) and cIAP-2 (Keane et al., 2001) have been

reported in neurons following brain injury. The potential role of survivin following TBI

has not been investigated.









Cellular Proliferation Following TBI

In addition to pro-survival factors, new cell production plays a pivotal role in the

brain following injury. Large pools of neural progenitor cells have recently been

identified in the germinal centers of the dentate gyms subgranular zone (SGZ) and

subventricular zones (SVG) of the adult brain (Gage et al., 1998; Magavi et al., 2000;

Gage, 2002; Sanai et al., 2004). Following both ischemia and TBI, these neural

progenitor cells proliferate (Gould and Tanapat, 1997; Yagita et al., 2001) and

differentiate into mature neurons (Gage et al., 1998; Doetsch et al., 1999; Magavi et al.,

2000; Cameron and McKay, 2001; Dash et al., 2001; Kemie et al., 2001; Yagita et al.,

2001; Peterson, 2002), astrocytes (Dash et al., 2001; Gould et al., 2001; Chirumamilla et

al., 2002; Chen et al., 2003) and oligodendrocytes (Gould et al., 2001). Consistent with

these findings, many cell cycle proteins (e.g., cyclins A, B and Dl, cdk4 and PCNA) are

also up-regulated after brain injury (Miyake et al., 1992; Kaya et al., 1999a; Chen et al.,

2003; McPherson et al., 2003).

Cellular proliferation following TBI can have both beneficial and detrimental

consequences to the recovery of the damaged brain. These consequences can also vary

by cell type. Neuronal progenitor cells have been shown to proliferate following brain

injury (Parent, 1997; Hill-Felberg et al., 1999; Dash et al., 2001; Kernie et al., 2001;

Yagita et al., 2001; Chirumamilla et al., 2002; Rice et al., 2003) but the functional

viability and therefore significance of these newly formed neurons is not clear. New

neurons appear to migrate away from the germinal centers of the subventricular zone

(SVZ) and subgranular zones of the dentate gyms (SGZ) but not towards areas of injury

(Rice et al., 2003). Additionally, as many as 80% of all newly formed neurons undergo

apoptosis within two weeks of their formation in normal conditions (Morshead and van









der Kooy, 1992; Morshead et al., 1994). The proliferation of neurons following TBI may

be advantageous but their inability to survive and contribute to recovery requires

additional clarification.

Glial cell proliferation, specifically astrocytes and oligodendrocytes, can serve to

both support and inhibit natural recovery processes. Adult oligodendrocyte precursor

cells (OPC) can develop into both astrocytes and oligodendrocytes following injury.

Furthermore, their distribution in the adult brain is not as restricted as the neuronal

precursor populations (Dawson et al., 2000). An increase in both the astrocyte and

oligodendrocyte population may contribute positively to the post-injury milieu.

Astrocytes metabolize extracellular glutamate, neutralize free radicals, modulate the

immunological response by production of cytokines and modulating nitric oxide activity

(Gabryel and Trzeciak, 2001; Bambrick et al., 2004; Heales et al., 2004). Similarly,

oligodendrocytes can help re-myelinate damaged axons. Furthermore, glial cell

proliferation may contribute to formation of the glial scar. This barrier can act to protect

non-damaged brain regions from advancing secondary injury processes (Ridet et al.,

1997; Bush et al., 1999; Smith et al., 2001). However, the glial scar also produces

chondroitin sulfate proteoglycans which may then act to form an impermissible

environment for axonal growth (Fawcett and Asher, 1999; Chen et al., 2002).

With developmental origins as hematopoietic cells, microglia are one of the few

mature neural cell types that retain the ability to divide (Simard and Rivest, 2004). After

various types of brain injury, microglia proliferate rapidly (Liu et al., 1998a; Csuka et al.,

2000; Liu et al., 2000; Grady et al., 2003) to remove cellular debris, protect injured

neurons and promote functional recovery (Giulian, 1991). However, microglia have been









documented as a major source of proteases and inflammatory cytokines following various

CNS injuries (Nakajima and Kohsaka, 1993; Streit and Kincaid-Colton, 1995; Streit,

1996; Aldskogius et al., 1999; Fawcett and Asher, 1999; Gong et al., 2000).

Functional replacement of injured and dying cells may contribute to more complete

recovery following TBI. Because the neural environment becomes hostile for new cells

to survive during the injury state, the identification of proteins that promote both cellular

proliferation and survival in compromised cellular environments may prove useful in

treating the injured brain. A very delicate balance between proliferation and cell death

inhibition is desired. Survivin is a protein that has recently been identified as having

roles in both mitosis regulation and apoptosis inhibition in other non-central nervous

system (CNS) pathological conditions and may contribute to this balance following TBI.

Survivin: Mitosis and Anti-apoptosis Protein

Survivin was discovered in 1997 as a protein expressed only by rapidly dividing

cells during development (Ambrosini et al., 1997). As its expression is prominent in

apoptosis-resistant tumor cells, survivin became an intensely studied protein in cancer

research. These studies demonstrated that survivin functioned to inhibit apoptosis and

was essential for the proper completion of mitosis. Because it has an integral role in

cellular proliferation and apoptotic cell death, both of which contribute to the

pathophysiology of TBI, survivin may have an important role in the secondary injury

cascade.

Survivin Protein Structure

The survivin protein is composed of 142 amino acids with a molecular weight of 17

kDa per monomer (Ambrosini et al., 1997). Cellular survivin exists as a homodimer

bound together by an intermolecular Zn atom giving the complex a "bow-tie "









appearance and is the only IAP known to homodimerize in solution (Chantalat et al.,

2000; Muchmore et al., 2000; Verdecia et al., 2000) (Fig. 1-2). Survivin is the smallest

IAP to have anti-apoptotic properties, containing only a single BIR domain and

microtubule-binding coiled coil domain (Ambrosini et al., 1997).

A distinct subcellular pool of survivin exists in the cytoplasm and nucleus of the

cell (Conway et al., 2000; Li, 2003; Badran et al., 2004) with a ratio of 6:1, respectively

(Fortugno et al., 2002). Recent evidence suggests that the subcellular localization of

survivin may designate its role. The nuclear pool appears to be associated with cellular

proliferation while cytoplasmic survivin appears to be more predictive of caspase

inhibition (Moon and Tarnawski, 2003). Survivin is a relatively short-lived protein with

a half-life of approximately 30 minutes (Zhao et al., 2000), though phosphorylation may

enhance its stability (O'Connor et al., 2000a; O'Connor et al., 2002). Survivin is removed

from the cell by polyubiquitination and proteasomal destruction (Zhao et al., 2000).

Survivin Expression and Mitosis

As a protein found almost exclusively in apoptosis-regulated embryonic and fetal

tissue (Adida et al., 1998; Kobayashi et al., 1999), survivin is not normally found in

differentiated adult tissues. However, it is present at very low levels in adult cells with a

high mitotic index (Ambrosini et al., 1997). The function of survivin during mitosis is

intimately related to its ability to bind microtubules. Survivin is required for the

assembly of a bipolar mitotic apparatus by controlling microtubule stability (Altieri,

2001, 2003b). Homozygous deletion of the survivin gene causes defects in microtubule

assembly, mitotic spindle formation and cell division resulting in multi-nucleation and

total lethality of the organism by E3.5-4.5 in knockout mice (Uren et al., 2000).









Beyond development, survivin is prominently expressed in many cancers and is

linked to poor survival prognosis, higher rates of cancer reoccurrence and elevated

mortality rates (Altieri, 2003a). Many neural derived cancer cell lines have been shown

to over-express survivin including astrocytes gliomaa), neurons neuroblastomaa) and

oligodendrocytes (oligodendroglioma) (Shankar et al., 2001; Borriello et al., 2002; Sasaki

et al., 2002; Kajiwara et al., 2003; Kleinschmidt-DeMasters et al., 2003; Jiao et al.,

2004), indicating that mature, albeit abnormal, neural cells retain the ability to express

survivin beyond differentiation. Additionally, proliferating neural stem cells express

mitosis proteins after brain injury (Cameron and McKay, 1998; Doetsch et al., 1999;

Cameron and McKay, 2001; Song et al., 2002) indicating that non-transformed neural

cells may also express survivin following TBI.

Survivin and Apoptosis Inhibition

The ability of survivin to inhibit apoptosis is known to occur in conjunction with

the cell cycle but also has been shown to be independent of mitosis. For example, many

tumor cells express survivin when not actively dividing and can inhibit apoptosis caused

by chemotherapeutic agents (Li et al., 1998). Beyond cancer, survivin expression has

been reported in non-proliferating, non-tumor cells after ischemic brain injury without

activating mitosis and with the ability to inhibit cell death (Blanc-Brude et al., 2002; Tran

et al., 2002; Conway et al., 2003). Therefore, survivin expression may occur without

activation of the cell cycle.

It has been demonstrated repeatedly that survivin over-expression can inhibit

apoptosis in cancer cells (Ambrosini et al., 1998; Grossman et al., 1999; Muchmore et al.,

2000; Shin et al., 2001; Kim et al., 2004). Survivin expressing gastric and esophageal

squamous cell cancers exhibit significantly lower rates of apoptosis compared to









1 14 BIR 8o 9s Coiled coil 140142 Su
SSurvivin









Coiled Coil 142




BIR
Domain


Survivin Dimer

Figure 1-2: Survivin protein structure. Survivin is a 142 amino acid (17 kDa) protein that
contains a single baculovirus IAP repeat (BIR) domain (red) and a C-terminus
a-helical coiled coil domain (orange). In solution, survivin homodimerizes
and is held together by a zinc ion interaction. The survivin BIR domain has
been shown to bind activated caspase-3 and inhibit apoptosis induced by
many factors. The coiled coil domain can bind and stabilize microtubules
during assembly of the bipolar mitotic apparatus and keep it in close
proximity to caspase activity as mitosis progresses. 3-D survivin structure
adapted from Verdecia et al 2000.

survivin-negative cancer cells (Lu et al., 1998). Molecular antagonists of survivin (e.g.,

siRNA, antisense, dominant negative mutants) cause caspase-dependent cell death and

magnify the effects of other pro-apoptotic signals in vitro and in vivo (Li et al., 1999;

Kanwar et al., 2001; Kasof and Gomes, 2001; Shankar et al., 2001; Xia et al., 2002a;

Zhou et al., 2002; Choi et al., 2003). In addition, survivin has been shown to protect cells

from a variety of apoptotic stimuli including IL-3 withdrawal (Ambrosini et al., 1997),

Fas stimulation (Tamm et al., 1998; Jiang et al., 2001), anoikis (Papapetropoulos et al.,

2000), cytochrome c administration (Takahashi et al., 1998; Tamm et al., 1998), Bax









over-expression (Deveraux et al., 1997; Tamm et al., 1998), active caspase-3 (Tamm et

al., 1998), active caspase-7 (Tamm et al., 1998; Jiang et al., 2001), Taxol (Li et al., 1998),

and etoposide (Tamm et al., 1998; Jiang et al., 2001).

In these models, survivin appears to exert its anti-apoptotic effects by directly

binding to active caspase-3 (Tamm et al., 1998; Kobayashi et al., 1999). It is possible,

however, that survivin may also act at other, less clearly defined points in the apoptotic

cascade (Suzuki et al., 2000; Grossman and Altieri, 2001; Grossman et al., 2001a;

Fortugno et al., 2003) or outside of apoptotic caspase activation to prevent cell death

(Shankar et al., 2001; Chakravarti et al., 2004).

Potential Role for Survivin in TBI Pathology

There are currently no comprehensive studies of survivin in neural cells following

CNS injury. Moreover, the potential involvement of survivin in TBI pathophysiology is

unknown. The role of survivin in apoptosis inhibition and cellular proliferation in

various in vitro and in vivo models supports the hypothesis that survivin may also

contribute to the pathophysiology of TBI. Both apoptosis and cellular proliferation occur

following traumatic brain injury and create an environment where survivin expression

may be important in balancing two contrasting yet related processes. From the literature,

it is clear that survivin is ubiquitously expressed by all cells early in development and

that this expression may be restored in certain mature cells following CNS injury.

Therefore, based on the existing data described above from the areas of cancer,

mitosis and apoptosis, a thorough investigation of survivin following TBI was warranted.

Thus, the main goal of this work is to reveal and characterize potential roles for survivin

in neural cell responses following traumatic brain injury. The general hypothesis of the

study is that survivin is up-regulated following TBI and plays a role in anti-apoptotic and






14


cell cycle activation mechanisms to oppose TBI pathogenesis. Specifically, I propose that

(i) survivin up-regulation inhibits caspase-3 mediated DNA fragmentation in a cell-

specific manner following TBI, and (ii) survivin plays a role in cell cycle progression

following TBI.














CHAPTER 2
METHODOLOGY

Induction of Controlled Cortical Impact Brain Injury

The surgical and cortical impact injury procedures were conducted as previously

described (Dixon et al., 1991; Pike et al., 1998). Briefly, adult male Sprague-Dawley rats

(250-300 g) were anesthetized with 4% isoflurane (Halocarbon Laboratories; River Edge,

NJ) in 1:1 O2/ N20 for 4 minutes and maintained during surgery with 2.5% isoflurane.

Core body temperature was continuously monitored using a rectal thermistor probe and

maintained at 36.5-37.5 C using an adjustable heating pad. A unilateral craniotomy

(ipsilateral to injury) was performed over the right cortex between the sagittal suture,

bregma and lambda while leaving the dura intact. Traumatic insult was generated by

impacting the exposed cortex with a 5 mm diameter aluminum tip at a velocity of 4

m/sec, a 150ms dwell time and 1.6 mm compression. Craniotomy control animals

received the craniotomy but not the impact injury. All procedures were performed

according to guidelines established by the University of Florida Institutional Animal Care

and Use Committee (IACUC) and the National Institutes of Health (NIH). In the

following studies, "ipsilateral" refers to the same side as the impact injury whereas

"contralateral" refers to the opposite side of the injury. "Craniotomy control" refers to

animals that received the craniotomy but did not receive the impact injury.

Quantitative Reverse Transcriptase Polymerase Chain Reaction (Q-PCR)

Survivin primers were generated using GeneBank locus AF 276775: forward

primer 5' TAAGC CACTT GTCCC AGCTT 3', and reverse primer 5' AGGAT GGTAC









CCCAT TACCT 3'. GAPDH: forward primer 5' GGCTG CCTTC TCTTG TGAC 3'

and the reverse primer 5' CACCA CTTCG TCCGC CGG 3'. Cortical and hippocampal

tissues from the ipsilateral and contralateral hemispheres were rapidly excised at either 1

day, 2 days, 3 days, 5 days, 7 days or 14 days and 'snap-frozen' with liquid nitrogen.

Total RNA was isolated from the samples using TRIzol reagent (Invitrogen, Carlsbad,

CA, USA) according to the manufacturer's instructions. Final RNA concentrations were

determined via spectrophotometry and were stored at -200 C in diethyl pyrocarbonate

(DEPC) water for future cDNA preparation.

cDNA synthesis was performed using 1 |jg of total RNA with the SuperScriptTM

First-Strand Synthesis System for RT-PCR kit (Invitrogen/Life Technologies, Carlsbad,

CA) according to the manufacturer's instructions. Any DNA contamination was detected

in the RNA samples by "no reverse transcriptase" reactions that were performed in

conjunction with the cDNA synthesis reaction.

Q-PCR was performed as previously described (Tolentino et al., 2002) using the

LightCycler-FastStart DNA Master SYBR Green I reaction mix (Roche Diagnostics,

Indianapolis, IN) in combination with 0.5 [LM primers, 2.5 mM MgC12 in the Light

Cycler rapid thermal cycler system (Roche Diagnostics, Indianapolis, IN). Briefly, the

products were amplified then continuously quantified by online monitoring. Each PCR

reaction has its kinetics represented by an amplification curve. Each amplification curve

(fluorescence vs. cycle number) is assigned a crossing point value (CPV), which is the

exact time point at which the logarithmic linear phase could be distinguished from the

background. A lower CPV indicates a more rapid increase in the level of fluorescence

indicative of a higher concentration of specific message present in the sample. Therefore,









those samples with a lower CPV have more amplified message than those with a higher

CPV.

The survivin primer sets were subjected to serial dilution and linear regression

analysis of the logarithm of the dilution factor vs. the CPV generated a standard curve for

each transcript-specific template. The specificity of the amplified products were

confirmed using melting curve analysis and gel electrophoresis. The relative amounts of

RNA from the unknown samples were extrapolated from its calculated CVP in relation to

the generated standard curve. Results are presented as percentage of craniotomy control.

Data were analyzed by ANOVA with a post-hoc Bonferroni-test and are given as mean +

SEM. Differences were considered significant at the level of p < 0.05.

Rat-Specific Survivin Polyclonal Antibody Production

Commercially available survivin antibodies were not adequate to label survivin in

tissue sections. Therefore, a new rat-specific antibody was developed for use in

fluorescent immunohistochemistry. Two rat-specific survivin sequence peptides were

synthesized using the protein sequence from GeneBank, accession number AF276775

(Swissprot Q9JHY7), for antibody production. The two peptides corresponded to regions

in the conserved BIR domain (CPTENEPDLAQC) and from the C-terminus coiled coil

domain (CFKELEGWEPDDNPIEE). The peptides used to develop the survivin antibody

(R51) are specific to survivin and do not recognize other IAP family proteins according

to SDSC Biology Workbench BLASTP (2.2.2) (Altschul et al., 1997) and CLUSTAL W

(1.81) analysis (Higgins et al., 1992; Thompson et al., 1994) resulting in the survivin

antibody's specificity. Alignment scores for CLUSTALW (1.81) were computed with

the following multiple alignment parameters: Matrix: Gonnet, Gap Open Penalty: 10.00,









% Identity for Delay: 30, Penalize End Gaps: on, Gap Separation Distance: 0, Negative

Matrix?: no, Gap Extension Penalty: 0.20, Residue-Specific Gap Penalties: on,

Hydrophilic Gap Penalties: on, Hydrophilic Residues: GPSNDQEKR.

Rabbits were immunized with these peptides, allowed to produce antibodies to the

peptides and finally serum was extracted from the immunized rabbits. The rat specific

survivin antibodies were removed and affinity purified using a SulfoLink kit (Pierce Inc;

Rockford, IL) as per the manufacturers instructions.

Survivin Polyclonal Antibody Characterization

The specificity of the survivin antibody (R51; Dr. G. Shaw) was compared to other

commercially available survivin antibodies (Chemicon; Temecula, CA and Novus

Biologicals; Littleton, CO) on western blots and in cell culture. On western blots using

dividing cell culture lysates (HeLa and SY5Y) and injured tissue lysates, R51 and the

Novus survivin antibody show a similar labeling pattern and recognized the 17 kDa

monomer of survivin. For IHC, R51 showed characteristic staining of the cleavage

furrow between dividing HeLa and SY5Y cells consistent with other reports (Li et al.,

1998; Li et al., 1999; Uren et al., 2000) (Figure 2-1). In addition, dual-labeling in

dividing cell cultures of both HeLa and SY5Y cells with R51 and the Chemicon survivin

antibody showed co-localization at the cleavage furrow.

Western Blot Analyses

The cortex and hippocampus from each set of brain tissues was excised, rinsed with

cold PBS, snap frozen in liquid nitrogen and homogenized in ice-cold triple detergent

lysis buffer containing a CompleteTM protease inhibitor cocktail (Roche Biochemicals,

Indianapolis, IN). Protein concentration was determined by bicinchoninic acid (BCA)















4M 17.

Ik~a


Figure 2-1: IHC characterization of the rat-specific survivin antibody. The survivin
antibody (R51) reveals a characteristic and previously described labeling
pattern on western blot (A) and in IHC (B). Western blot analysis revealed a
classic 17 kDa band in cell culture lysates (lanes 1-3) and in injured rat tissue
lysates (lanes 6-7) but not in un-injured rat tissue lysates (lanes 4-5). IHC
using the survivin antibody revealed a well-characterized survivin (green)
staining pattern around the nuclei (DAPI, blue) of proliferating SY5Y cells in
various stages of mitosis including G2/M, interphase (I), pro-metaphase (PM),
anaphase (A) and telophase (T).

micro protein assays (Pierce, Inc., Rockford, IL). Forty micrograms of protein per well

was loaded and separated by SDS-PAGE, transferred to PVDF membranes and probed

with either goat-anti-rabbit survivin antibody (Novus Biologicals; Littleton, CO; 1:1000)

or goat-anti-rabbit active caspase-3 (Cell Signaling; 9661L; 1:100). After incubation

with goat anti-rabbit HRP-labeled secondary antibody (Biorad, Hercules, CA), the

membranes were developed using Enhanced Chemiluminescence Plus reagents (ECL

Plus; Amersham, Arlington Heights, IL). For further PCNA analysis, developed PVDF

membranes were incubated in stripping buffer, rinsed twice in TBST and incubated with

PCNA (Santa Cruz Biotech; Santa Cruz, CA; 1:1000) antibody with goat-anti-mouse









HRP conjugated secondary antibody. Semi-quantitative, densitometric analysis was

performed using the AlphalmagerTM 2000 Digital Imaging System (San Leandro, CA).

The blots were not labeled with an antibody, such as actin or GAPDH, to act as an

internal standard because our previous studies found that many "stable" proteins are the

targets of proteolytic cleavage and thus could not act as a proper internal control.

Transformed data (experimental densitometry value/ craniotomy control densitometry

value x 100) was evaluated by ANOVA and a post-hoc Dunnet-test. Values were

expressed as percentage of craniotomy controls and are reported as mean + SEM.

Differences were considered significant at the level of p 0.05.

Preparation and Sectioning of Tissue for Immunohistochemistry (IHC)

Tissue was prepared and sectioned for vibratome and cryostat sectioning. For

vibratome sectioning, animals were transcardially perfused with 2% Heparin (Elkins-

Sinn, Inc.; Cherry Hill, NJ) in 0.9% saline solution (pH 7.4) followed by 4%

paraformaldehyde in 0.1M phosphate buffer (pH 7.4). The brains were post-fixed in 4%

paraformaldehyde and stored in 0.1M PBS or cryobuffer. Sections were cut on a Series

1000 vibratome (Ted Pella; Redding, CA) at forty microns. For cryostat sectioning,

animals were anesthetized with 4% isoflurane (Halocarbon Laboratories; River Edge, NJ)

in 1:1 02/ N20 for 4 minutes, then the head was removed. The brains were blocked in

O.C.T. (Ted Pella; Redding, CA), snap frozen in liquid nitrogen and cut on a Leica

CM3050 cryostat. Five micron sections were attached to Fropen (Ted Pella; Redding,

CA)-treated coverslips, fixed in cold methanol for 20 minutes at -200 C.









Dual Label Fluorescent Immunohistochemistry (IHC)

Sections were fluorescent immunolabeled with two primary antibodies in the

following experiments: survivin (1:500)/GFAP for astrocytes (Sternberger; Lutherville,

MD; 1:1000), survivin/NeuN for mature neurons (Chemicon; Temecula, CA; 1:1000),

survivin/PCNA (Santa Cruz Biotech; Santa Cruz, CA; 1:200), PCNA/GFAP,

PCNA/NeuN, active caspase-3 (1:100)/GFAP, active caspase-3 /NeuN and active

caspase-3 /survivin (G. Shaw; 1:250). In addition, sections were labeled with the

Apoptag Cell Death Labeling kit (terminal deoxynucleotidyl transferase-mediated

biotinylated dUTP nick-end labeling or TUNEL) to mark double stranded DNA breaks as

per the manufacturer's instructions. This kit was used in conjunction with the following

antibodies: TUNEL/GFAP, TUNEL/NeuN and TUNEL/survivin. The nuclear dye DAPI

(in Vectashield; H-1200; Vector Laboratories; Burlingame, CA) was used to label the

nuclei in all sections. The first primary antibody was incubated at 4 C for 24- 48 h in a

2% goat serum/ 2% horse serum/ 0.2% Triton-X 100 in 0.1 M PBS (block) solution

followed by the second primary antibody at 4 C for 1 h in block solution. Fluorescent-

tagged secondary antibody (Molecular Probes; Eugene, OR) was used for visualization.

Sections were viewed and digitally captured with a Zeiss Axioplan 2 microscope

equipped with a SPOT Real Time Slider high-resolution color CCD digital camera

(Diagnostic Instruments, Inc., Sterling Heights, MI). A Bio-Rad 1024 ES confocal

microscope was used to confirm single cell localization of the label pairings. The

settings for these images were as follows: power = 100%; for the red field: iris = 2.7 -

5.2, gain = 1400, Blev = -3; for the green field: iris = 3.0 5.7, gain = 1400, Blev = -3.

The number of animals used for each label pairing for dual-labeling IHC was four (n=4).









Dual Label Fluorescent IHC for Same-Species Antibodies

Two systems were used for dual-labeling using same species antibodies; the

tyramide signal amplification (TSA) kit (PerkinElmer Life Sciences, Boston, MA) and a

biotin/streptavidin antibody protocol. Both techniques rely on steric hindrance to block

same-species binding sites. Control sections showed the secondary/tertiary complex was

sufficient for steric hindrance of same species sites for both protocols (Figure 2-2).

Tyramide signal amplification (TSA) was accomplished using the TSA kit

(PerkinElmer Life Sciences, Boston, MA) according to the manufacturer's instructions

and as previously described (Stone et al., 2002). Biotin/streptavidin same species dual-

labeling begins with an endogenous biotin blocking step (Vector Laboratories;

Burlingame, CA) followed by incubation of the first primary antibody as described above

followed sequentially by a biotin-conjugated secondary antibody and fluorescent-labeled

streptavidin (Molecular Probes; Eugene, OR), both steps at room temperature for 1 h in

block solution. The second antigen was then labeled as described above.

Experimental Group Sizes

The number of animals used for western blot analysis is as follows (per time point):

survivin = 6, PCNA = 6, active caspase-3 = 6. The number of animals used for dual-

labeling IHC is as follows (presented as 5 days post injury or both 5 / 7 days post injury):

survivin x PCNA = 4, survivin x GFAP = 6, survivin x NeuN = 4, PCNA x GFAP = 4,

PCNA x NeuN = 4, active caspase-3 x survivin = 4/4, active caspase-3 x GFAP = 4/4,

active caspase-3 x NeuN = 4/4, TUNEL x GFAP = 4/4, TUNEL x NeuN = 4/4 & TUNEL

x survivin = 4/4.




























Figure 2-2: Control section for biotin/streptavidin same-species dual labeling IHC.
Biotin/streptavidin same-species dual labeling IHC is a technique based on
steric hindrance of same species binding sites to prevent the second
fluorescent-labeled secondary from binding to the first primary antibody. To
ensure that this process was sufficient, mouse antibodies for astrocytes
(GFAP, green) and neurons (NeuN, red) were used to label a section of brain.
These two protein targets were chosen because of their abundance and distinct
labeling patterns in adult rat brain. On the left (30 Control), neurons were
labeled with only the primary and biotin secondary with no fluorescent
streptavidin tertiary antibody while astrocytes were labeled with a primary and
fluorescent-tagged secondary antibody. On the right (Complete), neurons
were labeled with a primary, the biotin conjugated secondary and a
fluorescent-tagged streptavidin tertiary while astrocytes were labeled as
described previously. The absence of red fluorescent labeling in the picture
on the left indicates that the biotin secondary antibody was sufficiently large
to prevent the green fluorescent-labeled secondary antibody to bind to it thus
confirming steric hindrance of NeuN binding sites.

Cell Quantification and Statistical Analysis

Cell counts were obtained by comparing the number of dual-labeled cells to total single-

labeled cells in the following groups: survivin/NeuN positive cells to total NeuN positive

cells, survivin/PCNA positive cells to total PCNA positive cells, PCNA/NeuN positive

cells to total NeuN positive cells, survivin/GFAP positive cells to total GFAP positive

cells, active caspase-3/NeuN positive cells to total NeuN positive cells, active caspase-









3/survivin positive cells to total survivin positive cells, active caspase-3/GFAP positive

cells to total GFAP positive cells, TUNEL/NeuN positive cells to total NeuN positive

cells, TUNEL/survivin positive cells to total survivin positive cells and TUNEL/GFAP

positive cells to total GFAP positive cells. Percentages were calculated by dividing the

number of dual-labeled cells with the total number of single-labeled cells. For each

group, representative photomicrographs were selected and counted. Cells were counted

in a total area of 188,000 am2 for each label pairing in both cortical and hippocampal

regions. These numbers were then transformed into percentage of either total cells or

total cell type. Individual comparisons between groups were made using an unpaired

Student t-test. Results were considered significant at p<0.05. Two additional blinded

observers were used to count representative samples. Inter-rater reliability was calculated

using the inter-rater reliability formula created by R.L. Ebel (Ebel, 1951). The intra-class

correlation (ICC) value achieved was 0.97 indicating little variation between raters.














CHAPTER 3
SURVIVING EXPRESSION FOLLOWING TRAUMATIC BRAIN INJURY

Induction of Survivin Expression After TBI

Q-PCR analysis revealed an initial increase in survivin mRNA at 2 days post injury

in the ipsilateral cortex and hippocampus. These transcripts remained elevated in both

regions, reached maximum levels at day 5 post-injury and declined at 7 days in the cortex

and at 14 days in the hippocampus. All experimental animals remained alive and

exhibited slightly impaired motor and cognitive impairments (data not shown). Cortical

mRNA levels reached a maximum of 448 10.0%, whereas hippocampal mRNAs

attained 606 10.0% compared to craniotomy control values (Figure 3-1). To determine

if the induction of survivin mRNA resulted in corresponding increases in survivin

protein, western blot analysis was performed. Survivin (17 kDa protein) was readily

detectable in the ipsilateral cortex and hippocampus of TBI rats, while it was negligible

in contralateral cortex and hippocampus (Figure 3-2A). Survivin was expressed in a time-

dependant manner with a maximum increase at 5 days after injury followed by a gradual

decline by 14 days. Specifically, the levels of survivin in cortical tissue were at 616

257% at 3 days and at 839 339% at 5 days compared to craniotomy controls (Figure 3-

2B). Similar increases of survivin protein in the ipsilateral hippocampus were detected at

3 days and 5 days post injury: 464 196% and 545 102% compared to craniotomy

control, respectively (Figure 3-2C).










I ipsilateral cortex
S00 ipsilateral hippocampus
-0-0
o **
0 500** ** *

E 400 o
0
200



0 -0
100


cr~a y 1 2 3 5 7 14
Contr
Time After Injury (Days)


Figure 3-1: Survivin mRNA induction in rat brain after TBI. Rats were subjected to
craniotomy followed by controlled cortical impact brain injury. Total RNA
was isolated from injured (ipsilateral) cortex (ic) and hippocampus (ih) at
indicated post-injury times. cDNA was synthesized, and quantitative PCR
using survivin primers was performed as described in detail under Materials
and Methods. Data are given as percent of survivin expression over
craniotomy controls; each time point represents mean + SEM of 4
independent measurements in craniotomy control or TBI group. ** p<0.01
versus craniotomy control (one-way ANOVA test with post hoc Bonferroni
analysis).

PCNA Expression After TBI.

For detection of proliferating cell nuclear antigen (PCNA), PVDF membranes

immunostained for survivin were stripped and re-probed using a PCNA-specific

antibody. PCNA (36 kDa protein) was significantly detectable in the ipsilateral cortex

and hippocampus of TBI rats, but only negligible amounts were observed in the

contralateral cortex and hippocampus (Figure 3-3A). The temporal patterns exhibited by

PCNA protein were similar to that of survivin protein. Namely, PCNA expressed in a

time-dependant fashion with a maximum increase at 5 days after injury followed by a












IPsiIatpraI.Cpdpx




Contralateral Cortex




B

Corte%
~ 0 ps~ilteraI


E7oo
WO_

7300-
ODO

TiI

Crawm"o~o~ Id 2d
contrC9
Time.
C

Hippocampus
0 ipsilateraI
N Contrqlateral
E 4w
U
E'ua


U

,1WT


Insat~atral HirPpcamnous


&a17k 17

Contcalateral ippocamDus

I_ --o-I17 l


feny
After Injury (Days)


CrIi&oWrny 14 W 5d 74 i4.
CoT A Iroi
Time After Injury iDai''i


Figure 3-2: Expression of survivin protein after TBI in rats. Brain tissue homogenate
proteins (40 [pg) were separated using SDS-PAGE, immunoblotted with
survivin antibody and visualized as described in detail under Materials and
Methods. A-Representative western blot of survivin (17 kDa protein) in
ipsilateral cortex (ic) and hippocampus (ih), contralateral cortex (cc) and
hippocampus (ch) obtained from injured rats, and from craniotomy control
rats without cortical impact (craniotomy control.). Densitometry analysis
representation of survivin-positive bands in ipsilateral (ic) and contralateral
(cc) cortex (B) and ipsilateral (ih) and contralateral (ch) hippocampus (C)
after TBI is shown as percent of craniotomy control values. Each data point
represents the mean + SEM of 4 to 6 independent experiments. *p<0.05, **
p< 0.001 versus craniotomy control (one-way ANOVA test with post hoc
Bonferroni analysis).











gradual decline by 14 days. The levels of PCNA in ipsilateral cortical tissue were raised

over craniotomy control by 919+ 459% at 3 days, 2263 333% at 5 days, and 1035

356% at 7 days post injury (Figure 3-3B). Similar increases of PCNA protein in

ipsilateral hippocampus were detected at 5 days post injury with a maximum of 1006 +

229% compared to craniotomy controls (Figure 3-3C). No significant increase was found

in the contralateral regions when compared to craniotomy controls (Figure 3-3A).

Co-Expression of Survivin and PCNA Following TBI

To examine spatial co-localization of survivin and PCNA, dual-label

immunohistochemistry was performed on five-day post injury brain tissue sections, when

peak expression of these proteins was observed.

Survivin and PCNA immunoreactivity was found in the ipsilateral cortex (Figure 3-

4A) and ipsilateral hippocampus (Figure 3-4B) consistent with data obtained using

Western blot analyses. Within both regions, focal co-expression patterns of survivin and

PCNA in single cells were detected, which was demonstrated by both separate

fluorescent visualization of individual proteins and by merging the images of dual-stained

slides (Figure 3-4C-E). However, the dual expression of survivin and PCNA occurred

infrequently as survivin and PCNA immunoreactivity could readily be found separately

(Figure 3-4C-E). Approximately 12% of the total number of PCNA-positive cells also

labeled with survivin. The nuclear morphology of dual survivin and PCNA-positive cells

was ambiguous as indicated by DAPI staining (Figure 3-4F). Therefore, DAPI staining

was simply used for cell identification in all subsequent experiments.












Isilateral Cortex




Contralateral Cortex
I


Ipsilaterar Hippocampus


Now- 1-4-36kD


Contralateral HippoDampus


- 38 kDa


U










E
0






C



0

U



0
U


ia4


Cortex
[ ipsilateral
* contralateral


Cranioonry id 2d 3d d 7d 14d
Coimll
Time After Injury (Days)


Craniotmy id 2d Sd 6d 7d 14d
Coftrol
Time After injuryy (Days)


Figure 3-3: Expression of PCNA after TBI in rats. PVDF membranes visualized for
survivin were stripped and re-probed with PCNA antibody as described in
Materials and Methods. Representative western blots showing PCNA (36
kDa) (A) and densitometry analysis of PCNA-positive bands (B, C) are
presented. Experimental conditions, sample size and abbreviations are
identical to those in Fig. 3-2. *p<0.05, ** p< 0.01 versus craniotomy control
(one-way ANOVA test with post hoc Bonferroni analysis). Values are mean
+ SEM with n=6.
































Figure 3-4: Immunohistochemistry of survivin and PCNA. Dual-label fluorescent
immunostaining for survivin (red) and PCNA (green) was performed in the
ipsilateral cortex (A) and hippocampus (B) at 5 day post-injury as described in
detail under Materials and Methods. Survivin is expressed in the cytoplasm
(C, red) while PCNA is expressed in the nucleus (D, green). The white arrow
indicates the typical focal co-expression of survivin and PCNA as shown in
merged survivin and PCNA images (E). PCNA expression was co-incident
with DAPI staining (F, blue, white arrow). Magnification: 200x, scale bar 50
[tm (A and B); 400x, scale bar 20 [tm (C-F).

Survivin and PCNA are Expressed in Astrocytes After TBI

To determine the cell types expressing survivin and PCNA, dual-label

immunohistochemistry for these proteins and GFAP, a marker of astrocytes, was

performed in five-day post injury tissue. In accordance with western blot data, survivin-

positive immunoreactivity was observed in the ipsilateral cortex and hippocampus

proximal to the injury cavity (Figure 3-5A & G, green) but not in the contralateral areas

(Figure 3-5B & H). Survivin was co-localized with GFAP in the cells of injured cortex

and hippocampus, which strongly suggested primary accumulation of survivin in cells of








Cortex Hippocampus









541

U H

Figure 3-5: Co-localization of survivin and GFAP in brain tissue after TBI. Fluorescent
immunohistochemistry for survivin (green) and GFAP (red) was performed in
the ipsilateral and contralateral cortex (A, B) and in the CA1 and dentate
gyms regions of the hippocampus (G, H) at 5 day post-injury as described in
Materials and Methods. The injury has completely destroyed the cortex in G
leaving only the hippocampus in this picture. Survivin was expressed in the
cytoplasm (D, J, green) of GFAP-positive astrocytes (C, I, red) of the
ipsilateral cortex and hippocampus and was found to co-localize to these cells
as shown in merged C/D and I/J images (E, K, respectively, yellow). White
arrows indicate typical survivin-positive astrocytes. Nuclei are shown using
DAPI (F, L, blue). Magnification: 100x, scale bar 50 tm (A, B, G, H); 400x,
scale bar 20 tm (C F, and I L).
astrocytic lineage (Figure 3-5C-E, I-L). It was further observed that survivin was
uniformly distributed in the cytoplasm and processes of astrocytes in both cortex and
hippocampus (Figure 3-5D & J). DAPI staining is shown in Figures 3-5F & L.
Approximately 88% of the total number of GFAP-positive cells also labeled with
surviving.
PCNA-positive immunoreactivity staining was observed in the ipsilateral cortex
(Figure 3-6A, green) and hippocampus (Figure 3-6G, green) of injured brain, while
contralateral cortex and hippocampus exhibited negligible PCNA immunoreactivity









1-invocatn pus


Figure 3-6: Co-localization of PCNA and GFAP in brain tissue after TBI. Dual-label
immunostaining for PCNA (green) and GFAP (red) was performed in the
ipsilateral and contralateral cortex (A, B) and the CA1 and dentate gyms
regions of the hippocampus (G, H) at 5 day post-injury. PCNA is present in
GFAP positive cells of ipsilateral cortex (C, D) and, to a lesser extent
hippocampus (I, J). E and K depict merged C/D and I/J, respectively. White
arrows indicate typical PCNA-positive astrocytes. PCNA expression was co-
incident with DAPI staining (F, L, blue). Magnification: 100x, scale bar 50
tm (A, B, G, H); 400x, scale bar 20 tm (C F, and I L).

(Figure 3-6B & H). PCNA (Figures 3-6C & I) was partially co-localized with GFAP

(Figures 3-6D & J, red) in both regions, and was characteristically distributed in the

nucleus of the cells in both cortex and hippocampus (Figures 3-6E & K). DAPI staining

is shown in Figures 3-6F & L.

Taken together, dual-label immunohistochemistry data provides evidence that both

survivin and PCNA can be detected in GFAP-positive astrocytes following traumatic

insult. Since survivin and PCNA immunoreactivity was not exclusively localized in


GFAP-positive cells, other cell types must also express survivin.


Carter









Survivin Co-localized


I
NeuN PCNA Co-localized DAPI

























Contralateral Ipsilateral


Figure 3-7: A sub-set of NeuN-positive neurons express survivin and PCNA after TBI.
Dual-label fluorescent immunohistochemistry for survivin (green) and NeuN
(red) in the ipsilateral cortex (A & B) and the CA1 pyramidal layer of the
contralateral hippocampus (E & F) was performed as described in Materials
and Methods. Survivin is expressed in the cytoplasm and, to a limited extent,
in the processes of NeuN-positive neurons (merged images C & G). Dual
staining for PCNA (green) and NeuN (red) is shown in the ipsilateral cortex (I
& J) and the CA1 pyramidal layer of the ipsilateral hippocampus (M & N).


NeuN


DAPI









The nuclei are shown using DAPI staining (D & H, blue). PCNA is expressed
in the nucleus of NeuN-positive neurons (merged images K & O). PCNA
expression was co-incident with DAPI staining in these examples (L & P,
blue). White arrows indicate focal co-localization of survivin/NeuN and
PCNA/NeuN. Survivin/NeuN co-localization of survivin (green) and NeuN
(red) was seen only in TBI rats as opposed to either hemisphere of craniotomy
control (Q & R). (Magnification of A-P = 400x, scale bar = 20 atm;
magnification of Q & R 50x, scale bar = 100,000 atm).

Survivin and PCNA are Expressed in a Sub-Set of Neurons After TBI

As can be seen in Figure 3-7, survivin and PCNA were each co-expressed with NeuN, a

marker of mature neurons. NeuN-positive cells were found to express survivin in the

ipsilateral cortex distal to the injury cavity (Figure 3-7A-D) and in the contralateral

hippocampus (Figure 3-7E-H). It should be noted, however, that NeuN-positive cells that

also expressed survivin occurred infrequently. For example, the number of dual

survivin/NeuN positive cells was estimated at 0.1% to 1.5% of the total number of NeuN-

positive cells in these regions. Survivin immunoreactivity was negligible in either

hemisphere of craniotomy control brains (Figures 3-7Q & R). No co-localization of

survivin and NeuN was observed in ipsilateral hippocampus (data not shown). As can be

seen in Figures 3-7B & F, survivin was predominantly localized to the cytoplasm and

axons of NeuN-positive neurons. DAPI staining is shown in Figures 3-7D & H.

PCNA-positive neurons were found in the ipsilateral cortex (Figures 3-7I-L) and

hippocampus after TBI (Figure 3-7M-P), whereas craniotomy control tissue exhibited

only trace amounts of PCNA (data not shown). Similar to the survivin/NeuN co-

localization data, dual PCNA/ NeuN immunostaining was a rare event accounting for

approximately 4% of the total number of NeuN positive cells. PCNA was distributed in

the nuclei of these neurons (Figures 3-7K & O) although the nuclear morphology of these

cells was not clearly resolved by DAPI staining (Figures 3-7L & P).
























Figure 3-8: Survivin expression is absent in oligodendrocytes and microglia following
TBI in rats. Dual-label fluorescent immunohistochemistry for survivin
(green), CNPase (red, A) and OX42 (red, B) in the ipsilateral cortex and
hippocampus was performed as described in Materials and Methods.
Negligible co-localization was seen with survivin, CNPase and OX42 in the
ipsilateral cortex (A, B respectively) and hippocampus (data not shown).
Higher magnification photomicrographs (inset, A and B) show survivin-
positive cells (white arrowheads) surrounded by oligodendrocytes (white
arrows, A) and microglia (white arrows, B) that do not show co-localization.
(Magnification of A and B = 400x, scale bar = 20 tm).

Survivin is Not Expressed in Microglia and Oligodendrocytes

To further determine the neural cell types expressing survivin, dual-label

immunohistochemistry for survivin, OX42, a marker of microglia, and CNPase, a marker

for oligodendrocytes, was performed in five-day post injury tissue sections. No co-

localization with survivin and either CNPase (Figure 3-8A) or OX-42 (Figure 3-8B) is

observed following traumatic brain injury. In addition, attempts were made to localize

survivin with the neuronal progenitor cell markers nestin, doublecortin, a-internexin and

P-III-tubulin. However, these antibodies did not prove to be of acceptable quality to use

in western blot and IHC analyses in this model leaving proliferating progenitors

undetected.


/ Survivin


/ SU( B~









Discussion of Chapter 3

Traumatic brain injury (TBI) initiates various biochemical cascades that induce

neural tissue injury and cell death. To counteract these cascades, several proteins

expressed in neural cells after TBI are directed to resist cell death and promote recovery

in the injured CNS (Ridet et al., 1997; Chen and Swanson, 2003). Survivin is a multi-

functional protein that inhibits apoptosis and is also required for the proper completion of

mitosis. Anti-apoptotic and pro-mitogenic roles for survivin have been documented in

proliferating cells of neural origin in vitro, such as in neuroblastoma and glioma cells

(LaCasse et al., 1998; Tamm et al., 1998; Deveraux and Reed, 1999; Conway et al., 2000;

Shin et al., 2001; Sasaki et al., 2002). However, no studies have investigated the

potential role of survivin in the adult brain after TBI, when a sub-population of CNS cells

may initiate a cell cycle-related process in response to injury.

These data demonstrate the induction of survivin expression in rat brain subjected

to TBI. The expression of survivin was time-dependent, cell-specific and was present in

astrocytes and, to a much lesser extent, in neurons in ipsilateral cortex and hippocampus.

Induction of survivin in these cells was accompanied by occasional expression of PCNA,

a cell cycle protein involved in mitotic G1/S progression. These data are the first to show

that survivin mRNA and protein are significantly up-regulated after TBI in rats. PCNA

expression after TBI has been described previously (Miyake et al., 1992; Chen et al.,

2003), suggesting its role in mechanisms of brain recovery after injury. The concurrent

up-regulation of survivin with a similar temporal profile as PCNA shown herein further

suggests that survivin may play a role in cellular proliferation after TBI.

Brain injury evoked the expression of survivin and PCNA in a time-dependent

manner (Figures 3-2 & 3-3). Western blot analysis revealed maximal co-expression of









both survivin and PCNA at five days post injury. Immunohistochemistry demonstrated

co-localization of these proteins (Figure 3-4), although most cells were labeled separately

with PCNA and survivin. In fact, only 12 % of the total number of PCNA-positive cells

were also survivin positive. It has been reported that PCNA is expressed predominantly

in G1/S (Bravo et al., 1987), while survivin is found at the G2/M phase of the cell cycle

(Bravo et al., 1987; Otaki et al., 2000). Hence, a lack of strict co-localization of survivin

and PCNA in this study may be explained by their expression at different points in the

cell cycle. To determine if the differing expression patterns of survivin and PCNA

contributed to lower incidence of co-localization, other cell cycle proteins were

investigated including cdk4 (G1), cyclin B (G2), cyclin D (G1) and AIM-1 (M). Only

PCNA provided clear results in both western blot and IHC analyses.

Survivin-positive and PCNA-positive astrocytes were observed in the proximal

area of the injury and in the ipsilateral hippocampus. Proliferation of astrocytes is well

documented after TBI as shown by cell labeling with BrdU as well as expression of

PCNA (Latov et al., 1979; Dunn-Meynell and Levin, 1997; Carbonell and Grady, 1999;

Norton, 1999; Csuka et al., 2000; Kernie et al., 2001; Chen et al., 2003). Because

survivin and PCNA were expressed in astrocytes following TBI (Figures 3-5 & 3-6), it is

possible that survivin plays an important role linking astrocyte survival and proliferation

after traumatic insult. Astrocyte proliferation has been implicated in the formation of the

glial scar observed after injury (Latov et al., 1979) and creates a non-permissive

environment for repair (Sykova et al., 1999). However, glial proliferation may also

enhance neuronal survival (Smith et al., 2001; Wei et al., 2001).









Of particular interest is a sub-set of NeuN-positive neurons found to express

survivin only after TBI (Figure 3-7). These cells were much less abundant than survivin-

positive astrocytes and their functional significance is currently unknown. However, both

neurons and astrocytes have been documented previously to express cell cycle proteins

after various insults such as exposure to P-amyloid activated microglia (Wu et al., 2000),

TBI (Kaya et al., 1999a; Kaya et al., 1999b), chlorin e6 toxicity, (Magavi et al., 2000) or

as a consequence of Alzheimer's Disease (Yang et al., 2001). The ramifications of cell

cycle protein expression in mature neurons is still controversial and may be a marker of

cell death rather than cell proliferation (Herrup and Busser, 1995; Li et al., 1997; Kaya et

al., 1999a; Kaya et al., 1999b; Wu et al., 2000; Yang et al., 2001). These papers

underscore the significant controversy that exists regarding the function of cell cycle

proteins such as PCNA in neurons after different types of injury.

It should be noted that dual staining of survivin and PCNA could not be directly

attributed to a specific cell type due to the technical difficulties of triple labeling

antibody-based IHC. Therefore, other cell types, such as endothelial (Conway et al 2003),

inflammatory cells (Hill-Felberg et al., 1999) or neural progenitor cells (Ignatova et al.,

2002), may also contribute to survivin and PCNA expression after TBI. The appearance

of survivin and PCNA separately in neurons (NeuN-positive) and astrocytes (GFAP-

positive) along with co-localization of survivin with PCNA in the same cells provide

correlative data to suggest an activation of cell cycle-like program in astrocytes and

possibly in a small subtype of neurons after TBI. In these experiments, survivin co-

localization with PCNA does suggest that survivin may be associated with a pro-mitotic

process. In an attempt to clarify these protein's roles after TBI, the nuclear morphology









of survivin-positive cells was analyzed to define the apoptotic or mitotic architecture of

nuclei. DAPI staining proved too ambiguous in identifying apoptotic versus mitotic

phenotypes likely due to the thickness of the brain sections (40 atm). Further studies using

direct markers of mitosis such as BrdU incorporation as well as simultaneous labeling

with cell death related proteins is required to delineate anti-apoptotic and pro-mitotic

activities of survivin and PCNA in these cells.

To summarize, an induction of survivin was found in rat brain cortex and

hippocampus after TBI in a time-dependent fashion. Expression of survivin occurred

predominantly in astrocytes and a sub-set of neurons but not in oligodendrocytes or

microglia, and was occasionally accompanied by expression of PCNA. However,

survivin expression is only found in 12% of PCNA positive cells, which suggests that the

primary role of survivin after traumatic brain injury is not related to the cell cycle but

rather to apoptosis inhibition. Thus, the next specific aim of this study was to examine the

link between survivin, active caspase-3 and downstream DNA fragmentation following

traumatic brain injury in rats.














CHAPTER 4
SURVIVIN AND APOPTOSIS INHIBITION FOLLOWING TRAUMATIC BRAIN
INJURY

Caspase-3 is Activated in the Same Brain Regions as Survivin Following TBI

To determine the temporal and regional profile of caspase-3 activation, western blot

analysis was performed on cortical and hippocampal TBI samples. Active caspase-3 (19

kDa protein) was readily detectable in the ipsilateral cortex and hippocampus of rats

subjected to TBI (Figure 4-1A). Caspase-3 activation occurred in a time-dependant

manner in the ipsilateral cortex and hippocampus with prominent activation occurring

between five and fourteen days post-injury, with peak accumulation occurring at seven

days post-injury. In the ipsilateral cortex, significant increases in active caspase-3 levels

reached 3468 1088% at five days, 4019 1291% at seven days and 2984 1058%

fourteen days post-injury, compared with craniotomy controls. Similar increases in

caspase-3 activation were detected in the ipsilateral hippocampus with increases of 671 +

257% at five days, 2662 738% at seven days and 1487 405% at fourteen days post-

injury, compared with craniotomy controls (Figure 4-1B).

Survivin Expression Correlates with Decreased TUNEL Labeling but not Active
Caspase-3 Expression.

Immunohistochemistry (IHC) was performed on brain sections at five days post

injury to investigate the expression of active caspase-3 and TUNEL labeling at peak

survivin expression (Johnson et al., 2004). IHC revealed moderate co-localization of

survivin with active caspase-3 and TUNEL in the ipsilateral cortex (Figure 4-2A,F) and











Ipsilateral Cortex

s ^* a e I


[psilateral Hippocampus


-- .IO -a- 19 kODa


6000 -
Sipsilateral cortex
00 I Ig ipslateral hippocampus


** *


cranitomy id 2d 3d 5d 7d 14d
control
Time After Injury (Days)

Figure 4-1: Caspase-3 Activation in rat brain after traumatic brain injury. Rats were
subjected to craniotomy followed by controlled cortical impact brain injury.
Brain tissue homogenate proteins (40 tg) were separated using SDS-PAGE,
immunoblotted with antibody specific for active caspase-3 and visualized as
described in detail under Materials and Methods. Representative western
blots of active caspase-3 (19 kDa) in ipsilateral cortex and hippocampus
obtained from injured rats and from craniotomy control rats without cortical
impact revealed the accumulation of active caspase-3 after TBI (A).
Densitometry analysis of the active caspase-3 bands in ipsilateral cortex and
hippocampus after TBI show significant increases in a time dependent manner
(B). Data are given as percent of the active fragment of caspase-3 related to
craniotomy controls; each time point represents mean + SEM of 6
independent measurements in craniotomy control or TBI group. p<0.05, **
p<0.01 versus craniotomy control (one-way ANOVA test with post hoc
Dunnet analysis).

ipsilateral hippocampus (Figure 4-2B,G) of injured animals. These data are summarized

in Table 1 (pg 45). Survivin was found primarily in the cytoplasm and to a lesser extent

in the nucleus of the ipsilateral cortex (Figure 4-2C,H). A similar pattern was seen in the


0'


I








Survivin/ Active Caspase-3 Survivin/ TUNEL



CI
_tM
0


U.





Figure 4-2: Co-expression of survivin and apoptosis markers following TBI in rats.
Dual-label fluorescent immunostaining for survivin (red) and active caspase-3
(green, A-E) or TUNEL (green, F-J) was performed in the ipsilateral cortex
(A, F) and hippocampus (B, G) at 5 day post-injury as described in detail
under Materials and Methods. Survivin is expressed in both the cytoplasm
and in the nucleus (C & H, red) while active caspase-3 (D, green) and TUNEL
(I, green) label predominantly the nucleus. The white arrow indicates the
typical focal co-localization of survivin and active caspase-3 (E) and TUNEL
(J) as shown in the merged images. Magnification: 200x, scale bar 50 [m (A,
B, F, G); 200x, scale bar 10 [m (C-E, H-J).
ipsilateral hippocampus (data not shown). Active caspase-3 (Figure 4-2D) and TUNEL
(Figure 4-21) labeling were both found in the nucleus in both regions. Survivin co-
localization with active caspase-3 and TUNEL is shown at high magnification in Figure
4-2E and Figure 4-2J, respectively. Confocal microscopy was used to assure single cell
co-localization of survivin, active caspase-3 and TUNEL (data not shown).
Quantitative analysis revealed no significant difference in the accumulation of
active caspase-3 in survivin-positive cells compared to survivin-negative cells at five
days post injury in either the cortex or hippocampus (Figure 4-3A,B). However,
significantly higher percentage of TUNEL labeling was observed in survivin-negative











Cortex

p<0.01
I-------i


A soo
A 100-
I! 80-
m 60-

S40-


P
03
C 0







20-
60
1^30
s J


Hippocampus
p<0.01


Survivin (+) Survivin (-)


Hippocampus








posIUve negauve
TUNEL


Active Caspase-3 Positive
o TUNEL Positive
[ TUNEL Negative

Figure 4-3: Survivin expression decreases the accumulation of TUNEL but not active
caspase-3. Cells were quantified and visualized as described in detail under
Materials and Methods. The percentage of cells expressing active caspase-3
and labeling with TUNEL was compared in two cell populations, survivin-
positive and survivin-negative, in the ipsilateral cortex (A) and hippocampus
(B). No differences were found in the percentage of survivin-positive and
survivin-negative cells expressing active caspase-3 in either the cortex (A,
black bars) or hippocampus (B, black bars). Significantly more survivin-
negative cells were TUNEL-positive compared to survivin-positive cells in
both the cortex (p<0.01, A, white bars) and hippocampus (p<0.001, B, white
bars). No differences were found in the percentage of TUNEL-positive (C,
white bars) or TUNEL-negative cells (C, gray bars) that also labeled with
active caspase-3. Each bar represents mean + SEM of 4 (A, cortex) or 3 (B,
hippocampus) independent measurements using an unpaired Student t-test for
statistical analysis.


Survivin (+) Survivin (-)


C D
c 80


a
40

I 20

I 0


Cortex


1
potive negL ve
TUNEL









cells as compared to survivin-positive cells in both regions (p<0.01) (Figure 4-3A,B,

Table 1A). At seven days post injury, there was no significant difference in the

accumulation of both active caspase-3 and TUNEL labeling in survivin-positive cells

compared to survivin-negative cells in either the cortex or hippocampus (Table 1B).

To verify that caspase-3 activation does not necessarily result in irreversible cell

death, possibly due to survivin inhibition, quantification data was gathered on cells that

accumulate active caspase-3 and are TUNEL label-positive and compared to active

caspase-3-positive cells that do not label with TUNEL. Indeed, we found that the number

of dual-labeled active caspase-3 and TUNEL cells was not statistically different from

those cells labeled with active caspase-3 only (Figure 4-3C). This finding further

supports the notion that several counteractive factors, including survivin, may inhibit

active caspase-3 and diminish cell death following TBI.

Astrocytes and Neurons Demonstrate Cell Specific Differences in Active Caspase-3
and TUNEL Labeling

To determine cell types that express active caspase-3 or labeled with TUNEL, dual

immunohistochemistry of these apoptosis markers was performed with GFAP, a marker

of astrocytes, and NeuN, a marker of mature neurons. Co-localization of GFAP was

observed with both active caspase-3 and TUNEL labeling in the ipsilateral cortex (Figure

4-4A,F respectively) and ipsilateral hippocampus (Figure 4-4B,G respectively). Higher

magnification photomicrographs show a typical astrocyte expressing active caspase-3

(Figure 4-4C-E) or labeling with TUNEL (Figure 4-4H-J).

NeuN-positive cells exhibited modest accumulation of active caspase-3 and

considerable labeling with TUNEL in the ipsilateral cortex (Figure 4-5A,F) and

ipsilateral hippocampus (Figure 4-5B,G). These data are summarized in Table 1.











Table 1 Cell Quantification Data for Immunohistochemistry Labeling Pairs This table
summarizes the results from all cell count experiments investigating survivin,
cell type and the apoptosis markers. The numbers given are percentages
calculated as described in the Materials and Methods. Each percentage
represents the mean + SEM of 4 (cortex) or 3 (hippocampus) independent
measurements at either five days post-injury (A) or seven days post-injury
(B). ** p<0.01 versus survivin-negative cells, ## p<0.01, ### p<0.001 versus
NeuN-positive cells, $$ p<0.01 versus TUNEL-negative cells (unpaired
Student t-test).
A Survivin (+) Stuvivin (-) GFAP (+) NeuN (+) TUNEL (+) TUNEL (-)
Active coitex 37 2 26 6 50 7" 7 1 51 6 49 6
Caspase-3
(+) Hippocanipus 21 5 22 3 29 4 15 2 45 11 55 11

EL otex 252** 49 5 11 2 46 2
TUNEL
() Hippocanpus 30 4** 55 3 19 0.2" 60 3

B Stuvivin (+) Su-vivin (-) GFAP (+) NeuN (+) TUNEL (+) TUNEL (-)
Active Cortex 36 2 39 11 36 3" 11 1 484 524
Caspase-3
(+) Hippocanpus 37 2 22 6 31 3" 15 3 625" 385
205 2510 19+1"" 74 5
TUNEL Cortex
+) Hippocmnpus 20 3 31 10 28 7* 80 2


Specifically, both active caspase-3 (Figure 4-5D) and TUNEL (Figure 4-51) were present

in neuronal nuclei (Figure 4-5C,H), which was indicated by co-localization with NeuN

(Figure 4-5E, J). Confocal microscopy was used to assure single cell co-localization of

NeuN and GFAP with active caspase-3 and TUNEL (data not shown).

Quantitative analysis revealed a substantially different expression profile of active

caspase-3 and TUNEL in neurons and astrocytes. A significantly higher number of

astrocytes accumulated active caspase-3 as compared to neurons in the cortex, but not the

hippocampus (p<0.001) (Table 1). Conversely, a significantly greater number of neurons

were labeled with TUNEL compared to astrocytes in both the cortex and hippocampus

(p<0.001) (Figure 4-6, Table 1A). A similar expression profile of active caspase-3 and









GFAP/ Active CasDase-3


GFAP/ TUNNEL


Figure 4-4: Astrocytes express active caspase-3 and label with TUNEL following TBI in
rats. Dual-label fluorescent immunostaining for GFAP (red) and active
caspase-3 (green, A-E) or TUNEL (green, F-J) was performed in the
ipsilateral cortex (A, F) and hippocampus (B, G) at 5 day post-injury as
described in detail under Materials and Methods. GFAP is expressed in the
cytoplasm (C & H, red) while active caspase-3 (D, green) and TUNEL (I,
green) are expressed in the nucleus. The white arrow indicates the typical
focal co-expression of GFAP and active caspase-3 (E) and TUNEL (J) as
shown in the merged images. Magnification: 200x, scale bar 50 tm (A, B, F,
G); 200x, scale bar 10 tm (C-E, H-J).

TUNEL was observed in astrocytes and neurons seven days post injury (Table 1B).

Discussion of Chapter 4

Caspase-3 activation is a prominent feature of apoptosis and its role in DNA

fragmentation after TBI has been well documented (Nicholson et al., 1995; Tewari et al.,

1995; Pike et al., 1998; Tang and Kidd, 1998; Wolf et al., 1999; Beer et al., 2000; Buki et

al., 2000; Clark et al., 2000). Survivin is an inhibitor of apoptosis protein (IAP), which

can inhibit active caspase-3 and thereby moderate cell death in various tissues, including

CNS (Shankar et al., 2001; Sasaki et al., 2002; Van Haren et al., 2004). However, no









NeuN/ Active Caspase-3 NeuN/ TUNEL




0




U U
E






Figure 4-5: Neurons express active caspase-3 and label with TUNEL following TBI in
rats. Dual fluorescent immunostaining for NeuN (red) and active caspase-3
(green, A-E) or TUNEL (green, F-J) was performed in the ipsilateral cortex
(A, F) and hippocampus (B, G) at 5 day post-injury as described in detail
under Materials and Methods. NeuN is expressed primarily in the nucleus but
also in the cytoplasm (C & H, red) while active caspase-3 (D, green) and
TUNEL (I, green) are found in the nucleus. The white arrow indicates the
typical focal co-localization of NeuN and active caspase-3 (E) and TUNEL (J)
as shown in the merged images. Magnification: 200x, scale bar 50 tm (A, B,
F, G); 200x, scale bar 10 tm (C-E, H-J).

studies have investigated the potential anti-apoptotic role of survivin in the adult brain

after TBI.

These data characterize the relationship between survivin expression and two

apoptosis events: the accumulation of active caspase-3 and downstream DNA

fragmentation (as shown by TUNEL labeling) in rat brain subjected to TBI. The use of

TUNEL labeling in conjunction with active caspase-3 is considered a reliable tool to

assess apoptosis progression (Lei et al., 2004; Marciano et al., 2004; Nakase et al., 2004).

The appearance of active caspase-3 was time-dependent and region specific (Figure 4-1)










loo -Cortex Hippocampus

S80 p 'p<0.001
S70 p z 60 -
O
0
+- 50


30 -
13 *



10
Neurons Astrocytes Neurons Astrocytes
t I ,
TUNEL ) UNEL(+)
Figure 4-6: TUNEL labeling is cell specific following TBI in rats. Cells were quantified
and visualized as described in detail under Materials and Methods. The
percentage of cells labeling with TUNEL was compared in astrocytes (black
bars) and neurons (gray bars) in the ipsilateral cortex and hippocampus.
Significantly more TUNEL labeling was seen in neurons than in astrocytes in
both the cortex and hippocampus (p<0.001). Each bar represents mean +
SEM of 4 (cortex) or 3 (hippocampus) independent measurements using an
unpaired Student t-test for statistical analysis.

with a pattern similar to survivin expression after TBI which suggests that survivin may

inhibit caspase-3 activity to diminish the deleterious consequences of proteolysis after

TBI (Tamm et al., 1998; Kobayashi et al., 1999; O'Connor et al., 2000b; Shin et al.,

2001). Other IAPs are up-regulated in concert with activation of caspases after brain

injury (Keane et al., 2001). In addition, survivin expression is up-regulated by the pro-

survival PI3-kinase/Akt pathway which is activated after TBI (Kitagawa et al., 1999; Xia

et al., 2002b; Kim et al., 2004).

Survivin-positive cells expressed active caspase-3 and were labeled with TUNEL

(Figure 4-2) though survivin-positive cells showed no significant difference in

accumulation of active caspase-3 compared to survivin-negative cells (Figure 4-3).



















Survivin 4

Active
Caspase-3

Ae



DNA Cleavage (TUNEL)

Figure 4-7: Putative mechanism of apoptosis inhibition by survivin following TBI.
Traumatic brain injury induces activation of upstream caspases-8 and 9 that
can process pro-caspase-3 to its active form. Once activated, caspase-3 can
cleave several intracellular substrates and activate restrictases that may lead to
DNA fragmentation. Concomitant survivin expression is up-regulated in
response to the same TBI signals. Survivin has the ability to inhibit the
activity of active caspase-3, which results in the attenuation of DNA
fragmentation.

These data are in accordance with the ability of survivin (Tamm et al., 1998) and other

IAPs (Shankar et al., 2001; Maier et al., 2002) to inhibit the activity but not the activation

of caspase-3. In contrast, fewer survivin-positive cells exhibited DNA fragmentation

(TUNEL labeling) compared to survivin-negative cells at five days post injury (Figure 4-

3). These data suggest that survivin expression may attenuate the apoptotic cascade by

inhibiting the cleavage of caspase-3 specific substrates that result in DNA fragmentation.

Furthermore, the finding that active caspase-3 accumulation led to positive TUNEL


Traumatic --- --
Brain
Injury classe-9









labeling in 51% of all active caspase-3-positive cells (Figure 4-3) is consistent with the

observation that endogenous factors, including survivin, may inhibit active caspase-3 and

attenuate DNA fragmentation following TBI. In an attempt to more directly investigate

whether survivin could inhibit active caspase-3 activity, survivin was co-localized with

markers of caspase-3 activity including the cleaved species of PARP, DFF45/iCAD and

a-II-spectrin (120 kDa). Unfortunately, these markers proved to be of unacceptable

quality to use in western blot and IHC analyses in this model. Therefore, it remains

unknown whether survivin correlates with a decrease in active caspase-3 activity as

measured by caspase-3-specific breakdown products. The ability of survivin to inhibit

DNA fragmentation has been shown in gastric cancer cells (Lu et al., 1998). In addition,

survivin antisense treatment increases DNA fragmentation in neuroblastoma and

oligodendroglioma (Shankar et al., 2001). Taken together, these findings suggest that

survivin likely inhibits the proteolytic activity of caspase-3 after TBI to attenuate DNA

cleavage.

The labeling patterns of active caspase-3 and TUNEL in astrocytes and neurons

were investigated. Data described in Chapter 3 showed that a large majority of astrocytes

but few neurons express survivin after TBI. These data show that both astrocytes and

neurons expressed active caspase-3 and label with TUNEL post-injury but the prevalence

of this labeling was drastically different (Figures 4-4, 4-5). A higher percentage of

astrocytes accumulate active caspase-3 but fewer astrocytes label with TUNEL compared

to neurons (Figure 4-6). It is unclear at present why these experiments revealed few

neurons labeling with active caspase-3 (Table 1). Some active caspase-3-positive

neurons may not have been detected due to caspase-3 mediated loss ofNeuN antigenicity









(Unal-Cevik et al., 2004). Other groups have found prominent caspase-3 activation in

apoptotic neurons after TBI (Beer et al., 2000). In addition, caspase-independent necrosis

may also be a major contributor of neuronal cell death after TBI (Newcomb et al., 1999;

Wennersten et al., 2003).

Cell specific survivin expression may contribute to the lower incidence of TUNEL

labeling in astrocytes as compared to neurons (Table 1). Cell type specific expression of

IAPs after TBI has been previously shown. For example, XIAP is expressed primarily by

neurons (Lotocki et al., 2003) and a subset of oligodendrocytes (Keane et al., 2001)

following brain injury. NAIP is expressed in neurons after ischemia (Xu et al., 1997) and

TBI (Hutchison et al., 2001). Lastly, RIAP-2 is abundantly expressed in neurons as

opposed to astrocytes after kainic acid treatment in rats (Belluardo et al., 2002). Figure

4-7 shows a putative mechanism for apoptosis inhibition by survivin. Following

upstream caspase activation and cleavage of procaspase-3 to active caspase-3, survivin

acts to attenuate the apoptotic cascade and finally DNA cleavage and cell death by

inhibiting caspase-3 activity.

Taken together, data from Chapter 4 demonstrate that the activation of caspase-3 in

the rat brain after TBI follows the same timeframe, regions and cell type expression

patterns as survivin. Quantitative correlative analysis reveals that significantly fewer

cells expressing survivin undergo final stage of apoptosis and cell death. These data

suggest that DNA cleavage may be attenuated via inhibition of active caspase-3 by

survivin after traumatic brain injury in a cell-specific fashion. Namely, astrocytes have

significantly lower TUNEL labeling than neurons suggesting a more robust anti-apoptotic

role for survivin in astrocytes. Collectively, these results suggest that survivin plays a







52


role in diminishing apoptosis and DNA fragmentation following traumatic brain injury in

rats.














CHAPTER 5
CONCLUSIONS AND FUTURE DIRECTIONS

Conclusions

Traumatic brain injury (TBI) remains a major health care and economic issue in the

United States to this date. Despite continued education and improved first responder

care, the Centers for Disease Control estimate that there are more than 5.3 million

Americans living with disabilities from TBI with another 1.5 million new TBIs sustained

in the U.S each year. Cognitive and memory deficits resulting from TBI are especially

difficult to cure and no effective treatment options are available.

TBI is a complex injury that induces both apoptotic cell death and neural cell

proliferation. Apoptosis is opposed by up-regulation of inhibitor of apoptosis proteins

(IAPs) that attenuate apoptosis through direct inhibition of active caspases. Neural cell

proliferation may contribute to neural tissue healing by repopulating damaged regions

with new, functional cells but may also prevent normal recovery of the injured brain by

forming impermissible barriers for axonal growth.

The recently discovered protein, survivin, may play a significant role in these

processes following TBI. Survivin is a protein that is both integral for mitosis and is an

IAP with anti-apoptotic properties. Though not normally found in quiescent adult tissues,

survivin can be induced in certain mature, non-neural cells after CNS insult or cancer

transformation to resist apoptosis. Furthermore, brain injury can induce stem cell

proliferation, a process that likely requires the survivin protein for proper completion.









However, no study to date had investigated the temporal, regional and neural cell

expression patterns of this unique protein following TBI.

Chapter 3 of this dissertation critically examined the transcriptional and

translational expression of survivin following TBI in rats. This dissertation is the first to

demonstrate that survivin mRNA and protein are expressed in neural cells following TBI

and that this expression is cell type specific. QPCR analysis confirmed elevated levels of

survivin following TBI that peaked at five days post-injury in the ipsilateral cortex and

hippocampus. Immunoblot analysis confirmed survivin translation with peak expression

at five days post-injury in both regions. Survivin localization at this time point was

observed in approximately 88% of the astrocytes in the ipsilateral cortex and

hippocampus. Survivin expression was also observed in a much smaller sub-set of

neurons, where no more than 1.5% of neurons expressed survivin. Survivin expression

was not observed in microglia and oligodendrocytes. Like other IAP proteins, expression

of survivin appears to be cell type specific following TBI. In contrast to the IAPs XIAP,

NAIP, cIAP-1 and cIAP-2 which are predominantly expressed in neurons following brain

insult, survivin is expressed primarily in astrocytes. Attempts were made to localize

survivin with the neuronal progenitor cell markers nestin, doublecortin, a-internexin and

P-III-tubulin. However, these antibodies did not prove to be of acceptable quality to use

in western blot and IHC analyses in this model leaving proliferating progenitors

undetected.

Survivin has two biochemically distinct functions, that of apoptosis inhibition and

to properly separate DNA during mitosis. To reveal the relationship between survivin

and cellular proliferation, several cell cycle proteins were investigated including cdk4,









cyclin B, cyclin D, AIM-1 and PCNA. Only PCNA provided clear results in both

western blot and IHC analyses. Therefore, the regional, temporal and cell specific

protein expression of the previously characterized cell cycle protein, PCNA and its co-

localization with survivin was investigated. PCNA protein accumulated in the ipsilateral

cortex and hippocampus peaking at five days post-injury. In addition, astrocytes and a

small subset of neurons expressed PCNA in a pattern similar to survivin. However, only

12% of PCNA-positive cells also expressed survivin. The cellular identity of these dual-

labeled cells remains unknown due to difficulties with triple-label IHC. Therefore, the

ramifications of cell cycle protein expression in individual cell types remains unclear.

However, these data seem to indicate that the primary function of survivin following TBI

is not cellular proliferation. A more complete picture of survivin's role in cellular

proliferation following brain insult would require co-localization with other cell cycle

related proteins. Thus far, additional investigations with proteins such as AIM-1, cdk4,

cyclin D and cyclin B have been inconclusive. Further studies will be required to

determine the extent to which survivin acts as an indicator of cellular proliferation and

the cell types that may be actively proliferating following TBI.

Chapter 4 revealed the relationship between survivin and apoptosis inhibition by

investigating the accumulation of active caspase-3, the main executioner caspase in

apoptosis, and the appearance of DNA fragmentation (TUNEL) following TBI.

Immunoblot studies revealed that active caspase-3 did accumulate following TBI with

significant accumulation at five, seven and fourteen days post-injury in the ipsilateral

cortex and at seven and fourteen days post-injury in the ipsilateral hippocampus.

Because survivin and active caspase-3 levels peaked on different post-injury days, IHC









was performed at the peak expression times of five days (survivin) and seven days (active

caspase-3) post-injury. IHC analysis revealed that survivin and both active caspase-3 and

TUNEL did co-localize to the same cells at these time points. Attempts were made to co-

localize survivin with markers of caspase-3 activity to correlate the presence of survivin

to an absence in caspase-3 specific breakdown products. Antibodies for the caspase-3-

specific cleaved species ofPARP, DFF45/iCAD and a-II-spectrin (120 kDa) proved to be

of unacceptable quality to use in western blot and IHC analyses in this model leaving

markers of caspase-3 activity undetected.

Quantitative analysis revealed no significant difference in the accumulation of

active caspase-3 in survivin-positive cells compared to survivin-negative cells at five

days post injury in either the cortex (37 2% v. 26 6%) or hippocampus (21 5% v. 22

3%). Conversely, a significantly higher percentage of TUNEL labeling was observed

in survivin-negative cells as compared to survivin-positive cells in ipsilateral cortex (49 +

5% v. 25 2%) and hippocampus (55 3% v. 30 4%). These data are consistent with

my hypothesis that survivin expression may attenuate the apoptotic cascade by inhibiting

the cleavage of caspase-3 substrates. However, by seven days post injury, there was no

significant difference in the accumulation of either active caspase-3 or TUNEL labeling

in survivin-positive cells compared to survivin-negative cells. It is unclear why this

pattern was observed. In particular, the sharp decrease in survivin-negative, TUNEL-

positive cells from five days to seven days post-injury was unexpected. This decrease in

DNA fragmentation may be explained by death and removal of TUNEL-positive cells

between five and seven days as evidenced by progressive increases in cavity size seen

after brain injury (data not shown). The turnover from healthy to TUNEL-positive in the









survivin-negative cell population may not be steady, as indicated by the biphasic

fluctuations of cell death indicators after brain injury (Holmin and Mathiesen, 1995;

Domanska-Janik, 1996; Kampfl et al., 1996; Baskaya et al., 1997). Again because of the

difficulty of triple-label IHC, the cell type of survivin dual-labeled cells could not be

identified.

Because survivin is expressed in astrocytes and neurons, the accumulation of active

caspase-3 and TUNEL-labeling in these cells was investigated. Both cell types

accumulated active caspase-3 and labeled with TUNEL following TBI. Quantification

studies revealed a significantly greater percentage of neurons labeled with TUNEL

compared to astrocytes in both the cortex and hippocampus at both five and seven days

post injury. Taken with the observation that a majority of astrocytes express survivin

after TBI, it is possible that survivin expression contributes to the low DNA

fragmentation observed in these cells despite prominent caspase-3 activation. The

opposite appears to be the case with neurons. Few neurons express survivin following

TBI and many of these cells show prominent DNA fragmentation. Neurons are

particularly vulnerable to apoptosis signals following brain injury and absence of survivin

expression may contribute to this vulnerability. Therefore the hypothesis does not seem

to support an anti-apoptosis role for survivin in the majority of neurons following TBI.

Collectively, these data indicate that survivin, a developmental protein normally

absent in adult tissues, is up-regulated at both the transcriptional and translational level

following traumatic brain injury. Survivin is expressed predominantly by astrocytes and

a small sub-set of neurons. In addition, this is the first study to provide indirect evidence

that survivin functions as both an apoptosis inhibitor and as a cell cycle protein following









brain trauma. These data suggest that further studies are necessary to show the overall

ramifications of survivin expression on the secondary injury cascade following TBI.

Future Directions

Additional studies of survivin expression and function must be completed before

survivin may be considered a potential therapeutic agent/target for brain injury treatment.

First, survivin activities following TBI in the intact animal must be inhibited to observe

the histological and behavioral changes associated with its expression. Of interest are

heterozygous survivin knockout mice strains that appear to be particularly vulnerable to

even mild apoptotic stimuli (Conway et al., 2002). TBI experiments comparing the

histological and behavioral differences between heterozygous survivin knockouts and

their wild type littermates would give more direct evidence of survivin function following

brain insults. Additionally, a host of new techniques to inhibit survivin expression in the

whole animal are currently being developed for cancer treatment and can be readily

applied to a TBI model. Of practical interest to this model is "molecular antagonism"

and pharmacological inhibition. "Molecular antagonism" using siRNA, antisense and

dominant-negative survivin mutants are effective in vivo at interfering with survivin

expression and function (Grossman et al., 2001b; Kanwar et al., 2001; Yamamoto et al.,

2002; Williams et al., 2003). Pharmacological inhibition by flavopiridol (Zhai et al.,

2002) or Purv.A (Gray et al., 1998) can increase survivin turnover and reduce its

effectiveness as an apoptosis inhibitor by reducing the phosphorylation state of survivin

(O'Connor et al., 2000a; Grossman et al., 2001b). Based on the findings in this

dissertation, inhibition of survivin following TBI will likely increase apoptotic cell death,

cavity size and have a negative impact on behavioral recovery.









Second, the activities of survivin must be enhanced to see if increased survivin has

a beneficial role to the overall recovery of the organism. Both pharmacological and gene

transfection can be used to accomplish this task. Gene transfection has been successfully

used to temporarily up-regulate proteins of interest in the adult brain (Yenari and

Sapolsky, 2004) and may be used to increase survivin expression following TBI. A more

flexible approach may include pharmacological agents because they can be used in

established wild-type animal models. Compounds that enhance Cdk phosphorylation

activity such as PD0166285 (Li et al., 2002) may inhibit survivin turnover and promote

cell survival by increasing the phosphorylation state of survivin. Based on the findings

from this dissertation and work by other groups, enhancing survivin activity in the brain

following TBI will likely decrease cell death, reduce cavity size and enhance behavioral

recovery compared to untreated animals.

This set of proposed experiments will reveal the functional consequences of

endogenous survivin expression in injured brain tissues. With this knowledge, it can be

determined whether survivin is indeed a viable therapeutic agent for brain injury

treatment.















LIST OF REFERENCES


Adida C, Crotty PL, McGrath J, Berrebi D, Diebold J, Altieri DC (1998)
Developmentally regulated expression of the novel cancer anti-apoptosis gene
survivin in human and mouse differentiation. Am J Pathol 152:43-49.

Aldskogius H, Liu L, Svensson M (1999) Glial responses to synaptic damage and
plasticity. J Neurosci Res 58:33-41.

Altieri DC (2001) Cytokinesis, apoptosis and survivin: three for tango? Cell Death Differ
8:4-5.

Altieri DC (2003a) Blocking survivin to kill cancer cells. Methods Mol Biol 223:533-
542.

Altieri DC (2003b) Survivin in apoptosis control and cell cycle regulation in cancer. Prog
Cell Cycle Res 5:447-452.

Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ (1997)
Gapped BLAST and PSI-BLAST: a new generation of protein database search
programs. Nucleic Acids Res 25:3389-3402.

Alzheimer C, Werner S (2002) Fibroblast growth factors and neuroprotection. Adv Exp
MedBiol 513:335-351.

Ambrosini G, Adida C, Altieri DC (1997) A novel anti-apoptosis gene, survivin,
expressed in cancer and lymphoma. Nat Med 3:917-921.

Ambrosini G, Adida C, Sirugo G, Altieri DC (1998) Induction of apoptosis and inhibition
of cell proliferation by survivin gene targeting. J Biol Chem 273:11177-11182.

Badran A, Yoshida A, Ishikawa K, Goi T, Yamaguchi A, Ueda T, Inuzuka M (2004)
Identification of a novel splice variant of the human anti-apoptopsis gene
survivin. Biochem Biophys Res Commun 314:902-907.

Baldwin SA, Gibson T, Callihan CT, Sullivan PG, Palmer E, Scheff SW (1997) Neuronal
cell loss in the CA3 subfield of the hippocampus following cortical contusion
utilizing the optical director method for cell counting. J Neurotrauma 14:385-398.

Bambrick L, Kristian T, Fiskum G (2004) Astrocyte mitochondrial mechanisms of
ischemic brain injury and neuroprotection. Neurochem Res 29:601-608.









Baskaya MK, Rao AM, Dogan A, Donaldson D, Dempsey RJ (1997) The biphasic
opening of the blood-brain barrier in the cortex and hippocampus after traumatic
brain injury in rats. Neurosci Lett 226:33-36.

Beckman JS, Beckman TW, Chen J, Marshall PA, Freeman BA (1990) Apparent
hydroxyl radical production by peroxynitrite: implications for endothelial injury
from nitric oxide and superoxide. Proc Natl Acad Sci U S A 87:1620-1624.

Beer R, Franz G, Srinivasan A, Hayes RL, Pike BR, Newcomb JK, Zhao X, Schmutzhard
E, Poewe W, Kampfl A (2000) Temporal profile and cell subtype distribution of
activated caspase-3 following experimental traumatic brain injury. J Neurochem
75:1264-1273.

Belluardo N, Korhonen L, Mudo G, Lindholm D (2002) Neuronal expression and
regulation of rat inhibitor of apoptosis protein-2 by kainic acid in the rat brain.
Eur J Neurosci 15:87-100.

Blanc-Brude OP, Yu J, Simosa H, Conte MS, Sessa WC, Altieri DC (2002) Inhibitor of
apoptosis protein survivin regulates vascular injury. Nat Med 8:987-994.

Borriello A, Roberto R, Della Ragione F, Iolascon A (2002) Proliferate and survive: cell
division cycle and apoptosis in human neuroblastoma. Haematologica 87:196-
214.

Bravo R, Frank R, Blundell PA, Macdonald-Bravo H (1987) Cyclin/PCNA is the
auxiliary protein of DNA polymerase-delta. Nature 326:515-517.

Buki A, Okonkwo DO, Wang KK, Povlishock JT (2000) Cytochrome c release and
caspase activation in traumatic axonal injury. J Neurosci 20:2825-2834.

Bullock R, Maxwell WL, Graham DI, Teasdale GM, Adams JH (1991) Glial swelling
following human cerebral contusion: an ultrastructural study. J Neurol Neurosurg
Psychiatry 54:427-434.

Bush TG, Puvanachandra N, Horner CH, Polito A, Ostenfeld T, Svendsen CN, Mucke L,
Johnson MH, Sofroniew MV (1999) Leukocyte infiltration, neuronal
degeneration, and neurite outgrowth after ablation of scar-forming, reactive
astrocytes in adult transgenic mice. Neuron 23:297-308.

Buytaert KA, Kline AE, Montanez S, Likler E, Millar CJ, Hernandez TD (2001) The
temporal patterns of c-Fos and basic fibroblast growth factor expression following
a unilateral anteromedial cortex lesion. Brain Res 894:121-130.

Cameron HA, McKay R (1998) Stem cells and neurogenesis in the adult brain. Curr Opin
Neurobiol 8:677-680.

Cameron HA, McKay RD (2001) Adult neurogenesis produces a large pool of new
granule cells in the dentate gyms. J Comp Neurol 435:406-417.









Carbonell WS, Grady MS (1999) Regional and temporal characterization of neuronal,
glial, and axonal response after traumatic brain injury in the mouse. Acta
Neuropathol (Berl) 98:396-406.

Cervos-Navarro J, Lafuente JV (1991) Traumatic brain injuries: structural changes. J
Neurol Sci 103 Suppl:S3-14.

Chakravarti A, Zhai G, Zhang M, Malhotra R, Latham D, Delaney M, Robe P, Nestler U,
Song Q, Loeffler J (2004) Survivin enhances radiation resistance in primary
human glioblastoma cells via caspase-independant mechanisms. Oncogene
23:7494-7506.

Chan SL, Mattson MP (1999) Caspase and calpain substrates: roles in synaptic plasticity
and cell death. J Neurosci Res 58:167-190.

Chantalat L, Skoufias DA, Kleman JP, Jung B, Dideberg O, Margolis RL (2000) Crystal
structure of human survivin reveals a bow tie-shaped dimer with two unusual
alpha-helical extensions. Mol Cell 6:183-189.

Chen XH, Iwata A, Nonaka M, Browne KD, Smith DH (2003) Neurogenesis and glial
proliferation persist for at least one year in the subventricular zone following
brain trauma in rats. J Neurotrauma 20:623-631.

Chen Y, Swanson RA (2003) Astrocytes and brain injury. J Cereb Blood Flow Metab
23:137-149.

Chen ZJ, Negra M, Levine A, Ughrin Y, Levine JM (2002) Oligodendrocyte precursor
cells: reactive cells that inhibit axon growth and regeneration. J Neurocytol
31:481-495.

Chirumamilla S, Sun D, Bullock M, Colello R (2002) Traumatic Brain Injury Induced
Cell Proliferation in the Adult Mammalian Nervous System. Journal of
Neurotrauma 19:693-703.

Choi DW (1988) Calcium-mediated neurotoxicity: relationship to specific channel types
and role in ischemic damage. Trends Neurosci 11:465-469.

Choi DW, Rothman SM (1990) The role of glutamate neurotoxicity in hypoxic-ischemic
neuronal death. Annu Rev Neurosci 13:171-182.

Choi KS, Lee TH, Jung MH (2003) Ribozyme-mediated cleavage of the human survivin
mRNA and inhibition of antiapoptotic function of survivin in MCF-7 cells.
Cancer Gene Ther 10:87-95.

Clark RS, Kochanek PM, Watkins SC, Chen M, Dixon CE, Seidberg NA, Melick J,
Loeffert JE, Nathaniel PD, Jin KL, Graham SH (2000) Caspase-3 mediated
neuronal death after traumatic brain injury in rats. J Neurochem 74:740-753.









Cohen GM (1997) Caspases: the executioners of apoptosis. Biochem J 326:1-16.

Conti AC, Raghupathi R, Trojanowski JQ, McIntosh TK (1998) Experimental brain
injury induces regionally distinct apoptosis during the acute and delayed post-
traumatic period. J Neurosci 18:5663-5672.

Conway EM, Zwerts F, Van Eygen V, DeVriese A, Nagai N, Luo W, Collen D (2003)
Survivin-dependent angiogenesis in ischemic brain: molecular mechanisms of
hypoxia-induced up-regulation. Am J Pathol 163:935-946.

Conway EM, Pollefeyt S, Cornelissen J, DeBaere I, Steiner-Mosonyi M, Ong K, Baens
M, Collen D, Schuh AC (2000) Three differentially expressed survivin cDNA
variants encode proteins with distinct antiapoptotic functions. Blood 95:1435-
1442.

Conway EM, Pollefeyt S, Steiner-Mosonyi M, Luo W, Devriese A, Lupu F, Bono F,
Leducq N, Dol F, Schaeffer P, Collen D, Herbert JM (2002) Deficiency of
survivin in transgenic mice exacerbates Fas-induced apoptosis via mitochondrial
pathways. Gastroenterology 123:619-631.

Csuka E, Hans VH, Ammann E, Trentz O, Kossmann T, Morganti-Kossmann MC (2000)
Cell activation and inflammatory response following traumatic axonal injury in
the rat. Neuroreport 11:2587-2590.

Dash PK, Mach SA, Moore AN (2001) Enhanced neurogenesis in the rodent
hippocampus following traumatic brain injury. J Neurosci Res 63:313-319.

Dawson MR, Levine JM, Reynolds R (2000) NG2-expressing cells in the central nervous
system: are they oligodendroglial progenitors? J Neurosci Res 61:471-479.

Denecker G, Vercammen D, Steemans M, Vanden Berghe T, Brouckaert G, Van Loo G,
Zhivotovsky B, Fiers W, Grooten J, Declercq W, Vandenabeele P (2001) Death
receptor-induced apoptotic and necrotic cell death: differential role of caspases
and mitochondria. Cell Death Differ 8:829-840.

Deveraux QL, Reed JC (1999) IAP family proteins--suppressors of apoptosis. Genes Dev
13:239-252.

Deveraux QL, Takahashi R, Salvesen GS, Reed JC (1997) X-linked IAP is a direct
inhibitor of cell-death proteases. Nature 388:300-304.

Dietrich WD (1994) Morphological manifestations of reperfusion injury in brain. Ann N
Y Acad Sci 723:15-24.

Dixon CE, Clifton GL, Lighthall JW, Yaghmai AA, Hayes RL (1991) A controlled
cortical impact model of traumatic brain injury in the rat. J Neurosci Methods
39:253-262.









Doetsch F, Caille I, Lim DA, Garcia-Verdugo JM, Alvarez-Buylla A (1999)
Subventricular zone astrocytes are neural stem cells in the adult mammalian brain.
Cell 97:703-716.

Domanska-Janik K (1996) Protein serine/threonine kinases (PKA, PKC and CaMKII)
involved in ischemic brain pathology. Acta Neurobiol Exp (Wars) 56:579-585.

Dunn-Meynell AA, Levin BE (1997) Histological markers of neuronal, axonal and
astrocytic changes after lateral rigid impact traumatic brain injury. Brain Res
761:25-41.

Earnshaw WC, Martins LM, Kaufmann SH (1999) Mammalian caspases: structure,
activation, substrates, and functions during apoptosis. Annu Rev Biochem 68:383-
424.

Ebel RL (1951) Estimation of the Reliability of Ratings. Psycometrika 16:407-424.

Eldadah BA, Faden AI (2000) Caspase pathways, neuronal apoptosis, and CNS injury. J
Neurotrauma 17:811-829.

Enari M, Sakahira H, Yokoyama H, Okawa K, Iwamatsu A, Nagata S (1998) A caspase-
activated DNase that degrades DNA during apoptosis, and its inhibitor ICAD.
Nature 391:43-50.

Fawcett JW, Asher RA (1999) The glial scar and central nervous system repair. Brain
Res Bull 49:377-391.

Ferrer I, Planas AM (2003) Signaling of cell death and cell survival following focal
cerebral ischemia: life and death struggle in the penumbra. J Neuropathol Exp
Neurol 62:329-339.

Fortugno P, Beltrami E, Plescia J, Fontana J, Pradhan D, Marchisio PC, Sessa WC,
Altieri DC (2003) Regulation of survivin function by Hsp90. Proc Natl Acad Sci
USA 100:13791-13796.

Fortugno P, Wall NR, Giodini A, O'Connor DS, Plescia J, Padgett KM, Tognin S,
Marchisio PC, Altieri DC (2002) Survivin exists in immunochemically distinct
subcellular pools and is involved in spindle microtubule function. J Cell Sci
115:575-585.

Gabryel B, Trzeciak HI (2001) Role of astrocytes in pathogenesis of ischemic brain
injury. Neurotox Res 3:205-221.

Gage FH (2002) Neurogenesis in the adult brain. J Neurosci 22:612-613.

Gage FH, Kempermann G, Palmer TD, Peterson DA, Ray J (1998) Multipotent
progenitor cells in the adult dentate gyrus. J Neurobiol 36:249-266.









Gennarelli TA (1993) Mechanisms of brain injury. J Emerg Med 11 Suppl 1:5-11.

Giulian D (1991) Microglia-Neuron Interactions After Injury to the Central Nervous
System. In: Peripheral Signaling of the Brain: role in neural-immune interactions,
learning and memory (Frederickson R, McGaugh J, Felton D, eds), pp 73-82.
Lewiston, NY: Hogrefe and Huber.

Gong C, Hoff JT, Keep RF (2000) Acute inflammatory reaction following experimental
intracerebral hemorrhage in rat. Brain Res 871:57-65.

Gould E, Tanapat P (1997) Lesion-induced proliferation of neuronal progenitors in the
dentate gyrus of the adult rat. Neuroscience 80:427-436.

Gould E, Vail N, Wagers M, Gross CG (2001) Adult-generated hippocampal and
neocortical neurons in macaques have a transient existence. Proc Natl Acad Sci U
S A 98:10910-10917.

Grady MS, Charleston JS, Maris D, Witgen BM, Lifshitz J (2003) Neuronal and glial cell
number in the hippocampus after experimental traumatic brain injury: analysis by
stereological estimation. J Neurotrauma 20:929-941.

Graham DI, McIntosh TK, Maxwell WL, Nicoll JA (2000) Recent advances in
neurotrauma. J Neuropathol Exp Neurol 59:641-651.

Graham DI, Adams JH, Nicoll JA, Maxwell WL, Gennarelli TA (1995) The nature,
distribution and causes of traumatic brain injury. Brain Pathol 5:397-406.

Gray NS, Wodicka L, Thunnissen AM, Norman TC, Kwon S, Espinoza FH, Morgan DO,
Barnes G, LeClerc S, Meijer L, Kim SH, Lockhart DJ, Schultz PG (1998)
Exploiting chemical libraries, structure, and genomics in the search for kinase
inhibitors. Science 281:533-538.

Green DR, Reed JC (1998) Mitochondria and apoptosis. Science 281:1309-1312.

Grossman D, Altieri DC (2001) Drug resistance in melanoma: mechanisms, apoptosis,
and new potential therapeutic targets. Cancer Metastasis Rev 20:3-11.

Grossman D, McNiff JM, Li F, Altieri DC (1999) Expression and targeting of the
apoptosis inhibitor, survivin, in human melanoma. J Invest Dermatol 113:1076-
1081.

Grossman D, Kim PJ, Schechner JS, Altieri DC (2001 a) Inhibition of melanoma tumor
growth in vivo by survivin targeting. Proc Natl Acad Sci U S A 98:635-640.

Grossman D, Kim PJ, Blanc-Brude OP, Brash DE, Tognin S, Marchisio PC, Altieri DC
(2001b) Transgenic expression of survivin in keratinocytes counteracts UVB-
induced apoptosis and cooperates with loss of p53. J Clin Invest 108:991-999.









Gwag BJ, Canzoniero LM, Sensi SL, Demaro JA, Koh JY, Goldberg MP, Jacquin M,
Choi DW (1999) Calcium ionophores can induce either apoptosis or necrosis in
cultured cortical neurons. Neuroscience 90:1339-1348.

Heales SJ, Lam AA, Duncan AJ, Land JM (2004) Neurodegeneration or neuroprotection:
the pivotal role of astrocytes. Neurochem Res 29:513-519.

Hermann DM, Kilic E, Kugler S, Isenmann S, Bahr M (2001) Adenovirus-mediated
GDNF and CNTF pretreatment protects against striatal injury following transient
middle cerebral artery occlusion in mice. Neurobiol Dis 8:655-666.

Herrup K, Busser JC (1995) The induction of multiple cell cycle events precedes target-
related neuronal death. Development 121:2385-2395.

Higgins DG, Bleasby AJ, Fuchs R (1992) CLUSTAL V: improved software for multiple
sequence alignment. Comput Appl Biosci 8:189-191.

Hill-Felberg SJ, McIntosh TK, Oliver DL, Raghupathi R, Barbarese E (1999) Concurrent
loss and proliferation of astrocytes following lateral fluid percussion brain injury
in the adult rat. J Neurosci Res 57:271-279.

Holmin S, Mathiesen T (1995) Biphasic edema development after experimental brain
contusion in rat. Neurosci Lett 194:97-100.

Hutchison JS, Derrane RE, Johnston DL, Gendron N, Barnes D, Fliss H, King WJ,
Rasquinha I, MacManus J, Robertson GS, MacKenzie AE (2001) Neuronal
apoptosis inhibitory protein expression after traumatic brain injury in the mouse. J
Neurotrauma 18:1333-1347.

Ignatova T, Kukekov V, Laywell E, Suslov O, Vrionis F, Steindler D (2002) Human
Cortical Glial Tumors Contain Neural Stem-Like Cells Expressing Astroglial and
Neuronal Markers in vitro. Glia 39:online.

Iwata E, Asanuma M, Nishibayashi S, Kondo Y, Ogawa N (1997) Different effects of
oxidative stress on activation of transcription factors in primary cultured rat
neuronal and glial cells. Brain Res Mol Brain Res 50:213-220.

Jennett B (1996) Epidemiology of head injury. J Neurol Neurosurg Psychiatry 60:362-
369.

Jiang X, Wilford C, Duensing S, Munger K, Jones G, Jones D (2001) Participation of
Survivin in mitotic and apoptotic activities of normal and tumor-derived cells. J
Cell Biochem 83:342-354.

Jiao BH, Yao ZG, Geng SM, Zuo SH (2004) Expression of survivin, a novel apoptosis
inhibitor and cell cycle regulatory protein, in human gliomas. Chin Med J (Engl)
117:612-614.









Johnson EA, Svetlov SI, Pike BR, Tolentino PJ, Shaw G, Wang KKW, Hayes RL, Pineda
JA (2004) Cell-specific Upregulation of Survivin After Experimental Traumatic
Brain Injury in Rats. Journal of Neurotrauma 21:1183-1195.

Kajiwara Y, Yamasaki F, Hama S, Yahara K, Yoshioka H, Sugiyama K, Arita K, Kurisu
K (2003) Expression of survivin in astrocytic tumors: correlation with malignant
grade and prognosis. Cancer 97:1077-1083.

Kampfl A, Posmantur R, Nixon R, Grynspan F, Zhao X, Liu SJ, Newcomb JK, Clifton
GL, Hayes RL (1996) mu-calpain activation and calpain-mediated cytoskeletal
proteolysis following traumatic brain injury. J Neurochem 67:1575-1583.

Kanwar JR, Shen WP, Kanwar RK, Berg RW, Krissansen GW (2001) Effects of survivin
antagonists on growth of established tumors and B7-1 immunogene therapy. J
Natl Cancer Inst 93:1541-1552.

Kasof GM, Gomes BC (2001) Livin, a novel inhibitor of apoptosis protein family
member. J Biol Chem 276:3238-3246.

Kaya SS, Mahmood A, Li Y, Yavuz E, Chopp M (1999a) Expression of cell cycle
proteins (cyclin Dl and cdk4) after controlled cortical impact in rat brain. J
Neurotrauma 16:1187-1196.

Kaya SS, Mahmood A, Li Y, Yavuz E, Goksel M, Chopp M (1999b) Apoptosis and
expression of p53 response proteins and cyclin Dl after cortical impact in rat
brain. Brain Res 818:23-33.

Keane RW, Kraydieh S, Lotocki G, Alonso OF, Aldana P, Dietrich WD (2001) Apoptotic
and antiapoptotic mechanisms after traumatic brain injury. J Cereb Blood Flow
Metab 21:1189-1198.

Kernie SG, Erwin TM, Parada LF (2001) Brain remodeling due to neuronal and
astrocytic proliferation after controlled cortical injury in mice. J Neurosci Res
66:317-326.

Kim S, Kang J, Qiao J, Thomas RP, Evers BM, Chung DH (2004) Phosphatidylinositol
3-kinase inhibition down-regulates survivin and facilitates TRAIL-mediated
apoptosis in neuroblastomas. J Pediatr Surg 39:516-521.

Kitagawa H, Warita H, Sasaki C, Zhang WR, Sakai K, Shiro Y, Mitsumoto Y, Mori T,
Abe K (1999) Immunoreactive Akt, PI3-K and ERK protein kinase expression in
ischemic rat brain. Neurosci Lett 274:45-48.

Kleinschmidt-DeMasters BK, Heinz D, McCarthy PJ, Bobak JB, Lillehei KO, Shroyer
AL, Shroyer KR (2003) Survivin in glioblastomas. Protein and messenger RNA
expression and comparison with telomerase levels. Arch Pathol Lab Med
127:826-833.









Knoblach SM, Nikolaeva M, Huang X, Fan L, Krajewski S, Reed JC, Faden AI (2002)
Multiple caspases are activated after traumatic brain injury: evidence for
involvement in functional outcome. J Neurotrauma 19:1155-1170.

Kobayashi K, Hatano M, Otaki M, Ogasawara T, Tokuhisa T (1999) Expression of a
murine homologue of the inhibitor of apoptosis protein is related to cell
proliferation. Proc Natl Acad Sci U S A 96:1457-1462.

Kontos HA (1989) Oxygen radicals in CNS damage. Chem Biol Interact 72:229-255.

LaCasse EC, Baird S, Korneluk RG, MacKenzie AE (1998) The inhibitors of apoptosis
(IAPs) and their emerging role in cancer. Oncogene 17:3247-3259.

Lamer SF, Hayes RL, McKinsey DM, Pike BR, Wang KK (2004) Increased expression
and processing of caspase-12 after traumatic brain injury in rats. J Neurochem
88:78-90.

Latov N, Nilaver G, Zimmerman EA, Johnson WG, Silverman AJ, Defendini R, Cote L
(1979) Fibrillary astrocytes proliferate in response to brain injury: a study
combining immunoperoxidase technique for glial fibrillary acidic protein and
radioautography of tritiated thymidine. Dev Biol 72:381-384.

Lazebnik YA, Kaufmann SH, Desnoyers S, Poirier GG, Earnshaw WC (1994) Cleavage
of poly(ADP-ribose) polymerase by a proteinase with properties like ICE. Nature
371:346-347.

Lee SM, Wong MD, Samii A, Hovda DA (1999) Evidence for energy failure following
irreversible traumatic brain injury. Ann N Y Acad Sci 893:337-340.

Lei B, Popp S, Capuano-Waters C, Cottrell JE, Kass IS (2004) Lidocaine attenuates
apoptosis in the ischemic penumbra and reduces infarct size after transient focal
cerebral ischemia in rats. Neuroscience 125:691-701.

Li F (2003) Survivin study: what is the next wave? J Cell Physiol 197:8-29.

Li F, Ambrosini G, Chu EY, Plescia J, Tognin S, Marchisio PC, Altieri DC (1998)
Control of apoptosis and mitotic spindle checkpoint by survivin. Nature 396:580-
584.

Li F, Ackermann EJ, Bennett CF, Rothermel AL, Plescia J, Tognin S, Villa A, Marchisio
PC, Altieri DC (1999) Pleiotropic cell-division defects and apoptosis induced by
interference with survivin function. Nat Cell Biol 1:461-466.

Li J, Wang Y, Sun Y, Lawrence TS (2002) Wild-type TP53 inhibits G(2)-phase
checkpoint abrogation and radiosensitization induced by PD0166285, a WEE1
kinase inhibitor. Radiat Res 157:322-330.









Li Y, Chopp M, Powers C, Jiang N (1997) Immunoreactivity of cyclin D1/cdk4 in
neurons and oligodendrocytes after focal cerebral ischemia in rat. J Cereb Blood
Flow Metab 17:846-856.

Liu L, Rudin M, Kozlova EN (2000) Glial cell proliferation in the spinal cord after dorsal
rhizotomy or sciatic nerve transaction in the adult rat. Exp Brain Res 131:64-73.

Liu L, Persson JK, Svensson M, Aldskogius H (1998a) Glial cell responses, complement,
and clusterin in the central nervous system following dorsal root transaction. Glia
23:221-238.

Liu X, Li P, Widlak P, Zou H, Luo X, Garrard WT, Wang X (1998b) The 40-kDa subunit
of DNA fragmentation factor induces DNA fragmentation and chromatin
condensation during apoptosis. Proc Natl Acad Sci U S A 95:8461-8466.

Lotocki G, Alonso OF, Frydel B, Dietrich WD, Keane RW (2003) Monoubiquitination
and cellular distribution of XIAP in neurons after traumatic brain injury. J Cereb
Blood Flow Metab 23:1129-1136.

Lu CD, Altieri DC, Tanigawa N (1998) Expression of a novel antiapoptosis gene,
survivin, correlated with tumor cell apoptosis and p53 accumulation in gastric
carcinomas. Cancer Res 58:1808-1812.

Magavi SS, Leavitt BR, Macklis JD (2000) Induction of neurogenesis in the neocortex of
adult mice. Nature 405:951-955.

Maier CM, Chan PH (2002) Role of superoxide dismutases in oxidative damage and
neurodegenerative disorders. Neuroscientist 8:323-334.

Maier JK, Lahoua Z, Gendron NH, Fetni R, Johnston A, Davoodi J, Rasper D, Roy S,
Slack RS, Nicholson DW, MacKenzie AE (2002) The neuronal apoptosis
inhibitory protein is a direct inhibitor of caspases 3 and 7. J Neurosci 22:2035-
2043.

Marciano PG, Brettschneider J, Manduchi E, Davis JE, Eastman S, Raghupathi R,
Saatman KE, Speed TP, Stoeckert CJ, Jr., Eberwine JH, McIntosh TK (2004)
Neuron-specific mRNA complexity responses during hippocampal apoptosis after
traumatic brain injury. J Neurosci 24:2866-2876.

Maroni P, Bendinelli P, Tiberio L, Rovetta F, Piccoletti R, Schiaffonati L (2003) In vivo
heat-shock response in the brain: signalling pathway and transcription factor
activation. Brain Res Mol Brain Res 119:90-99.

Maxwell WL, Povlishock JT, Graham DL (1997) A mechanistic analysis of
nondisruptive axonal injury: a review. J Neurotrauma 14:419-440.









McCullers DL, Sullivan PG, Scheff SW, Herman JP (2002) Mifepristone protects CAl
hippocampal neurons following traumatic brain injury in rat. Neuroscience
109:219-230.

McIntosh TK, Saatman KE, Raghupathi R, Graham DI, Smith DH, Lee VM, Trojanowski
JQ (1998) The Dorothy Russell Memorial Lecture. The molecular and cellular
sequelae of experimental traumatic brain injury: pathogenetic mechanisms.
Neuropathol Appl Neurobiol 24:251-267.

McPherson CA, Kubik J, Wine RN, D'Hellencourt CL, Harry GJ (2003) Alterations in
cyclin A, B, and Dl in mouse dentate gyms following TMT-induced hippocampal
damage. Neurotox Res 5:339-354.

Miyake T, Okada M, Kitamura T (1992) Reactive proliferation of astrocytes studied by
immunohistochemistry for proliferating cell nuclear antigen. Brain Res 590:300-
302.

Moon WS, Tamawski AS (2003) Nuclear translocation of survivin in hepatocellular
carcinoma: a key to cancer cell growth? Hum Pathol 34:1119-1126.

Morganti-Kossmann MC, Rancan M, Otto VI, Stahel PF, Kossmann T (2001) Role of
cerebral inflammation after traumatic brain injury: a revisited concept. Shock
16:165-177.

Morshead CM, van der Kooy D (1992) Postmitotic death is the fate of constitutively
proliferating cells in the subependymal layer of the adult mouse brain. J Neurosci
12:249-256.

Morshead CM, Reynolds BA, Craig CG, McBurney MW, Staines WA, Morassutti D,
Weiss S, van der Kooy D (1994) Neural stem cells in the adult mammalian
forebrain: a relatively quiescent subpopulation of subependymal cells. Neuron
13:1071-1082.

Muchmore SW, Chen J, Jakob C, Zakula D, Matayoshi ED, Wu W, Zhang H, Li F, Ng
SC, Altieri DC (2000) Crystal structure and mutagenic analysis of the inhibitor-
of-apoptosis protein survivin. Mol Cell 6:173-182.

Nakajima K, Kohsaka S (1993) Functional roles of microglia in the brain. Neurosci Res
17:187-203.

Nakase T, Sohl G, Theis M, Willecke K, Naus CC (2004) Increased apoptosis and
inflammation after focal brain ischemia in mice lacking connexin43 in astrocytes.
Am J Pathol 164:2067-2075.

Newcomb JK, Zhao X, Pike BR, Hayes RL (1999) Temporal profile of apoptotic-like
changes in neurons and astrocytes following controlled cortical impact injury in
the rat. Exp Neurol 158:76-88.









Nicholson DW, Ali A, Thornberry NA, Vaillancourt JP, Ding CK, Gallant M, Gareau Y,
Griffin PR, Labelle M, Lazebnik YA, et al. (1995) Identification and inhibition of
the ICE/CED-3 protease necessary for mammalian apoptosis. Nature 376:37-43.

Nicotera P, Leist M, Single B, Volbracht C (1999) Execution of apoptosis: converging or
diverging pathways? Biol Chem 380:1035-1040.

Norton WT (1999) Cell reactions following acute brain injury: a review. Neurochem Res
24:213-218.

Nowak TS, Jr., Jacewicz M (1994) The heat shock/stress response in focal cerebral
ischemia. Brain Pathol 4:67-76.

O'Connor DS, Wall NR, Porter AC, Altieri DC (2002) A p34(cdc2) survival checkpoint
in cancer. Cancer Cell 2:43-54.

O'Connor DS, Grossman D, Plescia J, Li F, Zhang H, Villa A, Tognin S, Marchisio PC,
Altieri DC (2000a) Regulation of apoptosis at cell division by p34cdc2
phosphorylation of survivin. Proc Natl Acad Sci U S A 97:13103-13107.

O'Connor DS, Schechner JS, Adida C, Mesri M, Rothermel AL, Li F, Nath AK, Pober
JS, Altieri DC (2000b) Control of apoptosis during angiogenesis by survivin
expression in endothelial cells. Am J Pathol 156:393-398.

Otaki M, Hatano M, Kobayashi K, Ogasawara T, Kuriyama T, Tokuhisa T (2000) Cell
cycle-dependent regulation of TIAP/m-survivin expression. Biochim Biophys
Acta 1493:188-194.

Papapetropoulos A, Fulton D, Mahboubi K, Kalb RG, O'Connor DS, Li F, Altieri DC,
Sessa WC (2000) Angiopoietin-1 inhibits endothelial cell apoptosis via the
Akt/survivin pathway. J Biol Chem 275:9102-9105.

Parent A (1997) The brain in evolution and involution. Biochem Cell Biol 75:651-667.

Passineau MJ, Zhao W, Busto R, Dietrich WD, Alonso O, Loor JY, Bramlett HM,
Ginsberg MD (2000) Chronic metabolic sequelae of traumatic brain injury:
prolonged suppression of somatosensory activation. Am J Physiol Heart Circ
Physiol 279:H924-931.

Peterson DA (2002) Stem cells in brain plasticity and repair. Curr Opin Pharmacol 2:34-
42.

Pike BR, Zhao X, Newcomb JK, Posmantur RM, Wang KK, Hayes RL (1998) Regional
calpain and caspase-3 proteolysis of alpha-spectrin after traumatic brain injury.
Neuroreport 9:2437-2442.

Povlishock JT, Kontos HA (1985) Continuing axonal and vascular change following
experimental brain trauma. Cent Nerv Syst Trauma 2:285-298.









Raghupathi R, Graham DI, McIntosh TK (2000) Apoptosis after traumatic brain injury. J
Neurotrauma 17:927-938.

Rice AC, Khaldi A, Harvey HB, Salman NJ, White F, Fillmore H, Bullock MR (2003)
Proliferation and neuronal differentiation of mitotically active cells following
traumatic brain injury. Experimental Neurology 183:406-417.

Ridet JL, Malhotra SK, Privat A, Gage FH (1997) Reactive astrocytes: cellular and
molecular cues to biological function. Trends Neurosci 20:570-577.

Roy N, Deveraux QL, Takahashi R, Salvesen GS, Reed JC (1997) The c-IAP-1 and c-
IAP-2 proteins are direct inhibitors of specific caspases. Embo J 16:6914-6925.

Sahara S, Aoto M, Eguchi Y, Imamoto N, Yoneda Y, Tsujimoto Y (1999) Acinus is a
caspase-3-activated protein required for apoptotic chromatin condensation. Nature
401:168-173.

Sakahira H, Enari M, Nagata S (1998) Cleavage of CAD inhibitor in CAD activation and
DNA degradation during apoptosis. Nature 391:96-99.

Sanai N, Tramontin AD, Quinones-Hinojosa A, Barbaro NM, Gupta N, Kunwar S,
Lawton MT, McDermott MW, Parsa AT, Manuel-Garcia Verdugo J, Berger MS,
Alvarez-Buylla A (2004) Unique astrocyte ribbon in adult human brain contains
neural stem cells but lacks chain migration. Nature 427:740-744.

Sanz O, Acarin L, Gonzalez B, Castellano B (2001) Expression of 27 kDa heat shock
protein (Hsp27) in immature rat brain after a cortical aspiration lesion. Glia
36:259-270.

Sasaki T, Lopes MB, Hankins GR, Helm GA (2002) Expression of survivin, an inhibitor
of apoptosis protein, in tumors of the nervous system. Acta Neuropathol (Berl)
104:105-109.

Shankar SL, Mani S, O'Guin KN, Kandimalla ER, Agrawal S, Shafit-Zagardo B (2001)
Survivin inhibition induces human neural tumor cell death through caspase-
independent and -dependent pathways. J Neurochem 79:426-436.

Shin S, Sung BJ, Cho YS, Kim HJ, Ha NC, Hwang JI, Chung CW, Jung YK, Oh BH
(2001) An anti-apoptotic protein human survivin is a direct inhibitor of caspase-3
and -7. Biochemistry 40:1117-1123.

Simard AR, Rivest S (2004) Bone marrow stem cells have the ability to populate the
entire central nervous system into fully differentiated parenchymal microglia.
Faseb J 18:998-1000.

Slee EA, Adrain C, Martin SJ (2001) Executioner caspase-3, -6, and -7 perform distinct,
non-redundant roles during the demolition phase of apoptosis. J Biol Chem
276:7320-7326.









Smith C, Berry M, Clarke WE, Logan A (2001) Differential expression of fibroblast
growth factor-2 and fibroblast growth factor receptor 1 in a scarring and
nonscarring model of CNS injury in the rat. Eur J Neurosci 13:443-456.

Song H, Stevens CF, Gage FH (2002) Astroglia induce neurogenesis from adult neural
stem cells. Nature 417:39-44.

Sosin DM, Sniezek JE, Waxweiler RJ (1995) Trends in death associated with traumatic
brain injury, 1979 through 1992. Success and failure. Jama 273:1778-1780.

Sosin DM, Sniezek JE, Thurman DJ (1996) Incidence of mild and moderate brain injury
in the United States, 1991. Brain Inj 10:47-54.

Stennicke HR, Salvesen GS (1999) Catalytic properties of the caspases. Cell Death Differ
6:1054-1059.

Stone JR, Okonkwo DO, Singleton RH, Mutlu LK, Helm GA, Povlishock JT (2002)
Caspase-3-mediated cleavage of amyloid precursor protein and formation of
amyloid Beta peptide in traumatic axonal injury. J Neurotrauma 19:601-614.

Streit WJ (1996) The role of microglia in brain injury. Neurotoxicology 17:671-678.

Streit WJ, Kincaid-Colton CA (1995) The brain's immune system. Sci Am 273:54-55, 58-
61.

Sutton RL, Lescaudron L, Stein DG (1993) Unilateral cortical contusion injury in the rat:
vascular disruption and temporal development of cortical necrosis. J Neurotrauma
10:135-149.

Suzuki A, Ito T, Kawano H, Hayashida M, Hayasaki Y, Tsutomi Y, Akahane K, Nakano
T, Miura M, Shiraki K (2000) Survivin initiates procaspase 3/p21 complex
formation as a result of interaction with Cdk4 to resist Fas-mediated cell death.
Oncogene 19:1346-1353.

Sykova E, Vargova L, Prokopova S, Simonova Z (1999) Glial swelling and astrogliosis
produce diffusion barriers in the rat spinal cord. Glia 25:56-70.

Takahashi R, Deveraux Q, Tamm I, Welsh K, Assa-Munt N, Salvesen GS, Reed JC
(1998) A single BIR domain of XIAP sufficient for inhibiting caspases. J Biol
Chem 273:7787-7790.

Tamm I, Wang Y, Sausville E, Scudiero DA, Vigna N, Oltersdorf T, Reed JC (1998)
IAP-family protein survivin inhibits caspase activity and apoptosis induced by Fas
(CD95), Bax, caspases, and anticancer drugs. Cancer Res 58:5315-5320.

Tang D, Kidd VJ (1998) Cleavage of DFF-45/ICAD by multiple caspases is essential for
its function during apoptosis. J Biol Chem 273:28549-28552.









Tewari M, Quan LT, O'Rourke K, Desnoyers S, Zeng Z, Beidler DR, Poirier GG,
Salvesen GS, Dixit VM (1995) Yama/CPP32 beta, a mammalian homolog of
CED-3, is a CrmA-inhibitable protease that cleaves the death substrate poly(ADP-
ribose) polymerase. Cell 81:801-809.

Thompson C, Gary D, Mattson M, Mackenzie A, Robertson GS (2004) Kainic acid-
induced naip expression in the hippocampus is blocked in mice lacking TNF
receptors. Brain Res Mol Brain Res 123:126-131.

Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: improving the sensitivity
of progressive multiple sequence alignment through sequence weighting,
position-specific gap penalties and weight matrix choice. Nucleic Acids Res
22:4673-4680.

Thurman DJ, Alverson C, Dunn KA, Guerrero J, Sniezek JE (1999a) Traumatic brain
injury in the United States: A public health perspective. J Head Trauma Rehabil
14:602-615.

Thurman DJ, Alverson C, Browne D, Dunn KA, Guerrero J, Johnson R, Johnson V,
Langlois J, Pilkey D, Sniezek JE, Toal S (1999b) Traumatic Brain Injury in the
United States: A Report to Congress. In, p
http://www.cdc.gov/doc.do/id/0900f0903ec800101 Ic: National Center for Injury
Prevention and Control (NCIPC) date retrieved 08/23/2004.

Tolentino PJ, DeFord SM, Notterpek L, Glenn CC, Pike BR, Wang KK, Hayes RL
(2002) Up-regulation of tissue-type transglutaminase after traumatic brain injury.
J Neurochem 80:579-588.

Tran J, Master Z, Yu JL, Rak J, Dumont DJ, Kerbel RS (2002) A role for survivin in
chemoresistance of endothelial cells mediated by VEGF. Proc Natl Acad Sci U S
A 99:4349-4354.

Unal-Cevik I, Kilinc M, Gursoy-Ozdemir Y, Gurer G, Dalkara T (2004) Loss of NeuN
immunoreactivity after cerebral ischemia does not indicate neuronal cell loss: a
cautionary note. Brain Res 1015:169-174.

Uren AG, Wong L, Pakusch M, Fowler KJ, Burrows FJ, Vaux DL, Choo KH (2000)
Survivin and the inner centromere protein INCENP show similar cell- cycle
localization and gene knockout phenotype. Curr Biol 10:1319-1328.

Van de Craen M, Declercq W, Van den brande I, Fiers W, Vandenabeele P (1999) The
proteolytic procaspase activation network: an in vitro analysis. Cell Death Differ
6:1117-1124.

Van Haren K, van der Voorn JP, Peterson DR, van der Knaap MS, Powers JM (2004)
The life and death of oligodendrocytes in vanishing white matter disease. J
Neuropathol Exp Neurol 63:618-630.









Verdecia MA, Huang H, Dutil E, Kaiser DA, Hunter T, Noel JP (2000) Structure of the
human anti-apoptotic protein survivin reveals a dimeric arrangement. Nat Struct
Biol 7:602-608.

Vucic D, Kaiser WJ, Miller LK (1998) Inhibitor of apoptosis proteins physically interact
with and block apoptosis induced by Drosophila proteins HID and GRIM. Mol
Cell Biol 18:3300-3309.

Wang KK (2000) Calpain and caspase: can you tell the difference? Trends Neurosci
23:20-26.

Wei LH, Huang CY, Cheng SP, Chen CA, Hsieh CY (2001) Carcinosarcoma of ovary
associated with previous radiotherapy. Int J Gynecol Cancer 11:81-84.

Wennersten A, Holmin S, Mathiesen T (2003) Characterization of Bax and Bcl-2 in
apoptosis after experimental traumatic brain injury in the rat. Acta Neuropathol
(Berl) 105:281-288.

Williams NS, Gaynor RB, Scoggin S, Verma U, Gokaslan T, Simmang C, Fleming J,
Tavana D, Frenkel E, Becerra C (2003) Identification and validation of genes
involved in the pathogenesis of colorectal cancer using cDNA microarrays and
RNA interference. Clin Cancer Res 9:931-946.

Wolf BB, Schuler M, Echeverri F, Green DR (1999) Caspase-3 is the primary activator
of apoptotic DNA fragmentation via DNA fragmentation factor-45/inhibitor of
caspase-activated DNase inactivation. J Biol Chem 274:30651-30656.

Woo M, Hakem R, Soengas MS, Duncan GS, Shahinian A, Kagi D, Hakem A,
McCurrach M, Khoo W, Kaufman SA, Senaldi G, Howard T, Lowe SW, Mak
TW (1998) Essential contribution of caspase 3/CPP32 to apoptosis and its
associated nuclear changes. Genes Dev 12:806-819.

Wu Q, Combs C, Cannady SB, Geldmacher DS, Herrup K (2000) Beta-amyloid activated
microglia induce cell cycling and cell death in cultured cortical neurons.
Neurobiol Aging 21:797-806.

Xia C, Xu Z, Yuan X, Uematsu K, You L, Li K, Li L, McCormick F, Jablons DM
(2002a) Induction of apoptosis in mesothelioma cells by antisurvivin
oligonucleotides. Mol Cancer Ther 1:687-694.

Xia XG, Hofmann HD, Deller T, Kirsch M (2002b) Induction of STAT3 signaling in
activated astrocytes and sprouting septal neurons following entorhinal cortex
lesion in adult rats. Mol Cell Neurosci 21:379-392.

Xu DG, Crocker SJ, Doucet JP, St-Jean M, Tamai K, Hakim AM, Ikeda JE, Liston P,
Thompson CS, Korneluk RG, MacKenzie A, Robertson GS (1997) Elevation of
neuronal expression of NAIP reduces ischemic damage in the rat hippocampus.
Nat Med 3:997-1004.









Yagita Y, Kitagawa K, Ohtsuki T, Takasawa K, Miyata T, Okano H, Hori M, Matsumoto
M (2001) Neurogenesis by progenitor cells in the ischemic adult rat hippocampus.
Stroke 32:1890-1896.

Yakovlev AG, Faden AI (2001) Caspase-dependent apoptotic pathways in CNS injury.
Mol Neurobiol 24:131-144.

Yamamoto T, Manome Y, Nakamura M, Tanigawa N (2002) Downregulation of survivin
expression by induction of the effector cell protease receptor-1 reduces tumor
growth potential and results in an increased sensitivity to anticancer agents in
human colon cancer. Eur J Cancer 38:2316-2324.

Yang Y, Geldmacher DS, Herrup K (2001) DNA replication precedes neuronal cell death
in Alzheimer's disease. J Neurosci 21:2661-2668.

Yenari MA, Sapolsky RM (2004) Gene therapy in neurological disease. Methods Mol
Med 104:75-88.

Zhai S, Senderowicz AM, Sausville EA, Figg WD (2002) Flavopiridol, a novel cyclin-
dependent kinase inhibitor, in clinical development. Ann Pharmacother 36:905-
911.

Zhao J, Tenev T, Martins LM, Downward J, Lemoine NR (2000) The ubiquitin-
proteasome pathway regulates survivin degradation in a cell cycle-dependent
manner. J Cell Sci 113 Pt 23:4363-4371.

Zhou M, Gu L, Li F, Zhu Y, Woods WG, Findley HW (2002) DNA damage induces a
novel p53-survivin signaling pathway regulating cell cycle and apoptosis in acute
lymphoblastic leukemia cells. J Pharmacol Exp Ther 303:124-131.

Zipfel GJ, Babcock DJ, Lee JM, Choi DW (2000) Neuronal apoptosis after CNS injury:
the roles of glutamate and calcium. J Neurotrauma 17:857-869.















BIOGRAPHICAL SKETCH

Erik Andrew Johnson was born in Louisville, KY, and raised in Kansas City, MO.

He graduated high school from Lincoln College Preparatory Academy (Kansas City,

MO) in 1994. He attended Macalester College (St. Paul, MN) where he received a

Bachelor of Arts degree in 1998 with majors in biology, psychology and neuroscience.

After a year of graduate study at the University of Texas-Houston, he transferred to the

Interdisciplinary Program in Biological Sciences at the University of Florida

(Gainesville, FL) to complete his doctorate in the laboratory of Dr. Ronald Hayes. Erik

has been awarded top honors at the National and International Neurotrauma Society

Student Poster Competition in 2001. In addition, Erik has twice been awarded the B.W.

Robinson Research Endowment Grant-in-Aid Achievement Award in 2003 and 2004.

Erik finished his doctoral work with two peer reviewed first author papers and five total

papers to his credit.