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Magnetic Resonance Characterization of Skeletal Muscle Adaptations after Incomplete Spinal Cord Injury

Permanent Link: http://ufdc.ufl.edu/UFE0022047/00001

Material Information

Title: Magnetic Resonance Characterization of Skeletal Muscle Adaptations after Incomplete Spinal Cord Injury
Physical Description: 1 online resource (245 p.)
Language: english
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: magneticresonanceimaging, magneticresonancespectroscopy, skeletalmuscle, skeletalmuscleadaptations, spinalcordcontusion, spinalcordinjury
Rehabilitation Science -- Dissertations, Academic -- UF
Genre: Rehabilitation Science thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Spinal cord injury is one of the most disabling health related problems that often results in paralysis and paresis of body musculature below the lesion site. Persons with incomplete-SCI typically exhibit impaired motor performance and varying degrees of functional limitations. Despite the obvious motor dysfunctions, physiological muscle adaptations following incomplete-SCI are relatively unstudied. An understanding of the muscular adaptations following an incomplete-SCI will help in the development of therapies aimed at reducing the secondary effects of paralysis and paresis. The overall objective of this dissertation is to investigate skeletal muscle adaptations following incomplete-SCI using combinations of non-invasive MRI and MRS techniques. Findings from our human studies reveal that chronic incomplete-SCI is associated with significant muscle atrophy in the affected lower extremity that is uniform between limbs and somewhat influenced by mobility status. In addition, persons with incomplete-SCI demonstrate an increase in the total lipid, IMCL and extramyocellular EMCL content and enhancements in the T2 relaxation properties of the lower leg muscles. Moreover, repetitive locomotor training with body weight support and a treadmill are associated with significant increases in the plantarflexor muscle size. Data from our animal experiments reveal that the paralyzed rat hindlimb muscle show faster rates of PCr depletion, thereby suggesting that, after SCI, there is an increase in ATP requirement for similar demands in muscle contraction. In addition, a pronounced decrease in PCr recovery rates implies a less effective oxidative phosphorlyation and a reduction in the mitochondrial oxidative capacity of skeletal muscle. Collectively, findings from this dissertation work reveal that the paralyzed skeletal muscle shows drastic alterations in its morphological and metabolic properties after a SCI and that these adaptations can be successfully characterized by the use of non-invasive MR techniques. The present work will provide a foundation from which the relationship between skeletal muscle adaptations and function in this population can be further explored. Moreover, the use of sophisticated MR techniques will enable characterizing the paralyzed muscle non-invasively and with high resolution; while also allowing longitudinal follow-ups - all of which are crucial in assessing injury mechanisms, progression and therapeutic efficacy.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Thesis: Thesis (Ph.D.)--University of Florida, 2008.
Local: Adviser: Vandenborne, Krista.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0022047:00001

Permanent Link: http://ufdc.ufl.edu/UFE0022047/00001

Material Information

Title: Magnetic Resonance Characterization of Skeletal Muscle Adaptations after Incomplete Spinal Cord Injury
Physical Description: 1 online resource (245 p.)
Language: english
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: magneticresonanceimaging, magneticresonancespectroscopy, skeletalmuscle, skeletalmuscleadaptations, spinalcordcontusion, spinalcordinjury
Rehabilitation Science -- Dissertations, Academic -- UF
Genre: Rehabilitation Science thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Spinal cord injury is one of the most disabling health related problems that often results in paralysis and paresis of body musculature below the lesion site. Persons with incomplete-SCI typically exhibit impaired motor performance and varying degrees of functional limitations. Despite the obvious motor dysfunctions, physiological muscle adaptations following incomplete-SCI are relatively unstudied. An understanding of the muscular adaptations following an incomplete-SCI will help in the development of therapies aimed at reducing the secondary effects of paralysis and paresis. The overall objective of this dissertation is to investigate skeletal muscle adaptations following incomplete-SCI using combinations of non-invasive MRI and MRS techniques. Findings from our human studies reveal that chronic incomplete-SCI is associated with significant muscle atrophy in the affected lower extremity that is uniform between limbs and somewhat influenced by mobility status. In addition, persons with incomplete-SCI demonstrate an increase in the total lipid, IMCL and extramyocellular EMCL content and enhancements in the T2 relaxation properties of the lower leg muscles. Moreover, repetitive locomotor training with body weight support and a treadmill are associated with significant increases in the plantarflexor muscle size. Data from our animal experiments reveal that the paralyzed rat hindlimb muscle show faster rates of PCr depletion, thereby suggesting that, after SCI, there is an increase in ATP requirement for similar demands in muscle contraction. In addition, a pronounced decrease in PCr recovery rates implies a less effective oxidative phosphorlyation and a reduction in the mitochondrial oxidative capacity of skeletal muscle. Collectively, findings from this dissertation work reveal that the paralyzed skeletal muscle shows drastic alterations in its morphological and metabolic properties after a SCI and that these adaptations can be successfully characterized by the use of non-invasive MR techniques. The present work will provide a foundation from which the relationship between skeletal muscle adaptations and function in this population can be further explored. Moreover, the use of sophisticated MR techniques will enable characterizing the paralyzed muscle non-invasively and with high resolution; while also allowing longitudinal follow-ups - all of which are crucial in assessing injury mechanisms, progression and therapeutic efficacy.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Thesis: Thesis (Ph.D.)--University of Florida, 2008.
Local: Adviser: Vandenborne, Krista.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0022047:00001


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befdccf30690f374fc8a7647a1c6d3578a59f864







MAGNETIC RESONANCE CHARACTERIZATION OF SKELETAL MUSCLE
ADAPTATIONS AFTER INCOMPLETE SPINAL CORD INJURY




















By

PRITHVI KRISHNAKANT SHAH


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

UNIVERSITY OF FLORIDA




2008

































2008 Prithvi Krishnakant Shah






























To My Uncle, Dinesh 0 Shah









ACKNOWLEDGMENTS

I can no other answer make, but, thanks, and thanks.
-William Shakespeare

I take this opportunity to thank the numerous people involved in my journey towards

obtaining a doctorate degree in this foreign land. This work, to a great extent, is an outcome of

the excellent professional guidance of several educators at the University of Florida and the

unfaltering support, love and encouragement of my parents, family and friends.

The contribution of my dissertation advisor, Dr Krista Vandenborne in chiseling my

professional career throughout my doctorate studies is unparalleled. Equipped with her expertise

and experience in the field, Dr.Vandenborne has guided and encouraged me to pursue the

complex subjects of magnetic resonance (MR), muscle physiology and animal studies with great

persistence and simplicity. As a mentor, Dr. Vandenborne has introduced me to the concepts of

being relentless yet patient and prolific while producing high quality research. She has also been

most influential in inculcating into me the art of independent and scientific thinking. I next thank

my teacher Dr. Andrea Behrman for first, giving me the opportunity to be a part of the

Rehabilitation Science Program at the University and then, sharing with me her remarkable

enthusiasm in the field of spinal cord injury research. Without the assistance of Dr. Glenn

Walter, this dissertation work can be likened to a mechanic who possesses no knowledge of his

tools. Discussions about MR and muscle physiology with Dr. Walter have largely enabled me to

better understand these concepts and implement complex theoretical knowledge into practical

use. I also wish to thank Dr. David Fuller for his excellent ideas in enriching the quality of

research in my work related to animal studies. His generosity and support in providing me with

his lab resources are most appreciated. My committee member, Dr. Lorie Richards, has been









incredibly instrumental in directing me to think "outside the box" that helped this work take a

more interdisciplinary approach.

I wish to extend my special thanks to the past and present members in my laboratory To

Chris, Jennifer, Arun, Mike, Shiv, Neeti and Sean for their assistance with the human studies;

and to Min and Wendy for their excellent technical assistance with the animal experiments. The

cheerful presence of Sunita, Donovon, Nathan, Fan and Ravneet made the endless hours in the

laboratory far more enjoyable and science a lot more fun. I also wish to thank the Levine and

Reier laboratories in the College of Medicine at the University of Florida for offering their

resources to conduct part of the animal studies. I exclusively thank Milap, Ravi, Geisha, Todd

and Kevin for their help.

I am most grateful to the University of Florida for granting me the Grinter Scholarship for

International students; to Dr. Teitelbaum for providing me a research assistantship during my

first year as a doctorate student at the University of Florida; to the Department of Physical

Therapy for assigning me a teaching assistantship to complete a major portion of my doctoral

course work; and to the National Institutes of Health for the financial support awarded to Dr,

Vandenborne for the present dissertation studies. My heartfelt thanks also go to my research

subjects for their participation in the human studies. I extend my appreciation to the Department

of Physical Therapy's faculty and staff teams for creating an exceptionally dynamic and healthy

learning environment and for making my graduate student experience at the University most

enjoyable.

With genuine humbleness, I deeply thank all my friends who made the Unites States for

me a home away from home. Sailing with me through times of frustration and nostalgia, and

always attempting to encourage and inspire, their presence has proved matchless. I especially









wish to convey my gratitude to Seemant, Rakesh, Sheryl, Andrea, Shreya, Deepali, Avi, Jigesh,

Preeti, Sunita, Surjit, and Bijoy.

Without the unconditional love, strength and patience of my parents and sisters, this

seemingly impossible task would not be made possible. Their enthusiasm and continual support

often curtailed the perception of the "thousands of miles" distance from home. I wish to thank

my parents, sisters and relatives for having the confidence in me and relentlessly encouraging me

to remain persistent with my research efforts.

Lastly, I wish to thank my role model and uncle, Dr. Dinesh Shah, from the bottom of my

heart. I dedicate this dissertation thesis to him. In the truest sense, he has been my mentor, guide,

friend, and a parent cruising along with me through the ups and downs of my doctorate studies.

From him, I have learned much of life's philosophy and in him I have witnessed the essence of a

true scientist.









TABLE OF CONTENTS

page

A CK N O W LED G M EN T S ................................................................. ........... ............. .....

L IS T O F T A B L E S .........................................................................................12

LIST OF FIGURES ................................... .. .... .... ................. 13

L IST O F A B B R E V IA T IO N S .................................................................... ............................. 16

A B S T R A C T ............ ................... ............................................................ 18

CHAPTER

1 SPINAL CORD INJURY AND SKELETAL MUSCLE...................................................20

1 .1 S ig n ific a n c e ........................................................................... 2 0
1.2 Spinal C ord Injury in H um ans................................. ......... ............... ............... 21
1.3 Spinal Cord Injury in A nim al M odels ........................................ ......................... 22
1.3.1 Spinal Cord Injury in the Rat M odel ............................ ................................... 23
1.3.1.1 Spinal cord contusion m odel in rats .................................. ............... ..24
1.3.1.2 Spinal contusion by im action .......................................... ............... 25
1.3.2 Assessment of Locomotor Behavior in Rats ...........................................26
1.4 Skeletal M uscle Adaptations after SCI..................................... .......................... 28
1.4.1 Morphological Adaptations: Muscle Atrophy ..............................................28
1.4.2 Morphological Adaptations: Muscle Fiber Type Conversion.............. ...............31
1.4.3 Adaptations to Contractile Properties........................ ...............34
1.4.4 Metabolic Adaptations: Altered Oxidative Capacity ..........................................36
1.4.5 Metabolic Adaptations: Altered Glucose Homeostasis.............................39
1 .5 S u m m ary ...................................... .................................................... 4 4

2 SPINAL CORD INJURY AND LOCOMOTOR TRAINING........................................46

2.1. L ocom otor Function after SC I.......................................................................................46
2.1.1. Conventional Rehabilitation Therapies after SCI...................................... 47
2.1.2 Limitations of Compensatory Rehabilitation Strategies.............................49
2 .2 Spinal C ord P lasticity ............... ........ ..................................................... .. ....... 50
2.3 Locom otor Training: A Paradigm Shift.................... .................. ................. .... 54
2.3.1 A Historical Perspective and Locomotor Training in the Spinal Cord Injury
A nim al M odel .................................... .......... .. ............................... 55
2.3.2 Locomotor training in Individuals with Spinal Cord Injury................................58
2.3.3 Central Pattern Generators and Locomotion .................................. ............... 61
2.4 Locomotor Training Effects on Paralyzed Skeletal Muscle...........................................63
2 .5 Sum m ary ...............................................................................................65

3 MAGNETIC RESONANCE AND SKELETAL MUSCLE...............................................66









3 .1 In tro d u ctio n ................................................................................................................. 6 6
3.2 B asics of M agnetic R esonance ........................................................................... .... ... 66
3.2.1 M magnetic Property of Nuclear Spins ........................... .................................... 67
3.2.2 Larmor Frequency .............. ........................................... ... .. .. .... 68
3.2.3 Longitudinal and Transverse M agnetizations ................................ ............... 69
3.2.4 R relaxation Tim es .................. ........................ ....... ...... ............ .. 71
3.2.5 Fourier Transform .......................................... ............... .... ....... 73
3.3 M magnetic R resonance Im aging...................... .... .................. ................... ............... 73
3.3.1 T 1 and T2 W eighted Im ages ............................................................................ 74
3.3.2 Im age C construction ............................ ...................... ... .......... ................75
3.4 M RI A applications in Skeletal M uscle........................................ ........................... 78
3.5 M agnetic Resonance Spectroscopy ............................................................................ 82
3.5.1 Contrasting M RI and M R S......................... ......... ........................ ............... 82
3.5.2 N uclei Studied w ith M R S..................... ........ ........................ ............... 83
3.5.3 Spectral Components of MRS ............. ............ ............... 84
3.5.4 C hem ical Shift in M R S ................................................ .............................. 84
3.5.5 Correction of Saturation Effects............. .... ...................................... 86
3.5.6 Proton Spectroscopy (1H -M R S) ........................................ ......... ............... 86
3.5.7 Phosphorus Spectroscopy (31P-MRS).............................. ... ...............87
3.6 Application of Magnetic Resonance Spectroscopy in Skeletal Muscle .........................89
3.6.1 Role of 1H-MRS : Quantify Intramuscular Fat............... .... .................90
3.6.2 Role of 1H-MRS: Assess Muscle T2 Characteristics .......................... ..........90
3.6.3 Role of 31P-MRS: Quantify Resting Muscle Metabolites ...................................91
3.6.4 Role of 31P-MRS: Identify Fiber-type and Muscle Fatigue.............................. 92
3.6.5 Role of 31P-MRS: Measure Muscle Oxidative Capacity ......................................93
3.6.6 Relationship between PCr Recovery Rates and Muscle Oxidative Capacity.........95
3 .7 Su m m ary ............................................................................... 9 8

4 OUTLINE OF EXPERIM EN TS ............................................... ............................... 107

4.1 Experiment One .................. .................. ..................... ..... .... ....... ...... 107
4.1.1 Specific aim .....................................................................107
4.1.2 H ypotheses ..... ...................................................... .... ..... .. ............. 107
4.2. E xperim ent Tw o ....................................................... 107
4 .2 .1 S p e cific A im ................................................................................................10 7
4.2.2 H ypotheses ............................ ............ .............................. 108
4 .3 E xperim ent T three ...............................................................108
4.3.1 Specific A im .................................................... .......... .. ............ 108
4 .3 .2 H y p o th e sis .................................................................. .................................. 1 0 8
4.4. Experim ent Four ............... ............................................ ...... ...... .......... 108
4.4.1 Specific A im .................. .................. ................. ......... .. ............ 108
4 .4 .2 H y p oth eses ...............................................................109
4.5. Experiment Five ............... ................ .......... ................. ......... 109
4.5.1 Specific A im .................................................... ........... .. ............ 109
4 .5 .2 H y p oth eses ...............................................................109
4.6. Experiment Six ..................................................................... ......... 110
4.6.1 Specific Aim : ........................................................................... ...110


8









4 .6 .2 H y p o th e se s .................................................................. .................................1 10

5 M ETHODOLOGY .............. ................. ............ ............... .............. 111

5.1 H um an Studies................................................. 111
5.1.1 Subjects .................................. ...... .... ................. ............... 111
5.1.2 Clinical A ssessm ents ..................................... ....... ...... .. ........ .... 112
5.1.3 L ocom otor Training .............................. ....................... .... 113
5.1.4 Proton Magnetic Resonance Imaging (1H-MRI)............................................. 114
5.1.4.1 Muscle cross sectional area: data collection and analysis..........................115
5.1.4.2 T2 relaxation times: data collection and analysis..................................... 116
5.1.5 Proton Magnetic Resonance Spectroscopy (1H-MRS) .....................................116
5.1.5.1 M uscle lipid: data analysis .................................... ............. 117
5.1.5.2 Muscle T2 relaxation times: data analysis ......................................118
5.2 A nim al Stu dies ................................. ........................................................ 118
5.2 .1 A nim als ......................................................118
5.2.2 Gender Differences in Animal Models of SCI..........................................119
5.2.3 Spinal Cord Contusion Injury.......... ................................................. ........ 119
5.2.4 Experimental Electrical Stimulation Protocol ................................................ 120
5.2.5 Phosphorus Magnetic Resonance Spectroscopy (31P-MRS): Data Collection .....121
5.2.5.1 31P-M R S spectral analysis at rest ......................................................... 121
5.2.5.2 31P-MRS spectral analysis of electrical stimulation protocol.................122
5.2.6 B iochem ical A ssays............................................ ....................................... 123
5.2.6.1 A TP m easurem ents ............................................................................. 124
5.2.6.2 Total creatine measurements.............. .............. .... ............... 124

6 EXPERIMENT ONE LOWER EXTREMITY MUSCLE CROSS-SECTIONAL
AREA AFTER INCOMPLETE SPINAL CORD INJURY...............................................129

6 .1 S u m m ary ................................ ......................................................12 9
6 .2 In tro d u ctio n ............................................................................................................... 1 3 0
6 .3 M e th o d s .................................................................................................................... 1 3 2
6.3.1 Subjects .......... .. ............ ..... .. ......... ..........132
6.3.2 M maximum M uscle Cross-sectional Area ................... ...... ...... .....................133
6.3.3 Data Analysis ................................ ........................... ....... 133
6 .4 R esults....... ...........................................................134
6.5 Discussion........................ .............. ..................... 136

7 EXPERIMENT TWO NON-INVASIVE ASSESSMENT OF LOWER EXTREMITY
MUSCLE COMPOSITION AFTER INCOMPLETE SPINAL CORD INJURY ..............147

7 .1 S u m m a ry ........................ ................. ....................................................14 7
7.2 B background ..............................147............. ................
7 .3 M e th o d s .................................................................................................................... 1 4 9
7.4 R esults..... ...........................................................150
7 .5 D isc u ssio n ................................................................................................................. 1 5 1




9









8 EXPERIMENT THREE MAGNETIC RESONANCE ASSESSMENT OF MUSCLE
DAMAGE DURING LOCOMOTOR TRAINING IN PERSONS WITH
INCOMPLETE SPINAL CORD INJURY............... ........................... 162

8 .1 S u m m a ry .........................................................................................................................1 6 2
8 .2 In tro d u ctio n ............................................................................................................... 1 6 2
8 .3 M e th o d s ..........................................................................................................................1 6 3
8 .4 R esu lts....... ...........................................................164
8 .5 D isc u ssio n ....................................................................................................................... 1 6 4
8 .6 C o n c lu sio n ................................................................................................................ 1 6 5

9 EXPERIMENT FOUR IMPACT OF LOCOMOTOR TRAINING ON MUSCLE
SIZE AND INTRAMUSCULAR FAT AFTER SPINAL CORD INJURY ........................167

9 .1 Su m m ary ......... ...... .......... ...................................... ...........................16 7
9 .2 In tro d u ctio n ............................................................................................................... 1 6 7
9 .3 M e th o d s ..........................................................................................................................1 6 9
9 .4 R esu lts....... ...........................................................169
9 .5 D isc u ssio n ....................................................................................................................... 1 7 0

10 EXPERIMENT FIVE MONITORING ALTERATIONS IN INORGANIC
PHOSPHATE OF HINDLIMB MUSCLE AFTER SPINAL CORD CONTUSION IN
R A T S .................................................................................................................................... 1 7 6

10 .1 S u m m ary ...............................................................................17 6
10 .2 In tro d u ctio n .......................................................................................................... ... 17 6
10.3 Specific Aim s and Hypothesis ...................................................................178
10.3.1 Specific A im ............................................................................................ ..... 178
10 .3 .2 H y p oth eses ...............................................................17 8
10.4 M methods ...................... ............................................... 179
10 .4 .1 E x p erim ental D esign ..........................................................................................179
1 0 .4 .2 A n im a ls.............................................. ...................... ................... ............... 1 7 9
10.4.3 Magnetic Resonance Spectroscopy: Data Collection and Analysis ................ 179
10.4.3.1 31P-MRS spectral analysis at rest ....... .................. ..........180
10.4.4 B iochem ical A ssays..................................................... 181
10.4.5 Data Analysis ................................ ...............................181
10 .5 R e su lts......... ...........................................................18 1
10 .6 D iscu ssion ................................1 82.........................

11 EXPERIMENT SIX IN-VIVO ASSESSMENT OF SKELETAL MUSCLE
BIOENERGETICS AFTER SPINAL CORD CONTUSION IN RATS ..............................191

1 1 .1 S u m m a ry ................................ ...................................................19 1
11.2 Introduction ...................... ............... 191
11.3 Specific A im s and Hypothesis .................................. ........ ..............................194
11.3.1 Specific A im ......................................................................................... ............. 194
11.3.2 Hypotheses ................................ ............................. ........ 194



10









11.4 M methods .............. .......................................... 194
11.4.1 Experim ental D esign ................................................. ............................. 194
1 1.4 .2 A n im als............... .................................................................................................. 19 5
11.4 .3 D ata C collection ............... .. ........ ......................... ........... ... ......... 195
11.4.3.1 Experimental electrical stimulation protocol .......................................195
11.4.3.2 31P m agnetic resonance spectroscopy ..................................................... 196
11.4.4 31P magnetic resonance spectroscopy: data analysis ............... ................197
11.4 .5 B ioch em ical A ssay s............................................. ......................................... 19 8
11.4.6 Statistical Analysis .............. ................... ......................... ................ 198
11.5 R results ......................... .... .............. ......................................... ...............................198
11.6 D discussion ......... .......... .....................................200

12 C O N C L U S IO N ............................................................................................................... 2 18

LIST OF REFERENCES ......... .. ........ ..................................................................... 219

BIOGRAPHICAL SKETCH .................... ....................................245




































11









LIST OF TABLES


Table page

3-1 Longitudinal (T1) relaxation times (in milliseconds) of water and lipid components. .....99

3-2 Transverse (T2) relaxation times (in milliseconds) of water and lipid components..........99

6-1 Characteristics of subjects after income plete-SCI ...........................................................142

6-2 Percentage differences between the lower extremity maximum muscle CSA .............142

6-3 Relative proportions of muscles in the pooled i-SCI and control groups........................143

7-1 Characteristics of individuals with income plete-SCI........................................................ 157

7-2 Percent differences between the T2 relaxation times ....................................................... 157

8-I MR measures of T2 relaxation times of lower extremity muscles before and after
LT......................... .................................................. 166

10-1 Absolute phosphate metabolite concentrations after spinal cord contusion in rats.........187

11-1 Kinetic 31P-MRS data from the rat gastrocnemius muscle........ ..................211









LIST OF FIGURES


Figure page

3-1 Schematic representation of a nucleus and its behavior as a bar magnet ......................100

3-2 Application of a 90 RF pulse ...................................................................... 100

3-3 T 1 relaxation tim e ............................................................... .. ............ 101

3-4 T2 relaxation tim e ............................................................... .............. 101

3-5 Fourier transform of the FID signal....................................................... ............... 102

3-6 Schematic representation of two tissues with different T1 relaxation times .................102

3-7 Schematic representation of two tissues with different T2 relaxation times .................103

3-8 Bandwidth of frequencies excites a specific width of slice in the sample.......................103

3-9 Frequency and phase encoding gradient effects on spins.. ..............................................104

3-10 Representative H-MR spectrum of a healthy human soleus muscle at 1.5Tesla ..........104

3-11 Representative H-MR spectrum of a human skeletal muscle at 1.5Tesla .......... ......105

3-12 Representative typical 3p1-MR spectrum of a rat calf muscle at 11Tesla...................105

3-13 Schematic representation of the creatine-phosphocreatine shuttle and buffer role of
the creatine-kinase reaction m uscle. ...........................................................................106

5-1 Representative 1H-MRI coronal image of the calf muscles from a healthy control ........126

5-2 Representative 1H-MRI trans-axial image of the calf muscles from a healthy control ...126

5-3 A) Representative 1H-MRI T2 weighed images B) Individual pixel signal intensities
C ) R representative T2 m ap. ..................................................................... ...................127

5-4 Representative trans-axial image presents a voxel prescribed over the soleus muscle ...127

5-5 Decomposition of a lipid peak obtained from a healthy soleus muscle.........................128

5-9 Experimental-setup for electrical stimulation protocol during 31P-MRS data
acquisition ......... ...... ............ ..................................... ...........................128

6-1 Representative trans-axial proton magnetic resonance images obtained at 1.5Tesla......144

6-2 Muscle CSA in the pooled incomplete-SCI and control groups.................................... 145









6-3 Proportion ratios of muscle groups within the leg and thigh........................................146

6-4 Ratio of individual plantar flexor muscles to the max CSA of the posterior
com part ent of the leg .. ........................................................................ .................... 146

7-1 Representative T2 weighted trans-axial proton magnetic resonance images of the
low er leg ................... ........................................................................ 15 8

7- 2 Representative proton magnetic resonance spectra obtained from the soleus muscle.....159

7-3 Box-plot depicting variability in EMCL/water, IMCL/water and total soleus muscle
lipid/w after ratios. .......................................... ........................... 160

7-4 Individual data comparisons of IMCL to water ratio (IMCL/water) ............. ...............161

7-5 Individual data comparisons of EMCL to water ratio (EMCL/water) .............................161

9-1 Cross sectional area of lower leg muscles after nine weeks of locomotor training......... 174

9-2 Cross sectional area of thigh muscles after nine weeks of locomotor training.............. 174

9-3 Estimates of soleus muscle lipid before (Pre LT) and after (Post LT) nine weeks of
locom otor training ....................................................... .......... .. ...... ..... 175

10-1 Percent change in resting phosphate metabolites of the rat hind limb muscle one
w eek after spinal cord contusion............................................. ............................. 188

10-2 Change in phosphorylation ratios [ADP][Pi]/[ATP] before and after spinal cord
contu sion ........ ......... ......................................................................18 8

10-3 Representative 31P-spectra before and after one week SCI obtained at 11T .................189

10-4 Change in [PCr] before and after spinal cord contusion* Statistically significant difference. 189

10-5 [PCr] in individual rat hind limb muscle after one week of spinal cord contusion. ........190

11-1 Representative kinetic 31P spectra of rat gastrocnemius muscle at 11T.. .....................213

11-2 Average PCr depletion and recovery graphs in response to the stimulation protocol.....214

11-3 PCr depletion rates (1, 2, and 3min) before and after spinal cord contusion injuries
(SCI)................... .. .... ...................................... ......... 214

11-4 Expanded view of PCr kinetic data obtained at the onset of electrical muscle
stimulation (EMS)........................................... .......... 215

11-5 PCr recovery graphs obtained immediately after termination of electrical muscle
stim u latio n .............................................. .. .................... ................ 2 1 5









11-6 Maximum mitochondrial oxidative capacity (Vmax mM.min1) data from eight rats
(R 1-R 8) ........................................................................................2 16

11-7 Scatter-plot depicting relationship between maximum mitochondrial capacity
(Vmax) and rate of PCr depletion (Vdep)....................... ...............216

11-8 Relationship between end stimulation [ADP] and initial rates of PCr recovery (Vex). ...217

11-9 Maximal rates of oxidative capacity (Qmax) based on the [ADP] and
[A D P ][P i]/[A T P ] m odels.. .................................................................... .....................2 17










LIST OF ABBREVIATIONS


ASIA

[ADP]

[ATP]

[ADP][Pi]/[ATP]

CSA

EMCL

EMS

End ex

FID

GAS

1H-MRS

IMCL

kpCr

LT

mM

MR

MRI

MRS

[Pi]

[PCr]

31P-MRS

Qmax

Qmax-ADP


American spinal cord injury association

Absolute concentration of free cystolic adenosine diphosphate

Absolute concentration of adenosine triphosphate

Phosphorylation potential

Maximum cross sectional area

Extramyocellular lipid

Electrical muscle stimulation

End exercise (EMS)

Free induction decay

Gastrocnemius muscle

Proton magnetic resonance spectroscopy

Intramyocellular lipid

Rate constant of PCr recovery

Locomotor training

milimoles/liter of intracellular water

Magnetic resonance

Magnetic resonance imaging

Magnetic resonance spectroscopy

Absolute concentration of inorganic phosphate

Absolute concentration of phosphocreatine

Phosphorus magnetic resonance spectroscopy

Maximum oxidative ATP synthesis rate

Maximum oxidative ATP synthesis rates based on kPCr and [ADP]










Qmax-[ADP][Pi]/[ATP]


SCI

T2

TR

Vex

Vdep

Vmax-lin

Vmeas

APCr

([tmol/g wet wt)


Maximum oxidative ATP synthesis rates based on kPCr and
phosphorylation potential.
Spinal cord injury

T2 relaxation times

Repetition time

Extrapolated initial rates of PCr recovery (kpcr*APCr)

Rates of PCr depletion at onset of EMS

Maximal rate of PCr resynthesis

Initial rates of PCr recovery (first three points in recovery)

[PCr]rest- [PCr]end ex

micromol/gram wet weight











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

MAGNETIC RESONANCE CHARACTERIZATION OF SKELETAL MUSCLE
ADAPTATIONS AFTER INCOMPLETE SPINAL CORD INJURY
By

Prithvi Krishnakant Shah

May 2008

Chair: Krista Vandenborne
Major: Rehabilitation Science

Spinal cord injury is one of the most disabling health related problems that often results in

paralysis and paresis of body musculature below the lesion site. Persons with incomplete-SCI

typically exhibit impaired motor performance and varying degrees of functional limitations.

Despite the obvious motor dysfunctions, physiological muscle adaptations following incomplete-

SCI are relatively unstudied. An understanding of the muscular adaptations following an

incomplete-SCI will help in the development of therapies aimed at reducing the secondary

effects of paralysis and paresis. The overall objective of this dissertation was to investigate

skeletal muscle adaptations following incomplete-SCI using combinations of non-invasive MRI

and MRS techniques.

Findings from our human studies reveal that chronic incomplete-SCI is associated with

significant muscle atrophy in the affected lower extremity that is uniform between limbs and

somewhat influenced by mobility status. In addition, persons with incomplete-SCI demonstrate

an increase in the total lipid, IMCL and extramyocellular EMCL content and enhancements in

the T2 relaxation properties of the lower leg muscles. Moreover, repetitive locomotor training

with body weight support and a treadmill are associated with significant increases in the









plantarflexor muscle size. Data from our animal experiments reveal that the paralyzed rat

hindlimb muscle show faster rates of PCr depletion, thereby suggesting that, after SCI, there

is either an increase in ATP requirement for similar demands in muscle contraction or the

overall supply of ATP is compromised following the injury. In addition, a pronounced

decrease in PCr recovery rates implies a less effective oxidative phosphorlyation and a

reduction in the mitochondrial oxidative capacity of skeletal muscle. Collectively, findings

from this dissertation work reveal that the paralyzed skeletal muscle shows drastic alterations

in its morphological and metabolic properties after a SCI and that these adaptations can be

successfully characterized by the use of non-invasive MR techniques.

The present work will provide a foundation from which the relationship between skeletal

muscle adaptations and function in this population can be further explored. Moreover, the use of

sophisticated MR techniques will enable characterizing the paralyzed muscle non-invasively and

with high resolution; while also allowing longitudinal follow-ups all of which are crucial in the

assessment of injury mechanisms, disease progression and efficacy of therapeutic interventions.









CHAPTER 1
SPINAL CORD INJURY AND SKELETAL MUSCLE

1.1 Significance

Spinal cord injury (SCI) results in paralysis and paresis of muscles below the injury site

making movement difficult. Impairments in skeletal muscle contribute to a host of

musculoskeletal deficits that lead to secondary health related conditions that cost an estimated

$220,000 and $750,000 per person annually (NSCISC 2006). The clinically relevant

musculoskeletal and movement disorders in persons with SCI include muscle weakness and

paralysis of affected extremities (Gordon and Mao 1994; Kim, Eng et al. 2004), early muscle

fatigue and increased energy demands for simple functional activities (Hopman, Dueck et al.

1998; Ulkar, Yavuzer et al. 2003), diminished capacity or inability to ambulate (Burns, Golding

et al. 1997; Stein, Chong et al. 2002), and overall decreased endurance and dependence on

assistive aids for locomotion (Waters, Adkins et al. 1994; Ulkar, Yavuzer et al. 2003).

Furthermore, inactivation of paralyzed skeletal muscle interferes with implementation of

therapeutic interventions (Subbarao 1991). Inactivity and sedentary lifestyle in this patient cohort

leads to unloading of paralyzed muscles. As a result, a myriad of muscle adaptations including

morphological, contractile, metabolic and neural alterations ensue following SCI (Castro, Apple

et al. 1999; Shields 2002). Note worthily, most of these adaptations have been studied following

a complete SCI. Understanding these muscular adaptations following an incomplete SCI will

help in the development of therapies aimed at reducing the secondary effects of paralysis and

paresis. The purpose of this work is to investigate skeletal muscle adaptations following

incomplete SCI using non-invasive magnetic resonance imaging (MRI) and spectroscopy (MRS)

measures. Combinations of human and animal models of incomplete SCI are utilized to achieve

this goal.









1.2 Spinal Cord Injury in Humans

The current gold standard classification system for SCI, American Spinal Injury

Association Classification System -ASIA, is based on the presence, absence and degree of

normality of motor and sensory impairments (ASIA 2001). Since these impairments are highly

variable, classifying SCI has posed a major challenge for clinicians. According to the ASIA

classification system, human SCI is classified as either complete or incomplete. Complete SCI

involves "total absence of sensory and motor functions in the lowest sacral segments", whereas

an incomplete SCI is characterized by partially preserved motor and/or sensory function below

the level of lesion, including sparing of the lowest sacral segments (ASIA 2001). Irrespective of

the severity of injury, the majority of human spinal cord injuries leave the spinal cord tissue

relatively intact (Kakulas 1987). As a result, often after infarction, contusion and/or mechanical

deformation, much of the spinal cord tissue is spared. A severed cord is seen only in rare injury

types such as sword injuries, bullet wounds or penetration of bone fragments into the cord.

Interestingly, while some individuals with significant sparing of spinal cord tissue may present as

"clinically complete," others with very little spinal cord tissue sparing are considered

"incomplete". These varying clinical signs make classifying SCI difficult and leaves clinicians

"guessing" as to the integrity of the spinal cord tissue.

With technological advancements in the management of acute SCI, and with an increased

life expectancy of persons with an incomplete injury, there is an emerging healthcare trend in the

treatment of persons with chronic SCI (NSCISC 2006). The majority of new spinal cord injuries

(-53%) occurring annually are now classified as incomplete. Given their unique injuries and

varying degrees of tissue sparing, persons with incomplete SCI constitute an extremely

heterogeneous group. Individuals after this type of injury exhibit a continuum of ambulatory

abilities ranging from being completely wheelchair dependent to nearly normal walking without









the use of assistive devices (Waters, Adkins et al. 1994; Melis, Torres-Moreno et al. 1999).

Accordingly, due to their variable paralysis and paresis, they present with impaired motor

performance and varying degrees of functional limitations (Subbarao 1991; Tang, Tuel et al.

1994; Bums, Golding et al. 1997). Despite the obvious motor dysfunctions in persons with

incomplete SCI, muscle adaptations following the injury have not been well described.

1.3 Spinal Cord Injury in Animal Models

The benefits of utilizing experimental animals is reflected in the more than hundred years

of ongoing research in animal models of SCI. Current understanding of anatomical and

molecular changes in response to human SCI has largely been derived from parallel lesion

models of experimental SCI in animals that mimic human SCI, both histologically and

behaviorally. Animal models of SCI enable investigation of SCI at a level of detail that would

not be possible in human studies. A variety of experimental animal models of adult SCI

involving rats, mice, pigs, monkeys and cats have been utilized for this purpose (Wrathall 1992;

Anderson, Howland et al. 1995; Qayumi, Janusz et al. 1997; Stokes and Jakeman 2002;

Rosenzweig and McDonald 2004; Ni, Li et al. 2005). The majority of these studies have utilized

various injury paradigms ranging from sharp transactions to blunt contusion of the spinal cord

that simulate the variations in the severity of human SCIs. Animal models of SCI have provided

insights into, and continue to contribute greatly towards our current understanding of the human

spinal cord injury mechanisms, anatomical and pathophysiological sequels of the injury,

neuromuscular adaptations following the injury, regeneration and repair of spinal cord, and

effectiveness of novel therapeutic interventions following injury. Furthermore, investigations

have also attempted to answer the question of effective duration and time frames of

implementing these interventions (Rosenzweig and McDonald 2004). Goals and objectives of a

study dictate which animal and lesion model is best suited for a given study.









1.3.1 Spinal Cord Injury in the Rat Model

Of the various animal models mentioned above, research on studying the rat model of SCI

has gained tremendous momentum in the recent years (Wrathall 1992; Kwon, Oxland et al. 2002;

Young 2002). In the 1980s, the feline spinal cord model dominated the field of SCI research in

investigating the wide gamut of neurophysiological changes including biochemical,

physiological, vascular and metabolic changes following the injury (Young 2002). In fact, the cat

spinal model yielded several therapies that went to clinical trial in humans. One of the most

classic examples is the first clinical trial of methylprednisolone in 1985 (Bracken, Shepard et al.

1985). However, in recent years the trend has been towards the use of rats for SCI studies. Two

major interconnected determinants of this transition have been recognized. Firstly, greater

percentages of SCI in humans are now being classified as incomplete injuries. Consequently, the

weight drop device similar to that used for the feline spinal cord contusion model was developed

by Noble and Wrathall in 1987 for use in rats. This device induces spinal cord contusion in rats

that morphologically and behaviorally mimics human incomplete spinal cord injuries (Wrathall,

Pettegrew et al. 1985; Noble and Wrathall 1989) (see below for more details). Secondly, rat

models are advantageous because they are easily accessible and large groups of rats are easy to

manage in a laboratory setting. Rat models allow for practical post-operative animal care,

conduction of longitudinal studies in a feasible time frame due to relatively shorter lifespan of

animals, and considerably lower initial costs (Khan, Havey et al. 1999); while simultaneously

serving the purpose of mimicking an injury that corresponds to human SCI. Assessment of post-

injury symptoms in the rat model is reliably and validly tested using the Basso Bresnahan,

Beattie scale (BBB) (Basso, Beattie et al. 1995).

Lastly, with the explosion of genetic research in neuroscience, SCI mice models are

becoming more prevalent (Stokes and Jakeman 2002). However, this model has not as well been









validated and suffers from relatively larger variability in injury production and locomotor

functional assessments post-injury (Rosenzweig and McDonald 2004; IOM 2005).

1.3.1.1 Spinal cord contusion model in rats

The common experimental rat models of SCI include transaction, compression and

contusion (Kwon, Oxland et al. 2002; Rosenzweig and McDonald 2004). Each of these models

exhibit unique characteristics and are used as per the experimental objectives. A transaction rat

model produces complete injury to the spinal cord and is more commonly used to study the

neurophysiological processes/mechanisms above or below cord lesion sites. Transection model

studies overcome the confounding effects of spared neural pathways that are commonly present

in studies using incomplete injury models. A compression model, on the other hand, causes cord

compression and better simulates ischemic injuries to the spinal cord. This model is commonly

used to study the acute vascular pathophysiology following SCI (Kwon, Oxland et al. 2002;

Young 2002). Finally, the SCI contusion model produces mechanical impaction of the cord and

most closely imitates an incomplete SCI in humans both pathophysiologically and behaviorally

and can create graded focal injuries similar to humans (Metz, Curt et al. 2000). In the present

dissertation work, SCI contusion injury in rats is utilized as the model of choice.

Various approaches have been devised to induce an experimental contusion injury in the

rat model including weight drop impaction of an exposed spinal cord (Wrathall, Pettegrew et al.

1985), contusion by compressing the cord between the arms of an aneurysmal clip (Rivlin and

Tator 1978), arterial occlusion ((IOM 2005) or chemical toxicity (Magnuson, Trinder et al.

1999). Of these, induction of contusion injury to the cord by impaction has proven to be most

relevant to incomplete, traumatic SCI in humans. The mechanical impact by a weight drop on the

exposed cord reflects the dynamics of clinical injury that occurs in humans (Khan, Havey et al.

1999; Metz, Curt et al. 2000; Kwon, Oxland et al. 2002; Young 2002). The majority of human









incomplete spinal injuries are a direct result of trauma to the cord. Traumatic SCI in humans

causes an initial mechanical insult to the cord that involves a primary lesion, termed the umbra

region, followed by a penumbra or secondary injury that involves either further damage to

already damaged tissue or new damage to otherwise healthy tissue (Hall and Springer 2004).

The underlying molecular and pathophysiological mechanisms and the functional symptoms

following an incomplete SCI in humans are well reproducible in the contusion rat model. In fact,

electrophysiological outcomes such as motor evoked potentials (MEP) and somatosensory

evoked potentials (SSEP), spinal cord lesion morphology, relationship between the morphology

and functional measures such as functional locomotor capacity have been demonstrated to be

analogous in humans with SCI and spinally contused rats (Noble and Wrathall 1985; Noble and

Wrathall 1989; Metz, Curt et al. 2000). As a result, the contusion animal model of SCI has been

established as a valid model of SCI for comparison between rats and humans.

1.3.1.2 Spinal contusion by impaction

The weight drop device used to induce spinal contusion injury was first introduced by

Allen in 1911 where weight was dropped onto canine cords exposed by laminectomy (Young

2002). Unfortunately, Allen's death in World War II created a void in the use of this model. It

was only in the late 1960s, that investigators revived the contusion model in the primate and

feline spinal cord to describe the histopathological effects, evoked potentials and assess effects of

corticosteroids and hypothermia in response to SCI. However, owing to questionable

reproducibility of the weight drop technique, a standard graded and reproducible spinal

contusion injury procedure by impaction was warranted. By making definite alterations in the

height at which a 10g weight was dropped on the exposed dura, a reproducible injury to the rat

spinal cord was established by Wrathall et al in 1985 (Wrathall, Pettegrew et al. 1985).

Depending upon the desired severity of injury to the cord, the 10g weight is dropped from









heights of 2.5cm, 5cm or 17.5 cm on the exposed dura. Accordingly, injury to the cord is

considered mild, moderate or severe respectively, corresponding to similar severity of spinal

injuries in humans.

Before experimental therapeutic interventions can be implemented, standard protocols are

required to help minimize variability in injury between animals. Indeed, the magnitude of

mechanical injury produced by these methods have been validated and proven to closely

correlate with histological, behavioral, electrophysiological evaluations and functional

measurements following SCI (Gale, Kerasidis et al. 1985; Noble and Wrathall 1985; Metz, Curt

et al. 2000). Three standard devices are now used to produce this injury in rodents: Multicenter

Animal Spinal Cord Injury Study (MASCIS) impactor formerly called the New York impactor;

the Ohio State University impactor and the Infinite Horizons device (Wrathall 1992; Basso,

Beattie et al. 1996; Khan, Havey et al. 1999; IOM 2005).

1.3.2 Assessment of Locomotor Behavior in Rats

One of the first behavioral test scores to assess recovery in spinal injured animals was

established by Tarlov and Klinger in 1954. Briefly, the testing used a six-point scale to assess

motor function recovery after spinal injury in dogs (grade 0 = no spontaneous movement; grade

5 = normal motor function) (Tarlov and Klinger 1954). The Tarlov score, as it is commonly

named today, has emerged as a popular tool to assess motor recovery in a variety of spinal

injured animals including rats. In addition, various other motor scoring approaches have been

specifically formulated for the spinal injured rat. Bresnahan et al in 1987 compiled a behavioral

testing strategy that included assessments of general locomotor skill (in an open field), fine

locomotor skills (through grid walking task) and postural adjustment to displacement (in an

inclined plane) after SCI in rats (Bresnahan, Beattie et al. 1987). However, a valid and reliable









tool the Basso, Beattie and Bresnahan score, also commonly known as the BBB score was

launched only in 1995 (Basso, Beattie et al. 1995).

The BBB score is an operationally defined ordinal scale that assesses hind limb locomotor

recovery of thoracic spinal cord injured rats. It is the first assessment tool that has been validated

with testers from several laboratories across the United States (Basso, Beattie et al. 1996). Each

of the components of the 21 point scale is based on specific features of locomotor recovery after

spinal cord contusion in rats including joint movements, trunk posture, weight support, stepping

and weight support ability, coordination between forelimbs and hind limbs, and tail position.

The score order from 0 to 20 assumes progressive recovery with every score representing a

unique and sequential stage of locomotor recovery: a) scores 1 through 8 center around recovery

of joint movements unique to the early phase of recovery, b) scores 9-13 focus upon initial

recovery of stepping ability and coordination that represents an intermediate stage of recovery

and c) scores 14-21 focus on the progression in foot placement, toe clearance trunk stability and

tail position during stepping that represents the late and final phase of recovery. Testing consists

of placing the animal in an open field beginning as early as one day post injury and observing it

for 4 minutes. Behavioral scoring is done by two observers in real time with repeated testing

every week for 6-9 weeks. Used extensively throughout the neurotrauma literature, the scoring

system is sensitive enough to differentiate between severities of the spinal injury including mild

to severe spinal contusion and transaction (Basso, Beattie et al. 1996).

Though the versatility of the BBB score makes it a valuable locomotor assessment tool,

investigators have also questioned its accuracy in assessing specific categories such as

coordination. Accordingly, measures such as the Catwalk analysis have been proposed as

potential addendums to the standard BBB scale (Koopmans, Deumens et al. 2005). The Catwalk









analysis allows collection of data on dynamics of locomotion such as degree of coordination and

weight bearing, duration of gait cycles etc. In addition, various other measures have been

identified to assess locomotor recovery after SCI in the rat; the use of which depends on the

research question posed by investigators. The inclined plane test for example measures the

animals' ability to maintain position in an inclined plane and is commonly used to assess balance

and posture (Rivlin and Tator 1977; Bresnahan, Beattie et al. 1987). The crossway tests involves

crossing runways such as a beam or grid (Kunkel-Bagden and Bregman 1990). The substantial

motor control required to pass this test makes it suitable for assessing limb coordination. Video

recordings from these tests yield data such as time taken and number of errors made during

crossing. Treadmill walking is yet another measure of assessing locomotor recovery and is often

used for assessment of footprint and kinematical analysis of individual joint movements

(Kunkel-Bagden and Bregman 1990).

1.4 Skeletal Muscle Adaptations after SCI

Skeletal muscle adaptations following SCI are a direct consequence of neuronal damage

within the spinal cord and an indirect result of prolonged periods of muscle inactivation. In

addition, paralyzed skeletal muscle is subject to altered loading conditions and muscle length

(Alaimo, Smith et al. 1984; Gordon and Mao 1994; Dietz, Colombo et al. 1995; Shields 2002).

Ultimately, a myriad of muscle adaptations including morphological, contractile and metabolic

muscle alterations occur following SCI. The following discussion is focused on adaptations of

paralyzed muscles that are partially or completely devoid of a descending spinal drive and yet

have an intact peripheral nerve supply.

1.4.1 Morphological Adaptations: Muscle Atrophy

Like any other model of muscle disuse, such as cast immobilization (Vandenborne, Elliott

et al. 1998; Stevens, Walter et al. 2004), unilateral lower limb suspension (Berg, Dudley et al.









1991; Hather, Adams et al. 1992), bed rest (Berg, Larsson et al. 1997; Alkner and Tesch 2004),

space flight (Akima, Kawakami et al. 2000; Tesch, Berg et al. 2005) and inactivity secondary to

injury or disease processes (Arokoski, Arokoski et al. 2002; Johansen, Shubert et al. 2003), SCI

is accompanied by marked atrophy of the paralyzed muscles. The abovementioned human

models of disuse atrophy result in 15% 32% decrease in muscle size of the immobilized lower

extremity muscles, depending upon the muscles tested and time of testing. A number of studies

have reported effects of disuse ranging from immobilization periods of 1 week to almost 17

weeks. Generally, greater declines in muscle size are reported for postural and antigravity

muscles such as the soleus and vasti versus the knee and ankle flexors of the lower extremity

(Hather, Adams et al. 1992; Akima, Kawakami et al. 2000; Tesch, Berg et al. 2005). In

comparison to other disuse models, atrophy of the paralyzed muscles following SCI is markedly

higher. An overall 40-80% decline in the average CSA of human lower extremity muscles has

been reported 24 weeks after complete SCI. The degree of atrophy typically depends upon the

functional role, shortened muscle length related to posture assumed by paralyzed limbs and

anatomical location of muscles (Castro, Apple et al. 1999). Using magnetic resonance imaging

(MRI) techniques, Castro et al reported that the antigravity plantarflexor muscles show greater

decline in CSA as compared to the dorsiflexors (40% versus 20%). On the other hand, the thigh

antigravity knee extensors and knee flexors, unlike in other models of muscle disuse (Tesch,

Berg et al. 2005), show similar degrees of atrophy (-42%) (Castro, Apple et al. 1999). While

this initial study by Castro et al reported the muscle CSA inclusive of fat within the muscle, the

same research group subsequently reported an almost 38% decline in total thigh fat-free muscle

CSA of the injured group as compared to controls (Elder, Apple et al. 2004). Modelesky et al

also estimated a 38-44% decline in the fat-free muscle mass in individuals with complete SCI









two years after injury using MRI and Dual X-Ray absorptiometry (Modlesky, Bickel et al. 2004).

Other investigators have studied alterations in muscle fiber size based on needle biopsies.

Declines of 54-74% in muscle fiber size are observed by 24 weeks after SCI with maximum

declines seen in muscle fiber types that have a greater fiber CSA initially (i.e. fast twitch fibers)

(Scelsi, Marchetti et al. 1982; Castro, Apple et al. 1999). While maximum rates of decline in

whole muscle size (60%) and muscle fiber size (74%) are seen during the first 6 weeks after

injury, progressive atrophy is observed for as long as one year after SCI (Scelsi, Marchetti et al.

1982). Furthermore, atrophy appears to be muscle specific: the gastrocnemius muscle, for

example, continues to atrophy for as long as 6 months, whereas the tibialis anterior and

semimembranosus reach a plateau by as early as 6 weeks after injury (Castro, Apple et al. 1999).

While maximum emphasis has been placed on studying atrophic adaptations in the lower

extremity paralyzed muscles, a couple of studies have also reported atrophy of upper extremity

muscles (Thomas, Zaidner et al. 1997) and abdominal muscles (Estenne, Pinet et al. 2000)

following SCI.

Atrophy following animal models of SCI is in concurrence with humans with SCI where

almost 10% to 56% of whole muscle and fiber atrophy is observed depending upon the muscles

studied and the model of injury based on injury severity (Roy and Acosta 1986; West, Roy et al.

1986; Dupont-Versteegden, Houle et al. 1998; Gregory, Vandenborne et al. 2003; Otis, Roy et al.

2004). Similar to human studies, larger degrees of atrophy are observed in the slow antigravity

soleus muscle as compared to the fast flexor muscles such as the tibialis anterior or extensor

digitorum longus. Moreover, similar to humans, antigravity muscles continue to atrophy for at

least 6 months after SCI. However, unlike the paralyzed knee flexors in humans, minimal

declines are seen in the non-antigravity knee flexor muscles (semitendinosus) following SCI in









animal models (Roy and Acosta 1986; Roy, Talmadge et al. 1998; Otis, Roy et al. 2004). While

all the above-mentioned studies have focused on the complete SCI human and animal

models, no studies in humans and just few spinal contusion animal model investigations

have reported the impact on skeletal muscle after incomplete spinal cord injuries.

Hutchinson et al studied lower extremity muscle atrophy following contusion injury in rats over

the course often weeks (Hutchinson, Linderman et al. 2001). At one week, they found a 20-25%

decline in the wet weight of lower leg muscles soleuss, plantaris, gastrocnemius and tibialis

anterior) and no change in the EDL muscle wet weight. While spontaneous recovery occurred in

the soleus, plantaris and tibialis anterior muscles by three weeks, the gastrocnemius muscles

continued to show declines for as long as ten weeks (Hutchinson, Linderman et al. 2001).

Similarly, Liu et al have reported an 11%-26% decline in the triceps surae and tibialis anterior

cross sectional areas two weeks after spinal contusion injury. Spontaneous recovery in the

paralyzed muscle was seen by four weeks (Liu, Bose et al. 2008).

In the present work, muscle cross-sectional area of paralyzed lower extremity muscle is

studied in humans with incomplete SCI. In addition, the impact of ambulatory status of persons

with incomplete SCI on muscle size is determined. A detailed explanation of this study follows

in Chapter 6.

1.4.2 Morphological Adaptations: Muscle Fiber Type Conversion

Muscle fiber type conversion is another hallmark of chronically paralyzed skeletal muscle.

Muscle fibers can be categorized using several classification systems. The most common and

recent refinement for characterization of muscle fibers is based upon the myosin heavy chain

(MHC) expression of muscle fibers. MHC is a critical structural and enzymatic muscle protein

that possesses distinct molecular forms (isoforms). MHC isoforms control the pH of the myosin

ATPase reaction and accordingly are responsible for the degree of histochemical staining of









muscle fibers during immunocytochemistry (McComas 1996). Depending upon the staining, four

main isoforms of the MHC corresponding to I, IIa, IIx, lib fiber types have been identified in the

mammalian skeletal muscle. Thus, fibers that stain strongly at pH 9.4 are referred to as type II in

contrast to the poorly reacting type I fibers (McComas 1996). Type II fibers are further classified

as IIa, lib or IIx on the basis of the strength of staining. (Note: lib fiber type in humans is now

termed as type IIx fibers rendering humans without type lib fibers). Importantly, MHC isoforms

also determine the rate of cross-bridge reactions with actin filaments and therefore the speed of

muscle shortening. Accordingly, type I fibers generally are the slow twitch fibers that generate

twitch forces slowly and are less fatigable. By and large, type I fibers possess relatively higher

concentrations of oxidative phosphorylation enzymes, mitochondrial content, capillary density

and redox proteins; thereby making them more suitable for sustained periods of ATP production.

Type lib fibers on the other hand quickly generate peak tension and are easily fatigable. They are

generally equipped with relatively larger concentrations of glycolytic enzymes, phosphocreatine,

glycogen, calcium sequestrating proteins and sarcoplasmic reticulum; thereby making them more

suitable for burst activities that demand rapid production of ATP. Fast twitch fibers contain

relatively lower mitochondrial content, blood capillaries and hence redox proteins. Finally, Type

IIa and IIx muscle fibers possess intermediate characteristics. Many studies also report existence

of a mixed/hybrid phenotype such as type 1/IIa and IIx/IIb; especially in paralyzed muscles

(Roy, Talmadge et al. 1999). While the above-mentioned classification is confined to muscle

fibers, categorization of whole muscle is not uncommon. Whole muscles are generally termed as

slow or fast twitch based upon the proportion of fiber types, though both fiber types co-exist in

the same muscle. Thus, a slow muscle is generally recognized as having relatively greater

oxidative capacity, predominantly consists of type 1 fibers, is functionally more fatigue resistant,









has slow twitch muscle properties (see below) and participates in slow phasic muscle activities.

Based on the resting content of bioenergetically important metabolites, slow muscles also have

relatively higher Pi/PCr ratios as compared to white muscles; the differences being largely due to

a higher Pi content (Meyer, Brown et al. 1985; Kushmerick, Moerland et al. 1992). Examples of

such a muscle include the soleus and vastus medialis. A fast muscle in contrast, demonstrates an

overall faster contractile property, is more fatigable, with relatively lower mitochondrial

oxidative capacity and larger glycolytic capacity, predominantly consists of type II fibers and

plays a major role in fast tonic activities (such as sprinting). Metabolically, fast muscles have

relatively lower Pi/PCr ratios as compared to slow muscles; the differences being largely due to a

higher PCr content (Kushmerick, Moerland et al. 1992). Examples include the gastrocnemius

and vastus lateralis muscles. While skeletal muscles of animals are more homogenous in the

fiber type composition and characteristics, the human skeletal muscle is extremely

heterogeneous.

Usually, muscle inactivity due to disuse, immobilization or even decreased neural activity

causes muscle fiber type conversion from the slow to fast phenotype. Reverse expression of

muscle fiber type is observed with increased muscle activity. There is a decrease in the

expression of type I muscle fibers and an increase in the proportion of hybrid type I+IIa and pure

type IIb and/or IIx muscle fibers after chronic SCI in both animal models (Mayer, Burke et al.

1984; Lieber, Friden et al. 1986; West, Roy et al. 1986; Roy, Talmadge et al. 1999; Otis, Roy et

al. 2004) and in humans (Scelsi, Marchetti et al. 1982; Lotta, Scelsi et al. 1991; Burnham, Martin

et al. 1997; Castro, Apple et al. 1999). Similar to complete SCI, recent studies using the rat

model of incomplete SCI have also demonstrated a similar shift in fiber type expression with

declines in the levels of type IIa, elevations in type IIb MHC along with the presence of a









transitional type IIx MHC (that is normally not seen in the control soleus muscle) after

incomplete injuries (Hutchinson, Linderman et al. 2001; Stevens, Liu et al. 2006). In incomplete

models of SCI, this conversion occurs as early as 1-3weeks and is reversible (Hutchinson,

Linderman et al. 2001). This fiber type conversion after SCI leads to significant alterations in

the characteristics of paralyzed muscle including a change in contractile properties, metabolic

capacities and an impaired endurance capacity (Hopman, Nommensen et al. 1994; Bumham,

Martin et al. 1997).

1.4.3 Adaptations to Contractile Properties

After SCI, muscles with a greater proportion of type I fibers and slower contractile

properties (slow twitch muscles) generally exhibit characteristics analogous to those of muscles

with a greater proportion of type II fibers and relatively faster contractile properties (fast twitch

muscle). Most human and animal studies concur that the contraction and relaxation speeds of

slow twitch muscle, but not fast twitch muscles, are significantly faster following chronic SCI

(one year after injury) when compared to before injury (Lieber, Friden et al. 1986; Roy and

Acosta 1986; Gerrits, De Haan et al. 1999; Roy, Talmadge et al. 1999; Shields 2002).

In the spinalized animal model, contractile properties of the paralyzed soleus muscle (an

otherwise slow muscle) are consistent with those of a primarily fast muscle. Elevations in the

slow muscle contraction speeds after SCI are reflected as decreases in time to peak tension (by

almost 20ms), increase in specific tension (by almost 100%), and increases in fusion frequency

(by almost 100%). In addition, paralyzed muscle demonstrates faster twitch half-relaxation times

(Lieber, Johansson et al. 1986; Roy and Acosta 1986). While adaptations following a complete

SCI are quite drastic, contractile properties after an incomplete SCI are relatively less marked. A

decrease in half- relaxation time by 20% in the soleus muscle that recovers to control values by

ten weeks is reported in rodents with a moderate spinal cord contusion (Stevens, Liu et al. 2006).









Though conversion of muscle fiber type is generally purported as reasons for alterations in

contractile properties, various molecular mechanisms are suggested for this change. These

mechanisms include decreases in Ca2+ uptake required for the actin-myosin coupling mechanism

and change in endoplasmic reticulum functioning secondary to altered motor neuron activity and

muscle activation. Because the soleus muscle is well established to be more sensitive to

decreased neuromuscular activation (Roy and Acosta 1986; Roy, Talmadge et al. 1998), it is

studied more extensively to explore the contractile properties of the skeletal muscle following

SCI.

Similar to animal studies, chronically paralyzed individuals with complete SCI exhibit

faster contractile properties of skeletal muscles. Depending upon the muscle studied, rates of

force rise (contraction rates) have been reported to be significantly greater (+52%) and half-

relaxation times are markedly shorter (-19% to 2fold) as compared to a healthy muscle.

Similarly, contractile properties from chronically paralyzed upper extremity thenar muscles also

show fast muscle characteristics (Thomas 1997). Note worthily, at relatively earlier time points

after injury (within 6-24 weeks post SCI), contractile properties of the human paralyzed muscles

are rather variable. Time to peak tension of the paralyzed soleus and quadriceps muscles, for

example, has been demonstrated to remain the same or even slower than control values (Shields,

Law et al. 1997; Castro, Apple et al. 2000). This disparity in the contractile properties of acute

and chronic paralyzed muscles is attributed to the relationship between contractile speed and

MHC isoform expression (Burnham, Martin et al. 1997; Dupont-Versteegden, Houle et al. 1998).

At 6-24 weeks, fiber type conversion and hence MHC type expression has not occurred thereby

retaining the contractile characteristics of the paralyzed muscle (Shields, Law et al. 1997; Castro,

Apple et al. 1999). As far as the relaxation time is concerned, the half-relaxation times are slower









in the skeletal muscle of acutely injured subjects (-47%). Briefly, the rate of Ca+2 uptake or the

rate of the cross-bridge detachment is surmised to have been compromised thereby slowing the

overall muscle relaxation rates (Shields 2002).

Altered contractile properties of a paralyzed muscle have important functional

implications. Paralyzed human muscle fatigues much more rapidly than a healthy muscle.

Increased fatigability of skeletal muscle as characterized by force loss over repeated contractions

with otherwise "non-fatigable" electrical stimulation parameters is a common phenomenon of

chronically paralyzed slow muscles (Shields 2002). Declines in isometric force production over

repetitive bouts of contraction are as large as 60% in the paralyzed skeletal muscle as compared

to 40% declines in controls. Furthermore, muscle endurance, as measured by the muscle's ability

to maintain force levels when stimulated repeatedly, is drastically reduced following SCI. While

a healthy quadriceps muscle can maintain force levels of more than 30% of maximum isometric

contraction for as long as 10 minutes, a paralyzed quadriceps muscle can maintain similar force

levels for only less than 4 minutes (Gerrits, De Haan et al. 1999). Such weakness and fatigability

of locomotor muscles potentially limits the use of rehabilitation programs that emphasize using

functional electrical stimulation to achieve standing and walking.

1.4.4 Metabolic Adaptations: Altered Oxidative Capacity

Alteration in skeletal muscle mitochondrial oxidative capacity and subsequent impairments

in endurance are reflected by a variety of muscle adaptations following SCI. Mitochondrial

oxidative capacity is a function of mitochondrial volume, competence and oxygen delivery to the

mitochondria (McCully, Mancini et al. 1999; Kemp, Roberts et al. 2001). It is commonly used as

an estimate of oxidative energy supply to the muscle and reflects overall muscle endurance.

Mitochondrial oxidative capacity is altered following SCI (Jiang, Roy et al. 1990; Jiang, Roy et

al. 1990; Otis, Roy et al. 2004). Histochemical measurements of various oxidative enzyme









activities from muscle homogenates or biopsy samples serve as markers of muscle metabolic

capacities. Additionally, non-invasive measurements of metabolic capacities by magnetic

resonance spectroscopy have been well established and are found to significantly correlate with

mitochondrial enzymes activity measured with biopsy specimens (McCully, Fielding et al.

1993).

Generally, marked alteration in muscle oxidative capacity is reported following SCI in

humans. Martin et al and Rochester et al have shown that the chronically paralyzed human

tibialis anterior muscle shows marked decrease in succinate dehydrogenase (SDH) activity as

compared to able-bodied individuals (Martin, Stein et al. 1992; Rochester, Barron et al. 1995).

Kjaer et al have demonstrated a marked decrease in activity of a variety of oxidative enzymes

(almost 1.5 to two fold) from the vastus lateralis muscle of men who sustained a complete spinal

cord injury for almost 12 years (Kjaer, Mohr et al. 2001). Hartkopp et al report a significantly

lower oxidative capacity in the upper extremity wrist extensor muscles of persons with more than

five years of SCI (Hartkopp, Harridge et al. 2003). An overall decrease in mitochondrial

oxidative capacity, also measured by decreases in mitochondrial DNA content, (reflective of

mitochondrial protein content), capillary density and blood flow are reported following chronic

SCI in humans (Scelsi, Marchetti et al. 1982; Wang, Hiatt et al. 1999). Furthermore, as early as

11 weeks after complete SCI in humans, Gregory et al have shown decease in SDH enzyme

activity by almost 41% (Gregory, Vandenborne et al. 2003). However, in a follow up study at 24

weeks after injury in the same subjects the SDH activity returned to control values (Castro,

Apple et al. 1999).

Interestingly, oxidative enzyme activity in animal models of SCI is rather muscle specific.

After six months of both spinal cord transaction and spinal isolation, the cat soleus muscle either









enhances or maintains SDH activity in comparison to control rats implying maintenance of

muscle oxidative potential after chronic SCI in the slow skeletal muscle (Jiang, Roy et al. 1990;

Graham, Roy et al. 1992). Elevated SDH activity is also demonstrated in the spinalized rat soleus

muscle 6 months after injury (Otis, Roy et al. 2004). Though mechanisms to elucidate the

elevated oxidative enzyme activity are unknown, investigators have attributed the greater

oxidative capacity in slow muscle to the presence of different fiber type composition of the

paralyzed slow muscle. Apparently, the paralyzed slow soleus muscle possesses an unusual

proportion of hybrid fibers (Roy, Talmadge et al. 1999) that in turn are purported to have

inherently larger oxidative capacities than the typical type 1 muscle fibers (Rochester, Barron et

al. 1995; Otis, Roy et al. 2004). In fact, mixed muscle such as the gastrocnemius and the vastus

lateralis with predominantly fast muscle fibers (type IIx and lib) show significant declines in

SDH activity following spinalization in both cats and rats (Jiang, Roy et al. 1990; Gregory,

Vandenborne et al. 2003). In an animal model of spinal transaction, Durozard et al have used

non-invasive magnetic resonance spectroscopy to demonstrate declines in oxidative capacity of

the gastrocnemius muscle in rats. Based upon their data, the authors also suggest transition in

source of energy supply from oxidative to anaerobic pathways for muscle metabolism following

paralysis (Durozard, Gabrielle et al. 2000). Though change in enzyme activity is associated with

phenotypic alterations following SCI (Martin, Stein et al. 1992; Rochester, Barron et al. 1995),

change in muscle enzyme activities is also thought to occur independent of shifts in fiber types

composition (Castro, Apple et al. 1999; Gregory, Vandenborne et al. 2003). Accordingly, the

existence of any association between enzyme activity and fiber type alteration after SCI remains

a subject for debate.









Irrespective of the cause, decrease in skeletal muscle oxidative capacity following muscle

inactivity is purported to ultimately associate with sub-maximal endurance and exercise

performance. Decrease in overall muscle endurance as measured by the fatigue index (defined as

the decrease in force production with repeated muscle contraction) accompanies muscle

inactivity following spinalization (Roy 1998, Shields 2002, Gerrits 1998). Researchers have

partly attributed muscle fatigability following SCI to altered muscle oxidative capacity. This

premise is somewhat supported by the observation that reduced physical training in able-bodied

individuals causes significant declines in oxidative enzyme activity, which in turn drops long

term skeletal muscle endurance (Houston 1979, Hickson 1982 from Kjaer 2001). Secondly,

conversion to faster muscle fiber phenotype at the cost of slower muscle fibers predisposes the

paralyzed muscle to acquire faster contractile speeds thereby making it easily fatigable (Shields,

Law et al. 1997; Gerrits, De Haan et al. 1999; Hutchinson, Linderman et al. 2001). Lastly,

studies have also linked reduced skeletal muscle oxidative capacity in the development of a

major contributor of cardiovascular complications, namely altered insulin resistance (see below

for more on insulin resistance) (Brehm, Krssak et al. 2006; Schrauwen-Hinderling, Kooi et al.

2007).

1.4.5 Metabolic Adaptations: Altered Glucose Homeostasis

Cardiovascular complications were the second leading cause of death in individuals with

SCI until 1990 (Bravo, Guizar-Sahagun et al. 2004; Jacobs and Nash 2004). With increasing

survival rates of SCI and the development of multiple cardiovascular risk factors, it is now the

leading cause of death after chronic injury (Bravo, Guizar-Sahagun et al. 2004). Risk factors for

these complications include development of insulin resistance, dyslipidemia (elevated low-

density lipoproteins, depleted high-density lipoproteins and increase in total cholesterol), overall

obesity and diabetes, a sedentary lifestyle and limited exercise (Bauman and Spungen 2001;









Bravo, Guizar-Sahagun et al. 2004; Jacobs and Nash 2004). Of these, glucose intolerance due to

development of insulin resistance is suggested as the main cause of cardiovascular disease after

SCI (Bravo, Guizar-Sahagun et al. 2004). Insulin resistance is the resistance of target cells such

as muscle or liver tissue to insulin. The target cells (muscle and fat that take up glucose) are said

to be resistant to glucose uptake and/or are less sensitive to the peripheral uptake of blood

insulin. Elevated blood glucose and insulin levels subsequently precipitate as one of the earliest

hallmarks in the development of type 2 diabetes mellitus (Jacob, Machann et al. 1999). In fact,

persons with complete and incomplete SCI are predisposed to hyperinsulinemia and an

imbalance in glucose homeostasis with subsequent development of insulin resistance (Bauman,

Spungen et al. 1999; Bauman and Spungen 2001). Additional risk factors include relative

increases in adiposity, a sedentary lifestyle and muscle atrophy. The association of skeletal

muscle and insulin resistance is explained below.

Skeletal muscle tissue is responsible for the majority of the insulin mediated glucose

disposal in the body (Goodpaster and Kelley 1998; Schrauwen-Hinderling, Hesselink et al.

2006). The study of intramuscular fat content has gained considerable attention because of the

association of high levels of skeletal muscle lipid with insulin resistance. Existence of lipid in

skeletal muscle was first identified by Denton and Randle in 1967 (Denton and Randle 1967).

However, it is only since the last decade, that wide arrays of studies have reported associations

between intramuscular fat and insulin resistance (Perseghin, Scifo et al. 1999; Furler, Poynten et

al. 2001). Pan et al first established this relationship; they found that muscle triglyceride content

correlates with insulin resistance irrespective of total body adiposity (Pan, Lillioja et al. 1997).

Lipids are typically stored in the skeletal muscle in the form of intramyocellular lipid (IMCL) or

extramyocellular lipid (EMCL). EMCL is the plate or tube shaped fatty infiltrate located outside









the myocyte and between muscle fibers (Szczepaniak, Babcock et al. 1999; Boesch, Machann et

al. 2006). EMCL can also be located intramuscularly as adipose tissue along with fascia

separating fascicles of the same muscle, inter-muscularly along with fasciae between adjacent

muscles and in subcutaneous fat layers. EMCL is purported to be a long-term storage depository

and suggested as being metabolically relatively inert. It is utilized for energy production during

very low intensity exercises and has no known correlation with insulin resistance (Boesch,

Slotboom et al. 1997). In contrast, electron microscopic studies have established IMCL to be the

fat located within the cytoplasm of muscle cell close to the mitochondria (Boesch, Slotboom et

al. 1997; Schrauwen-Hinderling, Hesselink et al. 2006). Because of its proximity to the

mitochondria and its relatively larger content in oxidative fibers, IMCL is regarded as an energy

source for mitochondrial fat oxidation during rest and long-term endurance activities (Krssak,

Petersen et al. 2000; Boesch, Machann et al. 2006; Schrauwen-Hinderling, Hesselink et al.

2006). IMCL can be mobilized and utilized within hours of physical activity, recovers after

several hours following exercise and finally reaches normal levels within days (Boesch,

Machann et al. 2006). However, excessive accumulation of IMCL can have a negative impact on

insulin signaling to induce insulin resistance (see below). A variety of studies have confirmed the

correlation of IMCL with insulin resistance in healthy persons as well with obesity and Type 2

diabetes mellitus (Jacob, Machann et al. 1999; Kelley, Goodpaster et al. 1999; Krssak, Falk

Petersen et al. 1999; Perseghin, Scifo et al. 1999; Sinha, Dufour et al. 2002; Goodpaster and

Wolf 2004; Schrauwen-Hinderling, Hesselink et al. 2006). Investigators have reported moderate

to strong associations of IMCL with several markers of coronary artery disease (interleukin -6,

homocysteine) and pre-diabetes states (insulin, insulin resistance) (Krssak, Falk Petersen et al.

1999; Weiss, Dufour et al. 2003; White, Ferguson et al. 2006). Accordingly, IMCL content is









suggested to serve as a potential non-invasive marker of insulin resistance. However, one

limitation of using IMCL to describe insulin resistance is that IMCL is largely influenced by a

host of factors including age, gender, diet, obesity, physical inactivity, exercise, genetics,

ethnicity as well as the muscles studied (Forouhi, Jenkinson et al. 1999; Sinha, Dufour et al.

2002; Goodpaster and Brown 2005; Stettler, Ith et al. 2005; Schrauwen-Hinderling, Hesselink et

al. 2006; Boesch 2007). IMCL levels are reported to be elevated in older people compared to

young (Cree, Newcomer et al. 2004), females than males (White, Ferguson et al. 2006), people

with high fat diets compared to low fat diets (Stettler, Ith et al. 2005) and higher levels in obese

as compared to lean persons (Sinha, Dufour et al. 2002). Additionally, lipid content varies from

muscle to muscle with the slow soleus muscle, for example, showing almost three times the

IMCL content as compared to the fast tibialis anterior in the same subject (Rico-Sanz, Thomas et

al. 1999; Hwang, Pan et al. 2001; Vermathen, Kreis et al. 2004). Accordingly, large variations in

IMCL continue to exist even in non-diseased healthy individuals, making it difficult to relate it

with insulin resistance. These factors therefore make it important to have stringent inclusion

criteria even for healthy controls in a study design. Nevertheless, studies have attempted to

document inter and intra-subject variations in the IMCL content of more than one muscle,

making it feasible to still conduct these measures and reliably relate them with insulin resistance

(Boesch, Decombaz et al. 1999; Torriani, Thomas et al. 2005). Positive correlations between

IMCL and insulin resistance have been reported in sedentary individuals irrespective of sex,

body weight and physical fitness (Sinha, Dufour et al. 2002; White, Ferguson et al. 2006).

Presently, the advent of new spectroscopic techniques continues to encourage investigators to

explore the relationship between IMCL and insulin resistance. Accordingly, simple ratio









measurements that yield intramyocellular fat content has the potential to serve as an in vivo

biomarker of insulin resistance, which might prove beneficial in a variety of patient populations.

In persons with either complete or incomplete SCI, a three to four fold increase in thigh

intramuscular fat is reported as compared to able bodied adults (Elder, Apple et al. 2004; Gorgey

and Dudley 2006). These studies also reported that their patient group had high plasma glucose

and insulin levels that were positively correlated to intramuscular content. Moreover, a decrease

in skeletal muscle mass in this patient population was inversely correlated with plasma glucose

levels suggesting that skeletal muscle atrophy and intramuscular fat both contributed towards the

decrease in insulin sensitivity. Taken together, it appears that persons with SCI may be

predisposed to the development of this health related risk factor and may also be at a relatively

greater risk for the development of type 2 diabetes.

In the present work, an attempt is made to quantify IMCL in the skeletal muscle of persons

with incomplete SCI (Chapter 7).

Mechanisms linking decreased insulin sensitivity (or increases in insulin resistance) with

elevated IMCL remain ambiguous, but a variety of factors, either singly or in combination have

been surmised for this association.

An increased level of IMCL is a possible result of greater uptake of fatty acid in muscle

(Hegarty, Furler et al. 2003; Hulver and Dohm 2004). Studies have found strong negative

correlations between adiponectin protein and IMCL measures in both obese and non-obese

adolescent and adult populations (Stefan, Vozarova et al. 2002). Adiponectin, a protein released

from adipose tissue is suggested to maintain normal triglyceride levels in blood. Elevated IMCL

content from the skeletal muscle is established as a strong predictor of adiponectin levels

suggesting a potential role of the protein in the accumulation of triglycerides in skeletal muscle









tissue (Stefan, Vozarova et al. 2002; Weiss, Dufour et al. 2003). Elevated IMCL content, in turn,

increases intracellular fatty acids and their derivatives. Intermediates of fatty acid metabolism at

the intramyocellular level (triacylglycerols, diacylglycerol, ceramide and fatty acyl CoAs)

inactivate insulin action by inhibition of specific steps in the insulin-signaling pathway that is

normally responsible for glucose uptake in the myocyte (Itani, Ruderman et al. 2002; Hegarty,

Furler et al. 2003; Hulver and Dohm 2004). Studies report that the elevated diacylglycerol and

fatty acyl CoA levels secondary to increase in muscle triglyceride, increases the protein kinase C

activity. This enzyme can directly inactivate insulin receptors for uptake of insulin in muscle.

Similarly, elevated ceramide levels mediate inhibition of signaling pathways by release of

specific enzymes (Hegarty, Furler et al. 2003; Hulver and Dohm 2004). Another proposed

mechanism suggests that increase in IMCL content leads to a decrease in insulin receptor

synthesis in the skeletal muscle. Interference with insulin receptor function ultimately leads to

an increase in insulin resistance, which in turn stimulates hepatocytes to increase serum

triglycerides and decrease serum HDL (White, Ferguson et al. 2006). Therefore it appears that

accumulation of IMCL serves as a mediator rather than being a direct cause of decreased insulin

action.

Collectively, decreased insulin action (uptake in muscle) is proposed as the major

contributor of insulin resistance in the myocyte. Though elevated plasma fatty acid levels

ultimately increase IMCL content, which can lead to the development of insulin resistance, the

reverse route that insulin resistance can lead to an elevation in IMCL content cannot be ruled out.

1.5 Summary

With technological advancements in the efficient management of acute SCI, the proportion

of and life expectancy of persons with an incomplete injury has considerably increased. Varying

degrees of tissue sparing and muscle loading presents the incomplete SCI population with









variable degrees of paralysis and paresis and hence distinct degrees of functional limitations. A

host of skeletal muscle adaptations including muscle atrophy, muscle fiber type conversion,

declines in contractile function, predisposition to muscle injury, fatty tissue infiltration, altered

oxidative capacity and glucose homeostasis are well established after complete SCI in animals

and humans. Despite the obvious motor dysfunctions, physiological muscle adaptations

following incomplete spinal cord injury are relatively unstudied. With the advent of new

therapeutic strategies and advances in SCI research aimed at recovery of lost functions, it is

imperative that the machinery for movement and locomotion remain intact. The overall objective

of the present work was to elucidate muscle adaptations after incomplete spinal cord injury in

humans and in the rat model of incomplete SCI.









CHAPTER 2
SPINAL CORD INJURY AND LOCOMOTOR TRAINING

2.1. Locomotor Function after SCI

Initial insult to the spinal cord profoundly impacts almost all biological systems of the

body. Specifically, neuromusculoskeletal deficits following the injury tremendously impair the

motor performance and locomotor capabilities of individuals with SCI. Following incomplete

SCI; there always exists some degree of spontaneous recovery. As a result, persons with

incomplete SCI exhibit variable paralysis and paresis of affected muscles, typically resulting in

impaired motor performance and varying degrees of functional limitations (Subbarao 1991;

Tang, Tuel et al. 1994; Bums, Golding et al. 1997). Similar to persons with complete-SCI,

persons with incomplete-SCI exhibit a variety of clinically relevant motor and functional

deficits, including local muscle fatigue, weakness of affected muscles (Sloan, Bremner et al.

1994; Johnston, Finson et al. 2003) and diminished capacity to ambulate (Waters, Adkins et al.

1994; Ulkar, Yavuzer et al. 2003). Other studies have shown a significant reduction in

ambulatory capacity, with functional deficits in gait including a reduced gait speed, step

frequency; stride length and longer durations spent in the double support phase of gait cycle

(Melis, Torres-Moreno et al. 1999; van der Salm, Nene et al. 2005). Loss of ambulation is the

most obvious functional limitation associated with a SCI and regaining walking capability one of

the main aims of this patient cohort (Kilgore, Scherer et al. 2001). In a report by lezzoni et al,

"Walking holds profound symbolic importance. Nowadays, upright movement permeates

American aphorisms, connoting independence, autonomy, self-reliance and strength". Inability

to "walking tall" creates a physical need to restore mobility and an emotional need to restore

one's "core sense of value and place in the world" (lezzoni 1996). With the remarkable focus

that is now spent for new treatments for SCI, a lot of energy is spent in exploring the









neuromuscular physiological impairments associated with locomotion (Dobkin 2000; Wolpaw

and Tennissen 2001; Dobkin and Havton 2004; Fouad and Pearson 2004). This approach has in

turn held optimism in determining mechanisms associated with other functional impairments

seen in this patient population such as urination, sexual, and bowel function that at present are

less understood. Over the past two decades, researchers in the field of SCI have come to

recognize the remarkable potential of the central nervous system to regenerate following its

injury. Accordingly, investigators are discovering new therapies to improve the locomotor

capability of persons with SCI. The rest of this chapter includes a brief review of: a)

Conventional rehabilitation for persons with SCI b) limitations of current clinical practice in SCI

c) spinal cord plasticity and principles of neuroplasticity d) a potential paradigm shift in

enhancing locomotor function after SCI e) locomotor training in animal and humans

2.1.1. Conventional Rehabilitation Therapies after SCI

Present rehabilitation goals in a clinical setup for individuals with SCI are re-entry into

community, energy conservation and functional independence. To achieve these goals, much of

the rehabilitation is focused on task-oriented training along with compensation of lost abilities

(Thompson 2000; Dobkin and Havton 2004). Patients are trained to perform a functional task

that is specific to the goal to be achieved, but at the same time, achievement of the task

accompanies using spared residual function and/or modifications in a person's environment.

Thus, patients use whatever residual sensorimotor functions persist after the injury along with

use of assistive aids to maximize their self-care, mobility and community roles. These task

specific strategies are also called compensatory techniques in current practice (Mathiowetz and

Haugen 1994). Examples of compensatory strategies that are commonly seen and/or taught in

clinical practice for individuals with SCI include use of assistive aids (example:

orthoses/crutches/wheelchair), new movement strategies (example: hip hiking) (Bell 1955) and









electrical stimulation induced movements (Peckham, Keith et al. 2001), (Dobkin and Havton

2004).

Use of a wheelchair after a SCI is one of the most common compensatory strategies. A

lower extremity motor score (LEMS) of 20/50 or less indicates that persons with SCI are likely

to be limited ambulators (ASIA 2001). Clinically, a wheelchair prescription is a common choice

for such persons. However, sometimes, wheelchair use stems from the need to enhance the

mobility and independence in the community [for reference see (Minkel 2000).

Functional electrical stimulation (FES) is being used extensively in improving functional

motor performance of persons with SCI. Generally, FES devices are designed to comprise of a

control unit and stimulating electrodes. The control unit translates signals from sensors or a

voluntary movement onto the stimulating electrodes that are taped over the skin or surgically

implanted near the nerve for specific muscle contraction. Mild electrical stimulus elicits muscle

contraction that translates into muscle movement. By adequate alterations in stimulus parameters

of the FES device, persons with SCI gain assistance in movement initiation and control. FES has

been used widely to facilitate a variety of functional activities following SCI including

controlling upper extremity movements and grasps, improving cardiovascular conditioning and

breathing, training to walk for short distances, standing up for transfers and controlling bowel

and bladder function (Creasey, Grill et al. 2001; Johnston, Finson et al. 2003; IOM 2005).

Physiological effects of FES involve increase in muscle mass, aerobic metabolism and

maintenance of bone density (Postans, Hasler et al. 2004); which ultimately play an important

role in prevention of musculoskeletal complications after SCI. However, the role of FES in

improving the ambulatory capability after SCI is limited. Expense of the equipment, assistance

needed for set up, time taken from other daily activities, and the meager daily effects of FES









exercise deter patients from its use (Dobkin and Havton 2004). The gait patterns with use of FES

are unrefined and allow for short-distance ambulation. Moreover, two major deterrents to the use

of FES for functional performance include a) presence of an optimal degree of initial motor and

sensory control in the lower extremities and b) a relatively lower level of incomplete SCI (lower

thoracic); thereby making it suitable for use for only a limited population of SCI. Therefore, FES

devices for assisting in ambulation are limited in scope. Most importantly, though FES signals

are proposed to alter central activity dependent plasticity, sensory feedback induced by FES is

not likely to cause any persistent central sensori-motor activation or motor skills learning [for

review see (Dobkin and Havton 2004). So ultimately, FES still represents a compensatory

strategy that leads to non-use of the persons' neurological systems and added disability in the

long-term still persists.

[Note: Though numerous other compensatory strategies concerning other body systems

are commonly implemented in rehabilitation (5), in this discussion, the primary focus is made on

techniques aimed at enhancing locomotor performance].

2.1.2 Limitations of Compensatory Rehabilitation Strategies

Compensatory techniques are focused little, if any on the neural processes necessary or

neurophysiological adaptations occurring secondary to performance of a functional task. In fact,

"there is no evidence that programs of rehabilitation have any effect on restoring impaired

nervous system function or enhancing natural recovery following disease or injury" (McDowell

1994). It appears that by using compensatory strategies and provisions in the environment, one

completely ignores what a person's body is capable or might be capable of doing. Any potential

for the person to explore the capabilities of his or her body systems to regenerate or restore is

completely neglected. Today community environments are becoming easily accessible for people

using wheeled devices as their primary means of mobility. Though this approach definitely









permits successful mobility of individuals in the community, long-term effects of such therapy

can precipitate further disability. In fact, impact of long-term use of a wheelchair causes further

disuse and negative plasticity of the neuromuscular system, which in turn creates further

dependence on the use of a wheelchair for mobility. As it turns out, such interdependence leads

to a host of psychosocial and physiological consequences that further enhance disability.

For a therapist, wheelchair provision for his/her client might be perceived as an

opportunity of mobility, but a young man's opinion about his wheelchair has been "You might

as well stick me in a damn closet. That wheelchair just makes me think of how hopeless I am"

(Minkel 2000). Thus, psychologically, many people interpret dependence on assistive aids as an

indication of greater disability rather than an option for increased mobility (lezzoni 1996).

Physiologically, inactivity/disuse of the affected body part leads to more systemic complications

(muscle atrophy, more predispositions to fractures, contractures, etc) and further dependence on

walking aids for mobility. Overcompensating with spared motor function limits the capacity of

the central nervous system to adapt (Barbeau, Fung et al. 2002). As such, prolonged disuse

conforms to the frequently used adage-"use it or lose it". Few studies also associate disuse with

suppression of genes that attempt axonal regeneration (for reference see (Dobkin and Havton

2004).

2.2 Spinal Cord Plasticity

The main reason for use of compensatory techniques to recover function after SCI has

evolved from the doctrine that the spinal cord is nothing more than a hard-wired system that

simply serves as a conduit for ascending and descending axons. More than 3500 years ago, SCI

was referred to as "A disease that cannot be treated" (from (Vikhanski 2001). This dogma

dominated for several centuries. In fact, Ramon Cajal in 1906 was awarded the Nobel Prize for

discovering the regeneration capabilities of peripheral nerves while simultaneously confirming









that "as is well known, the central tracts are incapable of repair" (Vikhanski 2001). A true

paradigm shift in the field of spinal cord neuroscience occurred in the 1950s. Windle, Liu and

Chambers first suggested "sprouting" (referred to as growth of short shoots or sprouts from

healthy nerves to compensate for damaged fibers) of healthy nerves within the central nervous

system as a mechanism of recovery in experimentally spinalized animals (Windle 1954; Liu and

Chambers 1958). However, scientific proof for nerve repair and regeneration within the spinal

cord emerged only in the 1980s (Richardson, McGuinness et al. 1980). In the past two decades,

animal and human research has revealed the potential capability of the CNS to repair and

regenerate following its injury (Dobkin 2000; Thompson 2000; Rosenzweig and McDonald

2004). Currently, convincing evidence from spinal cord repair literature suggests that the most

important contributors inhibiting growth of neurons is the presence of an non-permissive

environment that consists of a mechanical barrier such as a scar tissue, insufficient neurotrophic

factors for axonal regeneration and the presence of inhibitory molecules within the scar tissue

(Fouad and Pearson 2004; Rosenberg, Zai et al. 2005). In the field of SCI research, vigorous

attempts have been made in understanding the basic mechanisms of neuronal cell growth, death

and repair; influence of central and peripheral neural signals on motor neuronal output;

identifying spinal pathways (such as the central pattern generators) for locomotor function and

strategizing new therapeutic modalities based on understanding of these mechanism. Neurons

within the spinal cord have a complex interaction with cortical neurons, sub-cortical neurons

(neurons within brainstem), motor neurons and afferent pathways. Indeed, the spinal cord has a

large potential for plasticity at multiple levels of its architecture including afferent neurons that

enter the cord, inter-neuronal population interposed between these inputs and the motor neurons,

ascending axons within the cord, descending axons within the cord, synaptic connections on the









motor neurons and motor neurons themselves. Furthermore, alterations in neurotransmitter and

neuro-modulator activity along with cortical changes accompany SCI (Bregman, Coumans et al.

2002; Dobkin and Havton 2004).

Rightfully, the term 'neuroplasticity" has also been extended to the spinal cord.

Neuroplasticity is a permanent change in the structure and function of the nervous system in

response to experience, ones' state of health and disease and experimental manipulation or

injury. It is important to note that persistent changes both in the peripheral and descending input

to the cord via myriad of factors such as practice, trauma, and disease will cause plasticity at

multiple levels of the nervous system both spinal and supraspinal. Therefore, acquisition of any

new behavior whether it is skilled learned behavior through prolonged practice (or disuse) or an

abnormal response associated with CNS disease or compensatory movements is inevitably

associated with neuroplasticity. Interestingly, the spinal cord is so flexible that spinal

neuroplasticity is a function of experiences throughout one's subsequent life (Wolpaw and

Tennissen 2001). In this section, major focus is made on a specific, activity-dependent plasticity

(walking) of the spinal cord. Activity dependent plasticity is the lasting change that occurs in the

spinal cord because of activity sensorimotorr or sensory inputs) from the periphery or the brain.

Such a change finally affects output of the spinal cord. However, to retain neuroplasticity, few

basic rules are essential for its expression. These rules, focused on physiological recovery

following neural injury eventually form the fundamental principles of current neurorehabilitation

techniques.

a) Repetition/practice: Repeated performance of a task is invariably associated with

learning. Thus, practice makes perfect. Physiologically, learning is but a reorganization of

neuronal mechanisms (Kandel 2001). Repetition stimulates multiple neurons and consequently









alters neural activity that persists for long durations even after the activity ceases. The

physiological mechanisms associated with learning range from neuronal pre-synaptic and/or

post-synaptic structural changes, modulation of neurotransmitters, expression of new proteins,

activation of silent synapses, growth of new neural spines (Kandel; Kandel 2001) and changes in

representation of the cortical homunculus (Byl and Melnick 1997; Byl 2003). Therefore, for such

permanent physiological changes to manifest, the stimulus that causes it must be repetitive.

Moreover, whether the repetitive experience is in the form of excess or diminished stimuli

(example activity versus disuse), plasticity is inevitable. Several studies have demonstrated the

critical role of repetition for neural plasticity (Kandel; Wolf and Segal 1996; Beaumont and

Gardiner 2003). For example: single shock of tactile electrical stimulus to the aplysia-siphon

gives rise to a memory lasting only minutes, while four or five spaced shocks gives rise to a

memory lasting several days (Kandel 2001). Soleus H-reflex modulation in monkeys was

demonstrated after 3000-6000 trials/day (Wolpaw 1987). Therefore, to promote learning of a

new motor task or for retraining, tremendous emphasis is placed on repetition as one of the major

treatment principles in neurorehabilitation research (Edgerton, Roy et al. 1992; Behrman,

Lawless-Dixon et al. 2005).

b) Task specificity: The nervous system will respond to whatever stimulus it is exposed

to. Specific sensory stimulus will stimulate plasticity of appropriate neuronal circuits. In fact,

only the activated neural pathways will respond to the stimulus. Moreover, any motor output is

generally associated with and/or is a result of precise sensory experience. For example: the

normal pattern of walking requires afferent sensory stimulus from the feet (Rossignol, Chau et al.

1996; Dietz and Harkema 2004; Duysens, Bastiaanse et al. 2004). To enhance firing of specific

neuronal circuits and hence strengthen their synaptic efficiency, the same activity needs to be









entrained. For example: spinalized cats that were trained to walk had faster walking speeds than

animals that were trained to stand only (Hodgson, Roy et al. 1994). In fact, locomotor training

that closely simulates normal walking pattern has been suggested to improve functional

locomotor outcomes while conventional rehabilitation approaches have not shown similar effects

of training (Barbeau, Norman et al. 1998; Dobkin BH 2003, Sep).

c) Pattern of practice: Whether stimulus of practice is continuous or intermittent clearly

dictates plasticity. Generally, intermittent practice is a more appropriate stimulus than continuous

under wide circumstances of plasticity. For example: intermittent and not continuous hypoxic

exposure elicits long term enhancement of inspiratory motor output known as long term

facilitation (LTF) (Baker and Mitchell 2000). Also, continuous sessions of tactile stimulus to the

aplysia-siphon leads to a short-term memory for habituation of the gill-withdrawal reflex, while

intermittent sessions produces a long-term memory (Kandel 2001). One of the probable reasons

for this pattern is the molecular neural mechanism associated with plasticity. Mechanisms of

phrenic motor neuron LTF for instance, involve intermittent release of serotonin, which in turn

initiates a cascade of events for protein synthesis. The elevated protein increases synaptic

strength and hence phrenic motor output (Baker-Herman and Mitchell 2002). In fact, continuous

release of serotonin (due to continuous hypoxia) may have an inhibitory effect on the pre-

synaptic receptors for protein synthesis, thereby hindering plasticity. Thus, based on findings in

lower animals, it appears that interval training might be better preferred over massed practice.

2.3 Locomotor Training: A Paradigm Shift

With the above background in mind, one can deduce that rehabilitation techniques aimed at

enhancing locomotor function following SCI should minimize the use of compensatory

strategies. As such, a physiologically based intervention program seems to be a more scientific

approach towards recovery. As is well recognized now, regaining neuromuscular functions









following a neurological injury largely depends upon recognizing the pathophysiology of injury.

The current scientist's perspective of recovery based therapy dwells from the notion "Much

needs to be done in the management of neurological disability and it will only be through basic

science clarifying the mechanisms of disability and the application of scientifically sound

outcome measures that interventions will make a real difference" (Thompson 2000).

Consequently, approaches for rehabilitation of the SCI cohort needs to be emphasized on task-

specific training that can trigger appropriate neural mechanisms. When developing therapeutic

interventions to enhance functional recovery after SCI, an understanding of the underlying

physiology of neuromuscular responses that occur after this type of injury will promote different

interventions that rely less heavily on compensatory rehabilitation. Locomotor training using

treadmill and overhead body weight support (LT) is one such rehabilitation strategy that is based

on the neuroplasticity principles of walking recovery and has gained tremendous momentum in

improving walking ability of persons with SCI (see below). The LT approach has proved more

beneficial than conventional therapy in chronic incomplete spinal cord injuries. The following

section includes a brief historical perspective and description of LT in animals and in individuals

with SCI.

2.3.1 A Historical Perspective and Locomotor Training in the Spinal Cord Injury

Animal Model

One of the most remarkable finding in spinal cord injury rehabilitation research is that

spinalized cats were able to step with their hind limbs on a moving treadmill. This phenomenon

evolved from the pioneering works of Shurrager and Dykman who emphasized the importance of

regular repetitive locomotor movements in spinalized mammals (Shurrager and Dykman 1951).

Later in the 1970s, Grillner et al and Edgerton et al were able to demonstrate the ability of the

isolated spinal cord to produce cyclic activity between agonist and antagonist muscle groups









with levels of coordination that mimicked normal locomotion (Grillner and Zangger 1975;

Edgerton, Roy et al. 1992). Since then, numerous investigators have demonstrated the stepping

phenomenon in spinalized mammals on a moving treadmill (Forssberg, Grillner et al. 1980;

Lovely, Gregor et al. 1986; Barbeau and Rossignol 1987; Edgerton, Roy et al. 1992). A typical

training protocol in the animal model involves walking of a thoracic spinal cord lesioned animal

on a treadmill. Initially stepping is possible only with manual assistance by trainers that assist

with stepping of the hind limbs and application of a non-specific sensory input from the

perineum, abdomen or the tail. Generally, after three months of daily weight supported treadmill

training, the amount of support required to step declines and hind limbs move into a more

rhythmic alternate pattern that closely resembles normal walking (Edgerton, Roy et al. 1992).

Comparative data show that trained spinalized cats show better ability to step than those that are

allowed to recover spontaneously (de Leon, Hodgson et al. 1998). Stepping is also associated

with recovery of EMG patterns in the paralyzed muscles close to the pre-spinalized patterns

(Lovely, Gregor et al. 1990; Hodgson, Roy et al. 1994). Note worthily, training outcomes in

incompletely injured cats differ from spinalized cats. Incompletely injured cats eventually

recover voluntary quadrupedal locomotion overground and on the treadmill within three days to

three weeks depending upon the severity of injury (Rossignol, Chau et al. 1996; Rossignol, Drew

et al. 1999).

As mentioned in Section 1.3.1, the SCI rat model has gained enormous recognition for use

in SCI research. Accordingly, though much of the basic concepts in the neuronal control of

walking were established in the cat model, these findings have also been translated to the spinal

rat model. Over the past few years, investigators have assessed the effects of treadmill training

on the recovery of rats following spinal transaction (Moshonkina, Avelev et al. 2002;









Moshonkina, Gilerovich et al. 2004) as well as contusion (Thota, Carlson et al. 2001; Multon,

Franzen et al. 2003; Fouad and Pearson 2004; Stevens, Liu et al. 2006). In rats spinalized at the

thoracic level as adults, nine weeks of LT (5days/week, minutes, starting on day one after

operation) has been demonstrated to hasten hind limb joint movements in the spinalized rats,

generate separate voluntary joint movements in the hind limbs and produce coordinated step-

wise limb movements on a treadmill. This locomotor function has been shown to coincide with

morphologically intact motor neurons in the spinal cord (Moshonkina, Avelev et al. 2002;

Moshonkina, Gilerovich et al. 2004). In spinally contused rats, few studies in recent years have

shown that LT improves overground hind limb locomotor function. Thota et al reported

improvements in lower limb joint kinematics of spinally contused rats that led to recovery of

coordinated locomotor function after 7 weeks, albeit with deformities in gait (Thota, Carlson

et al. 2001). In 2003, Multon et al induced spinal contusion by compression and found

significant functional gains in the trained animals throughout a training period of 9 weeks (30

min/day, 5/week). The training starting at 3 days post injury enabled the trained rats to

voluntarily support their body weight at the end of training; while the untrained group showed

spontaneous recovery just enough to move their hind limb joints (Multon, Franzen et al. 2003)

Stevens et al in 2006 have observed the effects of a relatively shorter duration of LT (1 week,

20min/trial, 2trials/day) starting one week after spinal contusion in rats. An overall 32% in

overall locomotor function (BBB score) was observed in the trained rats that correlated well with

peak muscle force measurements (Stevens, Liu et al. 2006). Though LT has enhanced the

functional locomotor capabilities after incomplete injuries, it is important to note that several

factors including the severity of injury, dose of therapy, initiation of training, etc. can largely

dictate the effect of LT in these incomplete injury models. Fouad et al for example, have shown









that LT (2*15min for 5 weeks) did not add to the locomotor functional improvements in the

trained rats versus the untrained group. Even untrained contused rats show functional

improvements as early as 7-14 days post training. However, compared to other studies of spinal

contusion, this group utilized a different degree and kind of incomplete injury to the cord

(relatively less severe dorsal incomplete transaction of the cord as versus cord contusion or

compression). The authors suggested that spontaneous recovery in the control group probably

curbed differences between the two groups and their findings could be attributed to either

insufficient amount of training and/or insensitive testing outcome measures (Fouad, Metz et al.

2000).

Collectively, these findings suggest that LT enhances locomotor capabilities in spinal cord

injured animal models. However, an important note to make is that though LT has shown to

evoke stepping patterns in the mammalian animal model of complete SCI on the moving

treadmill, studies have failed to demonstrate their translation into independent overground

walking. Nevertheless, animal studies have laid the foundations for a potential new therapeutic

strategy for locomotion recovery in humans with SCI and have yielded valuable information

about spinal cord plasticity and its response to training.

2.3.2 Locomotor training in Individuals with Spinal Cord Injury

Based on the principles of locomotor training established in the animal model of SCI, the

training has been transferred to humans with SCI. Barbeau et al performed one of the initial

studies in humans to study the effects of locomotor therapy in individuals with SCI (Barbeau,

Wainberg et al. 1987; Visintin and Barbeau 1989). Subsequently, various investigators

worldwide demonstrated impact of LT in the walking capability of this patient population

(Wemig and Muller 1992; Dietz, Colombo et al. 1995; Dobkin, Harkema et al. 1995; Harkema,

Hurley et al. 1997; Behrman and Harkema 2000). Briefly, a typical LT protocol in humans









comprises of placing subjects on a treadmill while 0-50% of their body weight is supported by an

overhead climbing harness. Therapists manually assist in stepping over the moving treadmill

while attempting to maintain appropriate joint kinematics. Note that LT uses "body weight

support" to support body weight during training; but the training involves utilizing numerous

principles of neuroplasticity to enhance locomotion. The principles of LT, in which the stepping

pattern is repeatedly trained while body weight support is provided, are based on basic science

research demonstrating the role of the spinal cord in controlling locomotion in animal models of

SCI. In humans, attempts are made to encompass several basic principles that facilitate the

kinetic and kinematical parameters associated with phases of stepping. Briefly, these include: a)

adequate limb loading on the stance limb; b) maintain appropriate body kinematics including

appropriate joint angles and an upright and extended trunk and head; c) generate stepping speeds

approximating normal walking speeds (0.75-1.25 m/s); d) coordinate timing of hip extension and

unloading of limb in stance with simultaneous loading of the contra lateral limb; e) avoid weight

bearing on the arms and facilitate reciprocal arm swing; f) aid symmetrical inter-limb

coordination; and g) minimize sensory stimulation that would conflict with sensory information

associated with locomotion. These principles not only facilitate balance control, but also provide

the ensemble of appropriate input for motor output (Harkema, Hurley et al. 1997; Behrman and

Harkema 2000; Behrman, Lawless-Dixon et al. 2005). Such an approach also ensures training

consistency across researchers. Thus, LT is not to be considered as a modality, but is rather a

training strategy that is based on neurophysiological principles derived from animal studies and

whose implementation necessitates adequate professional training, knowledge and skill.

Though investigators have demonstrated EMG activity from the lower extremity muscles

following LT in persons with complete SCI, unlike the animal spinalized model however, LT has









failed to demonstrate initiation of voluntary stepping pattern in humans with complete SCI

(Dobkin, Harkema et al. 1995). Nevertheless, the training has shown significant improvements in

the locomotor capabilities in selective populations of incomplete SCI. Accordingly; therapies

directed towards recovery of walking function have focused extensively on the use of LT in

persons with incomplete SCI (Wernig, Nanassy et al. 1998; Behrman, Lawless-Dixon et al.

2005; Behrman, Bowden et al. 2006). Considerable research efforts have been directed in

revealing the major characteristics of the training, potential neuronal mechanisms associated with

the training along with its potential role as a therapeutic strategy (Dobkin and Havton 2004;

Hicks, Adams et al. 2005). Locomotor training has been suggested to have a positive impact on

the walking ability, walking speed, kinematical parameters, distance walked, limb coordination,

functional independence, ability to walk with fewer assistive aids, transition from use of a

wheelchair to upright walking with assistive aids and subjective well being (Wernig and Muller

1992; Behrman and Harkema 2000; Hicks, Adams et al. 2005; Hannold, Young et al. 2006)).

Locomotor training has also shown to induce modulation of H-reflex and EMG patterns towards

close to control values that accompany improved walking capabilities (Dietz, Colombo et al.

1995; Trimble, Behrman et al. 2001). As a result of these behavioral and neurological benefits,

the training has also been subject to a large multi-center randomized clinical trial the SCILT

(Spinal cord injury LT). This trial involved participation of around 140 persons with incomplete

SCI with ASIA grades B, C or D within 8 weeks of injury. The experimental group received LT

and the second group a similar intensity of standing and overground mobility training. However,

no significant differences were observed in the primary outcome measures of walking speed,

distance walked in 6 minutes and the functional independence measure scores for lower

extremity (FIM-L) between the two groups (Dobkin, Apple et al. 2006). Nonetheless, LT has









shown tremendous potential as a therapeutic strategy in improving locomotor capabilities of

persons with chronic incomplete SCI. In fact, LT has gained a new direction in the treatment of

SCI in that: a) investigators are now proposing a strict patient selection criteria for conducting

studies aimed at functional recovery of a heterogeneous population such as the incomplete SCI

(Dobkin 2007) and b) various researchers are favoring combining LT with other training

strategies for maximum locomotor gains. LT along with micro-stimulation of the spinal cord,

pharmacological agents such as clonidine, and repair with nerve grafts are suggested as potential

future strategies to enhance locomotor function after SCI (Herman, He et al. 2002; Dobkin and

Havton 2004; Field-Fote 2004).

2.3.3 Central Pattern Generators and Locomotion

One of the principal neuronal recovery mechanisms promoting repair/regeneration of the

injured spinal cord following LT is suggested to be the reestablishment of inter-neuronal

connections known as the central pattern generators (CPG) within the spinal cord. The discovery

of CPG in the quadruped spinal cord has stimulated much interest in current research on

promoting neural regeneration following SCI. In quadrupeds, CPGs are established to exist as a

group of inter-neuronal networks within the spinal cord that produce a rhythmic motor pattern

resembling normal locomotion (Edgerton, Leon et al. 2001; Fouad and Pearson 2004). In

spinalized cats, an approximate 25% of total daily-integrated EMG activity can be elicited from

the soleus muscle during treadmill stepping; thereby implying the presence of a partial

functioning spinal network even after a complete SCI. This fictive locomotion is produced

independent of supraspinal and phasic afferent input (Grillner and Zangger 1975).

Though their presence is well known in rats and cats, presence of CPG in the human spinal

cord is debatable. Based on the inability of persons with complete SCI to generate stepping,

some investigators have refuted its presence in the human spinal cord. In contrast, support for the









existence of CPG in humans is well reported (Dimitrijevic, Gerasimenko et al. 1998; Lamb and

Yang 2000; Dietz and Muller 2004; Edgerton, Tillakaratne et al. 2004). Dietz et al have

demonstrated LT induced increase in leg muscle EMG activity when persons with complete SCI

are made to step on a treadmill with body weight support and manual assistance (Dietz and

Muller 2004). The authors suggest existence of neuronal networks within the spinal cord as the

source of this enhanced neuronal activity. Furthermore, direct electrical stimulation of the lumbar

spinal cord in humans with complete SCI has been shown to evoke locomotor-like rhythmic

activity. This seemingly alternating pattern of the lower limbs has been attributed to the presence

of a programmed inter-neuronal network within the spinal cord that via input from the electrical

stimulation receives its "drive" to produce the motor output (Dimitrijevic, Gerasimenko et al.

1998). Lamb and Yang in 2000 further show that infants at birth are capable of stepping

continuously when their feet are placed on a treadmill. Since infants do not have a functional

descending spinal pathway, this locomotor behavior is attributed to a functional CPG within the

spinal cord (Lamb and Yang 2000).

Note worthily, presence of some kind of sensory stimulation (peripherally or supra-

spinally) presents as an essential prerequisite for mammalian locomotion. It is well known that

descending pathways in the ventrolateral region of the spinal cord play a significant role in

transmitting voluntary commands from the motor cortex to the spinal cord and are involved in

initiation of locomotion (Noga, Kriellaars et al. 1991; Brustein and Rossignol 1998). These

pathways most likely provide the stimulus necessary for CPG stimulation during normal

walking. On the other hand, phasic afferent input has been demonstrated to play a key role for

stepping in spinalized cats (Bouyer and Rossignol 2003), rats (Timoszyk, De Leon et al. 2002),

as well as in humans with incomplete SCI (Dietz and Duysens 2000; Harkema 2001).









Investigators purport that one of the main sources of proprioceptive feedback during stepping is

probably provided by the stretch sensitive and load sensitive receptors in the lower extremity

muscles. While cutaneous receptors in animals are also influential in producing a motor pattern,

they are implicated to play a larger role in skilled locomotor activities such as beam walking or

paw placement on rungs of a horizontal ladder (Bouyer and Rossignol 2003).

Therefore, neuronal networks within the spinal cord (CPGs) can be regarded as

autonomous; but sensory input is nevertheless necessary for both normal and spinal locomotion.

In addition to peripheral input, the ensemble of supraspinal input is also necessary in the

initiation, maintenance, balance and vestibular control of locomotion so as to adapt to

environmental constraints. Locomotor function then is a consequence of neuronal interaction

between a wide ensemble of information coming from supraspinal centers, afferent signals and

CPGs (Edgerton, Tillakaratne et al. 2004).

2.4 Locomotor Training Effects on Paralyzed Skeletal Muscle

While locomotor training has proven to yield much neuronal plasticity, the following

paragraphs discuss the effect of LT on skeletal muscle morphology and function in animal and

human models of SCI. Locomotor training effects on the paralyzed skeletal muscle holds

importance for two major reasons: 1) with increase in new therapeutic interventions for SCI, it is

necessary that an intact machinery for limb movement is maintained; 2) current studies purport

that exercise in normal rats increases the level of neurotrophic factors and proteins associated

with neuronal growth and plasticity. Thus, exercise induced increase in neurotrophic factors

produced in the muscle might, via their retrograde transfer, promote neurite outgrowth or

synaptic plasticity within the injured spinal cord (Fouad and Pearson 2004; Hutchinson, Gomez-

Pinilla et al. 2004).









Locomotor training has an overall ameliorating effect on the spinal transaction or

contusion induced muscle alterations. Generally, maximum effect of the training is seen in the

slow extensor muscles of the lower extremities with minimal or no effect on the fast extensor or

flexor muscles (Roy and Acosta 1986). Five weeks of daily locomotor stepping with emphasis

on load bearing 30min/day, 5days/week, beginning one month after transaction has shown to

markedly alleviate the atrophic response in the lower extremity muscles of spinalized cats (Roy

and Acosta 1986; Roy, Talmadge et al. 1998). In addition, a relatively higher proportion of Type

la fibers are expressed in the paralyzed soleus muscle after LT as compared to before initiation

of the training. Such conversion is reflective of transition to a healthier muscle. Furthermore,

these studies show that the peak isometric forces produced by slow extensor muscles soleuss and

vastus intermedius) increases to control values at the end of the training period. In addition, there

is a concurrent increase in the overall oxidative enzyme activity after LT. Recently; Stevens et al

have elucidated the impact of one week of LT, starting at Iweek after contusion (20min/trial, 2

trials/day at 1 Impm) on the skeletal muscles of contused rats. Significant increases in soleus

muscle fiber cross-sectional area, peak tetanic force and decreases in muscle fatigue

measurements have been demonstrated in trained rats versus untrained injured rats.

Measurements of force improvement correlated well with functional performance (BBB score);

implying marked improvement in motor recovery by LT of as short as one week (Stevens, Liu et

al. 2006).

In humans with incomplete traumatic SCI (ASIA C), a couple of recent studies have

reported the morphological and metabolic effects of LT on skeletal muscle. In nine persons with

incomplete SCI, sixty-eight training sessions of LT spanned over six months increased the vastus

lateralis fiber cross-sectional area, an overall increase in the expression of Type Ia muscle fibers









and increases in muscle oxidative capacity (Stewart, Tamopolsky et al. 2004). These measures

were accompanied by concurrent increases in the walking speed and locomotor capacities of

injured subjects Subsequently, the same research group also observed increased vastus lateralis

muscle glycogen stores (suggestive of increase in the glycogenolytic capacity of the muscle) and

hexokinase enzyme activity (reflective of improvements in insulin sensitivity) in their subjects

(Phillips, Stewart et al. 2004).

2.5 Summary

Persons with both complete and incomplete SCI display significant locomotor deficits

following the injury. Current advances in the field of neuroscience and rehabilitation research are

providing a new dimension to therapeutic approaches in SCI. Accordingly; physiological based

restorative interventions have the tremendous potential to replace current conventional therapies.

Locomotor training, based on the principles of neuroplasticity is one such intervention and has

been used extensively to improve locomotor capabilities in select populations of incomplete SCI.

In addition to alleviating various functional deficits, LT has the potential to induce several

muscle adaptations including increases in muscle fiber size, fiber type conversion and

improvements in muscle oxidative capacity. In the light of maintaining an intact peripheral

machinery after SCI while also revealing potential effects of the trained muscular system on the

neurological system, more studies are necessitated that focus on studying muscle adaptations

following LT in persons with SCI.









CHAPTER 3
MAGNETIC RESONANCE AND SKELETAL MUSCLE

3.1 Introduction

Throughout the present work on both human and animal models of SCI, magnetic

resonance (MR) is used as a key methodological tool to characterize lower extremity skeletal

muscle. Skeletal muscle function depends both on its morphological and metabolic properties.

Traditionally, muscle properties are studied using non-specific measurements (for example,

anthropometric measures to determine muscle morphology), invasive techniques (for example,

muscle biopsy to estimate fiber types or muscle enzyme profile) and/or global measures (for

example, maximum oxygen consumption levels to reflect muscle oxidative capacity). In the past

decade however, MR has gained tremendous momentum in characterizing skeletal muscle in

healthy as well as a variety of patient populations. One of the most critical features of MR that

makes it an extremely valuable tool in studying muscle is that it is non-invasive. In addition, the

specificity, sensitivity and high resolution nature of MR makes it highly suitable for the

assessment of a wide range of skeletal muscle characteristics including structural, functional and

metabolic properties. Repetitive measurements enable users to determine disease progression and

allow for follow up of various therapeutic interventions. Lastly, obtaining information about a

physiological process from a functioning muscle in real time makes MR a unique non-invasive

measurement technique in modem science. The following sections review the basics of MR,

followed by distinct features of magnetic resonance imaging (MRI), magnetic resonance

spectroscopy (MRS) and their application in skeletal muscle.

3.2 Basics of Magnetic Resonance

The term Magnetic Resonance (MR) refers to the magnetic properties of the nucleus that is

utilized in magnetic resonance imaging (MRI) and magnetic resonance spectroscopy (MRS).









MRI is an imaging tool that is capable of imaging soft tissues in the body; MRS, on the other

hand, is used to study the metabolism of human tissues and organs.

Generally, a diagnostic image uses some physical property of the substance that is being

imaged. For example, a photograph uses reflected light from the object that is pictured while

ultrasonography uses reflected sound from the body part under study. Similarly, current MR

techniques are based on receiving and processing signals from atomic protons. The exact

molecular environment where these protons are located has a profound effect on the nature of the

magnetic resonance signals created and thus gives rise to the remarkable power and versatility of

MR. The following sections focus on the fundamentals of MR. However, it should be recognized

that various complexities including nuclear spin physics theories and mathematical calculations

that more completely explain MR are beyond the scope of this dissertation.

3.2.1 Magnetic Property of Nuclear Spins

An atom consists of a nucleus that contains positively charged protons and neutral

neutrons. Surrounding the atomic nucleus is a cloud of negatively charged electrons that are

located in orbits around this nucleus. In 1946, Nobel prize winners Felix Bloch and Edward

Purcell theorized that any spinning charged particle (for example, specific charged atomic

nuclei) when placed in a strong magnetic field creates an electromagnetic field around it (Bloch

1953). The fact that these spinning particles behave as tiny bar magnets and can emit signal

when subjected to a radiofrequency pulse has formed the basic concept of magnetic resonance

(Figure 3-1).

Existence of a nuclear magnetic field depends on the number of unpaired protons in an

atomic nucleus. According to quantum physics, protons in a nucleus are paired. For every proton

spin with a magnetic field in one direction, a paired proton aligns in the opposite direction

having an opposing magnetic field. Consequently, magnetic moments of a proton pair cancel









each other out and the net magnetic field is zero. In other words, when the number of protons in

an atomic nucleus is even, the magnetic moments created by the paired protons cancel out each

other and the net magnetic moment is zero. In contrast, when the number of protons is odd, there

is always one proton that is unpaired and this gives rise to a net magnetic field or non-zero

magnetic dipole moment (MDM) in the nucleus. The unpaired protons of an element can have

their nuclear MDM oriented in either a parallel or anti-parallel direction. This alignment follows

the Boltzmann distribution e-AE/kT (k stands for the Boltzmann constant) such that, at thermal

equilibrium, part of the nuclei aligns anti-parallel (higher energy state), and a larger part aligns

parallel (lower energy state). The net magnetization of a sample is equal to the sum of these

individual nuclear magnetic moments. Generally, a MDM exists in any nucleus that has an odd

number of protons. Nuclei of certain elements like Hydrogen (1H), Phosphorus (31P), Flourine (19

F), Sodium (23Na), Carbon (13C), Nitrogen isotopes (14N and 15N), Deutrium (2D) and Oxygen

isotope (170) have a MDM property (Garlick and Maisey 1992; Andrew 1994; Kevin K McCully

1994). Each of these nuclei can be used for MR purposes, but we use hydrogen atom for most

MRI purposes and hydrogen, phosphorus and carbon for most MRS purposes. Hydrogen is the

simplest and the most abundant element in the human body, since almost 60% of the human

body is made up of water. Every water molecule has two hydrogen atoms and larger biological

molecules such as lipids and proteins contain many hydrogen atoms. Sometimes enrichment

(adding an extra proton to the nucleus) of nuclei is required to enable them for use in MR. Thus,

enriching the 12C atom to 13C does not alter the chemical properties of the carbon atom much, but

enrichment gives it a nuclear magnetic moment (Mulkem and Chung 2000).

3.2.2 Larmor Frequency

Each proton behaves like a bar magnet and has its own magnetic axis. In nature, the

orientation of these axes is random. In the presence of a magnetic field (Bo) however, the axes









are aligned parallel to the axis of the main magnetic field. Spinning of protons around this axis

when placed in a magnetic field is called precession. The frequency at which the protons process

in the magnetic field is directly proportional to the strength of the main magnetic field. This

frequency is called the Larmor or precessional frequency. The Larmor equation expresses the

relationship between the precessional frequency and magnetic field strength.

g=6 x Bo

Where t is the Larmor frequency, Bo is the magnetic field strength and 6 is the gyro

magnetic constant. The gyro magnetic constant is a number without units that describes an

intrinsic characteristic of a nucleus in a given environment. For water protons, the gyro magnetic

constant is 42.6. At 0.5T the resonant frequency of water protons is therefore 21.3 MHz and at

1.5T it is around 63.9MHz. The gyro magnetic constant of protons within lipids and other non-

water molecules is somewhat different. Thereby, the resonant frequencies of protons in different

chemical environments are close, but not identical to that of water protons.

3.2.3 Longitudinal and Transverse Magnetizations

At the core of all MRI instruments is a homogenous magnetic field (Bo). The purpose of

the magnetic field is to cause magnetization of protons within it. The processing of protons gives

rise to small secondary magnetic fields, or magnetization. The average magnetization of protons

at a given time is referred to as net magnetization. At equilibrium, protons process with their net

magnetization align longitudinally along the axis of the main magnetic field and therefore the

magnetization is more precisely referred to as net longitudinal magnetization, Mz.

This equilibrium/net longitudinal magnetization (Mz) can be considered as potential

energy. Strength of Mz from tissues is dwarfed by the strength of main magnetic field Bo. Since

we can only transmit and receive signals that oscillate, and the longitudinal magnetization is not

an oscillating function, a receiver cannot read it. This severely limits detection of signal from









tissue protons at equilibrium. To detect MR signals for imaging, it is therefore necessary to

disturb this equilibrium (Ray H Hashemi 1997). An excitation pulse, called the radio-frequency

(RF) pulse is used to excite protons to emit radiations and cause disequilibrium. MR

electromagnetic radiations are called RF waves and the pulse is called RF pulse because these

are low frequency waves in the range of frequencies typically used by radio stations. These low

frequencies are not capable of any DNA or biological tissue damage that is otherwise associated

with high frequency ionizing radiations like X-Rays; a unique feature of MR that makes it

exceptionally useful for use in living tissues. Note worthily, to excite a proton processing within

a magnetic field, the frequency of the RF pulse has to be similar to the precession frequency at

which the proton is processing. A resonance condition is one in which "energy may be

transferred to and from a system very efficiently under unique conditions" (Gift, Pera et al.

1989). In absence of the unique conditions, energy transfer does not occur. Nuclear magnetic

resonance in particular, involves measurement of signals coming from the atomic nuclei in

response to radio waves that have the same natural frequency (precessional frequency) as the

nuclei themselves. That is, nuclei may absorb energy in the form of electromagnetic waves from

the RF pulse and give rise to a signal only when the frequency of the radio pulse exactly matches

the nuclear magnetic moment precessional frequency. The Larmor or precessional frequency is

called the resonance frequency because it is equal to the frequency of the radio pulse that induces

this resonance (reverberations/echoes) in the protons.

Application of the radio pulse causes the net longitudinal magnetization Mz to rotate into

the transverse plane, producing transverse magnetization Mxy (Figure 3-2). Since the transverse

magnetization continues to process in the x-y plane and is not obscured by the longitudinal

magnetization of the main magnetic field, it is now capable of inducing current in a coil placed in









its vicinity. The coil is called the RF receiver coil and the induced current in the coil (i.e. the

signal) is called an echo. Echo amplitude is greatest when it is first created. Once the excitation

pulse is switched off, the amplitude of this signal rapidly decays and becomes weaker with time.

Thus, the final signal is an oscillating decaying signal and is called free induction decay (FID).

This signal is recorded and stored in the computer to construct an image.

3.2.4 Relaxation Times

Immediately after application of the RF pulse, Mxy processes in the x-y plane around the

z-axis. This happens only as long as the pulse is on. As soon as the RF pulse is switched off, the

protons tend to regain their state of equilibrium, and hence their lowest energy state, by

realigning themselves in the direction of the main magnetic field, Bo. The net axis of proton

magnetization, Mz, eventually returns back to equilibrium. This return to equilibrium is referred

to as relaxation. Relaxation is the process in which the spins are relaxing back towards their

equilibrium state in the direction of the main magnetic field. Explanations of the two relaxation

times associated with MR follow.

Longitudinal relaxation time (T1 relaxation time): T1 or longitudinal relaxation time is

the time it takes to regain the longitudinal magnetization (Figure 3-3). As shown in the figure, at

time T1, 63% of Mz is regained. That is, T1 relaxation time can also be referred to as the time

required for the longitudinal magnetization to reach 63% of its original equilibrium level after

complete flip by a 900 pulse. T1 relaxation rate (1/T1) is the rate at which the longitudinal

magnetization Mz recovers along the z-axis after saturation by the RF pulse. Also, note that at

time 2*T1, the longitudinal magnetization has recovered to 91% of its original equilibrium value

and after three relaxation times, Mz has recovered to 97% of its original net magnetization

(Mitchell 1999). T1 is also called the spin-lattice relaxation time because it refers to the energy

that the protons have to give away to their surrounding (lattice) before gaining the equilibrium









state. Rapid transfer of energy to the surrounding lattice results in a shorter T1 and slow energy

transfer to the lattice produces a long T1.

Transverse relaxation time (T2 relaxation time): As soon as the RF pulse is switched

off, protons spin out of phase resulting in a rapid decay of the transverse magnetization Mxy. T2

relaxation time is the time it takes for the Mxy component to decay. Thus, in addition to T1

relaxation, a simultaneous but separate process is happening after the RF pulse (Gift, Pera et al.

1989). Mxy decay is a result of dephasing of protons once the RF pulse is removed. Dephasing

occurs because of a) spin-spin interaction: because of their proximity, protons within the same

molecule and between two molecules interact with each other. The spins dephase because of the

consequent spin-spin interactions; b) external magnetic field inhomogeneity: inhomogeneties in

the main magnetic field causes the protons to spin at slightly different frequencies.

T2 relaxation rate is the rate of decay of the transverse magnetization Mxy. T2 relaxation

time can also be referred to as the time required for the transverse magnetization to decay to 37%

of its signal (Figure 3-4). Like T1, T2 relaxation depends on inherent properties of the tissue and

is fixed for a specific tissue. T2 of a tissue depends on how fast the proton spins in the tissue

dephase. Rapid spin dephasing leads to a short T2 and slow dephasing causes a long T2. Tables

3-1 and 3-2 give a brief summary of the T1 and T2 relaxation times of human skeletal muscle

(bound water), muscle lipid, intramyocellular lipids (IMCL) and extramyocellular lipids (EMCL)

at various magnetic field strengths. Note that at a specific magnetic field strength, the T2

relaxation times of a tissue are 5-10 times smaller than the T1 relaxation times (Boesch 1999).

Moreover, both T1 and T2 are affected by magnetic field strengths such that T1 gets longer and

T2 decreases with increase in magnetic field strength).









3.2.5 Fourier Transform

Fourier transform is an extremely important and useful tool in MR that was introduced by

Jean Baptiste Fourier. The MR signal is acquired in the time domain i.e. a waveform that varies

with time. Hence, every signal is composed of a series of frequencies. The RF receiver coil

detects and records not one Larmor frequency, but sum of all the possible Larmor frequencies

produced from the frequency encoding gradients. To disentangle frequencies in the signal and

therefore determine precessional frequencies of nuclear spins in the x and y directions, a

mathematical manipulation of the signal called the Fourier transform (FT) is performed. FT

basically decomposes arbitrary signals of time into familiar sine and cosine waves. In MR, FT

enables one to determine which Larmor frequencies are present in any signal (Figure 3-5).

Fourier transform of the digitized MRI data is the final image and that of the raw MRS data the

final spectrum.

3.3 Magnetic Resonance Imaging

Magnetic Resonance Imaging (MRI) is a non-invasive, non-ionizing and powerful imaging

tool that is capable of imaging soft tissues in the body and is now being proposed as the imaging

modality of first choice for a wide array of diseases. MRI can produce contrasts between soft

tissues with great resolution. Contrast on MR images is a direct result of the different relaxation

times of protons in body tissues. Though these times per se cannot be altered, two important MR

parameters repetition time (TR) and echo time (TE) enable tissue contrast. Appropriate

adjustments of TR and TE allow putting more weight on T1 or T2 relaxation times of a tissue

thereby yielding the most common T weighted, T2 weighted and proton density weighted

(PDW) images. The choice of imaging is based upon the tissue desired for study.









3.3.1 T1 and T2 Weighted Images

Figure 3-6 shows T1 relaxation times of two tissues, one with a long T1 relaxation time

and the other with a short T1 relaxation time. When the repetition time (time between two RF

pulses; TR) is short, then the Mz component of the tissue with long T1 time possesses a smaller

magnitude of recovery as compared to Mz component of tissue with short T1. Consequently, the

magnitude of Mxy will differ largely between two tissues show larger differences in signal

intensities. It is however necessary that the TR should be at least close or similar to the T1 of one

of the tissues. This allows for best contrast between the two tissues. Also, a very short TR will

not produce any signal because of insufficient recovery of the Mz component of either tissue. On

the other hand, a very long TR will allow close to complete relaxation of both tissues such that

their signal intensities will be similar (not shown here). A long TR therefore prevents

differentiation between the two tissues. In this way, based on T1 of a tissue, tissue contrast is

obtained using an appropriate TR. Because T1 of tissues dictates final image contrast, such

images are called T1 weighted images. TE for such images is usually short.

Figure 3-7 shows T2 relaxation times of two tissues, one with a long T2 relaxation time

and one with a short T2. After the Mz component is flipped to the transverse plane, spin

dephasing of both tissue occurs. The tissue with long T2 will take longer to dephase as compared

to tissue with a short T2. When images are acquired at an optimal TE, adequate differences in

signal intensity exist between the two tissues. Therefore, differences in T2 relaxation will

produce contrast between two tissues if the signal is collected at an appropriate TE. When TE is

too short, the difference in SI is not much. For best T2 contrast, a TE is chosen that balances

sufficient decay of transverse magnetization from one tissue against adequate presence of

transverse magnetization from another tissue. Generally, images with TEs that approximate T2

relaxation times of tissues of interest have a significant T2 weighting. Lastly, very long TEs will









cause almost complete decay of transverse magnetization of both tissues and the ratios of their SI

and hence contrasts between the two tissues are lost. In this way, based on T2 of a tissue, tissue

contrast is obtained using an appropriate TE. Because T2 of tissues dictates final image contrast,

such images are called T2 weighted images. TR and TE for such images are usually long.

Lastly, consider a sequence utilizing a long TR and short TE that yields a proton density

image (Figure 3-7). When the TE is too short, enough time is not allowed for spin dephasing and

the differences in signal intensity are purely based on differences in proton concentration of

tissues. The T2 relaxation property of the tissue in this case is not explored. Furthermore, the

long TR eliminates differences in contrast based on T1 relaxation rates of tissues. This is a

proton density weighted image (PDW). For PDW image, one eliminates T1 effect by using a

long TR and eliminates a T2 effect by using a short TE (The two factors that determine the

respective image weighting). In such an image water will show up bright and fat will be less

bright.

3.3.2 Image Construction

MR signal, like other radio waves, does not possess any directional information.

Therefore, signals received contain information from the entire part of the body imaged. The

fundamental process used to determine the location of the sources of MR signal and hence

identify the specific body part imaged, is by application of magnetic field gradients. In a

homogeneous magnetic field, water protons resonate at the same frequency, regardless of

location. If a second magnetic field is now superimposed upon the main magnetic field, a

predictable variation is observed in the magnetic field along a predetermined axis. The resulting

magnetic field is highest at one end of the gradient and lowest at the other; between are

intermediate values along the axis of the gradient. Thus, protons at one end of the gradient spin

slower and protons at the other end spin faster. Gradients therefore create temporary









inhomogeneties in the main magnetic field to obtain spatial information. These signals from the

protons can now be measured and used to construct images. The three basic steps in an imaging

process are discussed below:

Slice selection: This process involves use of gradient during application of an excitation or

refocusing pulse. A slice is selected by simultaneous application of a selective RF-pulse and

gradient along the z-axis (conventionally defined) such that alignment of protons in a specific

width of tissue is disturbed. The pulse excites certain portion of tissue that has the same resonant

frequency as the gradient. At the end of the brief RF pulse, specific spins that are exposed to a

specific gradient in a sample are excited. Magnetization vectors representing out-of-phase

locations remain undisturbed and therefore do not contribute to the signal following the slice-

selective excitation process. Application of a gradient causes tipping of protons only in a specific

slice (Figure 3-8). The RF pulse has a specific frequency (called the central frequency) and a

range of frequencies around this central frequency (bandwidth of frequencies) that excites a

specific slice (as described above). The read out phase takes approximately 3msec. Though the

signal is obtained from the entire slice, the slice image is not yet seen because of lack of in-plane

spatial information about the slice. The signals are phase and frequency encoded for this

purpose(Gift, Pera et al. 1989; Ray H Hashemi 1997; Mitchell 1999; Mulkern and Chung 2000).

Phase encoding: Phase encoding conventionally defines application of magnetic field

gradients and hence image construction in the y-direction. To get spatial information in the y-

direction, a gradient is applied in this direction. The phase encoding gradient (Bruhn, Frahm et

al.) is turned on before application of the frequency encoding gradient (Gx) (explained next). It is

usually applied right after the RF pulse or just before the Gx gradient or anywhere in between.

Consider three rows of spins as in Figure 3-9. The left panel shows the spins before application









of Gy and the right panel shows the spins after application of Gy (arrows depict spin phases).

After application of Gy, spins in the upper row experience a higher magnetic field and process

faster, spins in the lowest row experience the least magnetic field and process slower. Spins in

the center row experience no change in magnetic field and therefore do not change their

precessional frequencies. Consequently, after a short time, protons in the three rows are left with

different phases of their spins. When the Gy gradient is switched off, the precessional frequency

of protons in each row becomes the same. However, the gradient has brought about a permanent

shift in the spin phases. So the protons process at same frequencies but are out of phase from

each other. Because the gradient brings about a change in the phase of proton spins this process

of image construction is termed "phase encoding". Note that a separate phase encoding step is

needed for each row of pixels that needs to be discriminated in the slice. Therefore to

discriminate between 256 rows in a slice, the process has to be repeated 256 times, each with a

unique Gy gradient (phase shift).

Frequency encoding: Frequency encoding conventionally defines application of magnetic

field gradients and hence image construction in the x-direction. This process occurs after the

application of Gy and it is during this cycle that the signal is recorded as a function of time and

stored in a computer. Consider the same matrix as defined above. To get spatial information in

the x-direction of this matrix, a gradient is applied in the x direction called the frequency-

encoding gradient (Gx). The left panel shows the spins before application of Gx and the right

panel shows the spins after application of Gx. Note that application of Gy had already caused a

phase shift. Application of Gx gradient varies the Larmor frequency of the spins in the three

columns so that the spins in the right column have a higher frequency than the spins in the left

column. Therefore, the central column of spins appears not to process at all, the one on the right









has the highest frequency and the one on the left has the smallest frequency. In addition, because

of the phase shift, each cell in the matrix ultimately experiences a unique phase shift and

frequency. The read out phase takes approximately 10msec. The different frequencies are

summed to form a signal that is detected by the receiver coil. The signal amplitude is a sum of all

the frequencies and is recorded as a function of time. Note that the signal is collected only once

during the read-out phase. That is, after one RF pulse, the Gy gradient is applied. This is then

followed by the Gx gradient and subsequent measurement of the signal. Each signal is a

representation of the entire image. However, a single RF pulse and hence one signal is not

enough to produce the image. To get spatial information from body-tissue a series of RF pulses

and phase encoding steps are necessary to resolve the image (Gift, Pera et al. 1989; Ray H

Hashemi 1997; Mitchell 1999; Mulkern and Chung 2000).

3.4 MRI Applications in Skeletal Muscle

Skeletal muscle function depends on the morphological and metabolic properties of the

muscle. MRI has the distinct ability to provide high-resolution spatial information and possesses

the ability to specifically measure muscle size, quantify muscle damage, and visualize muscle

recruitment patterns and intramuscular fat. The following paragraphs discuss the present and

potential role of MRI in characterizing skeletal muscle.

Muscle size significantly impacts muscle function (Berg, Dudley et al. 1991; Ploutz-

Snyder, Tesch et al. 1995; Stevens, Walter et al. 2004). Muscle size measurements include

anatomical and physiological cross-sectional area (CSA), muscle thickness and length, and

muscle volume. Traditional methods of assessing muscle size involve using anthropometric

measurements like skin fold thickness and limb circumference, ultrasonography and dual X-Ray

absorptiometry (DEXA). These measurements however, do not clearly differentiate between

muscle and non-muscle tissue. Consequently, inclusion of non-muscle tissue like intramuscular









fat and connective tissue in muscle size measurements can overestimate muscle size. Other

shortcomings include use of ionizing radiations (DEXA), limited field of view (ultrasonography)

and inability to distinguish between individual muscles and/or muscle groups (anthropometric

measures). MRI circumvents many of these disadvantages. MRI is non-ionizing, can clearly

contrast between fat and muscle tissue, differentiates between contractile and non-contractile

tissues within a muscle and can image the entire length of more than one muscle at the same time

(Boesch 1999; Mulkern and Chung 2000). These advantages have established MRI as a gold

standard of assessing skeletal muscle size. Numerous studies have used MRI as a standard

technique to assess skeletal muscle size following disease processes (Mulkern and Chung 2000),

progression of muscle atrophy following disuse (Vandenborne, Elliott et al. 1998; Kitahara,

Hamaoka et al. 2003) and effects of interventions (Stevens, Pathare et al. 2006). Moreover,

anatomical and physiological CSA measures by MRI have been shown to correlate significantly

higher with muscle strength measures such as maximum voluntary contraction than

anthropometric and DEXA indices (Bamman, Newcomer et al. 2000).

Muscle T2 relaxation properties are sensitive to muscle contraction and therefore possess

widespread applications in studying muscle characteristics. One of the initial studies reporting

exercise induced increase in signal intensity of skeletal muscle T2 in humans was conducted by

Fleckenstein in 1988 (Fleckenstein, Canby et al. 1988) Fleckenstein et al showed that

depending upon exercise intensity, contraction induced increase in T2 relaxation times can occur

as early as after two muscle contractions, reach a plateau by few contractions and return to

baseline values after 10-40 minutes of exercise. Interestingly, studies have successfully

demonstrated positive correlations between elevated T2 times following exercise with integrated

EMG patterns (Adams, Duvoisin et al. 1992). Furthermore, the exercise induced contrast









increases with exercise intensity so that the active area of the muscle is "mapped" to detect

muscle use (Ploutz-Snyder, Tesch et al. 1995). Consequently, if a muscle shows more contrast

shift, it reflects more use of the muscle and vice versa. (Adams, Duvoisin et al. 1992;

Fleckenstein, Watumull et al. 1993; Ploutz-Snyder, Tesch et al. 1995). This phenomenon of

muscle T2 has been used to infer which muscles are used during activity, the extent of their

contribution, presence of any substituted activity by other muscles; including contribution of

multiple and deep anatomical muscle groups at the same time. Thus, T2 changes are sensitive to

the activity status of the muscle and therefore hold tremendous potential in identifying muscle

activation patterns in both normal and diseased conditions.

Currently, studies indicate that the mechanisms of change in muscle T2 following rest to

work transitions are a consequence of increase in T2 relaxation times of muscle water (Saab,

Thompson et al. 2000; Patten, Meyer et al. 2003). This is probably a consequence of osmotically

driven shifts of water into intramyocellular spaces secondary to accumulation of end products of

muscle metabolism and/or intracellular acidosis (Ploutz-Snyder, Nyren et al. 1997;

Vandenbome, Walter et al. 2000; Patten, Meyer et al. 2003).

Note worthily, the increase in T2 values after normal activity is transient and typically

resolves within minutes to a couple of hours (Fleckenstein, Canby et al. 1988; Hayashi,

Hanakawa et al. 1998). However, enhancement of and subsequent persistence of T2 values for

longer periods (as long as two to three months) may indicate muscle damage. This is commonly

observed following eccentric exercise protocols. Several studies have reported that eccentric

exercise-induced muscle injury is associated with marked elevations in T2 values in both healthy

and patient populations (Ploutz-Snyder, Tesch et al. 1995; Ploutz-Snyder, Nyren et al. 1997;

Bickel, Slade et al. 2004). Elevated T2 values following strenuous exercises correlate with









specific markers of muscle damage including serum creatine kinase activity, plasma myosin

heavy chain fragments (a specific marker of slow fiber muscle damage), muscle soreness and

maximal isometric force (Foley, Jayaraman et al. 1999; Sorichter, Mair et al. 2001). Repeated

MRI measurements of skeletal muscle for as long as two to three months following the exercise

continue to show elevated T2 relaxation times; thereby suggestive of a long lasting change in the

muscle (damage). Interestingly, similar long lasting elevated T2 relaxation times reflective of

muscle damage have also been reported following reloading after hind limb immobilization,

spaceflight and disease processes in both humans and animals (Bryan, Reisch et al. 1998;

LeBlanc, Lin et al. 2000; Frimel, Walter et al. 2005). Muscle damage following reloading is

attributed to mechanical disruption of muscle fibers and inflammation in muscle (Kasper, Talbot

et al. 2002). Frimel et al have shown strong correlations between the elevated T2 relaxation

times and histological markers of muscle damage (Frimel, Walter et al. 2005). Relatively large

areas of T2 signal contrast have also been observed in the quadriceps muscle of persons with SCI

as compared to control subjects after electrically stimulated muscle contraction. The authors in

this study suggest that the increased recruitment of muscle in the SCI group not only implies

more use of the muscle to evoke force; but also that persistence of the elevated T2 values most

likely implies muscle damage (Slade, Bickel et al. 2004). In a follow up study, the same research

group found that the thigh muscle areas showed a similar enhancement of signal intensities after

a repeated bout of exercise that was followed 8 weeks after the initial bout. Since the controls did

not show similar areas of elevated pixel signal intensities, the authors surmised that the skeletal

muscle of persons with SCI remained injured and that probably the paralyzed muscles did not

develop a protective effect following the first bout of exercise (Bickel, Slade et al. 2004).









Lastly, based on the high-resolution feature and ability of MRI to contrast between tissues;

soft tissues within the muscle can be well demarcated using distinct MRI techniques.

Accordingly, MRI has served as a non-invasive measure to identify, quantify and monitor

progression of fatty tissue infiltration within skeletal muscle during diseased conditions (Huang,

Majumdar et al. 1994; Elder, Apple et al. 2004). However, similar to other imaging techniques,

T2 weighted MRI also suffers from partial volume filling, making it challenging to reliably

assess the inherent T2 of skeletal muscle in the presence of increased amounts of intramuscular

lipid.

Since elevated T2 values can reflect a variety of muscle processes including damage

(Bryan, Reisch et al. 1998; LeBlanc, Lin et al. 2000; Frimel, Walter et al. 2005), edema (Ploutz-

Snyder, Nyren et al. 1997; Patten, Meyer et al. 2003), fibrosis and fat (Huang, Majumdar et al.

1994), investigators are now making attempts to discriminate between these physiological

processes utilizing a variety of advanced MR techniques including diffusion weighted imaging,

magnetization transfer contrast and spectroscopy. Thus, the scope of MR in studying skeletal

muscle is on a constant rise.

3.5 Magnetic Resonance Spectroscopy

While MRI provides spatial information, magnetic resonance spectroscopy (MRS) has

been used for more than two decades to study the metabolism of human tissues and organs

(Heerschap, Houtman et al. 1999). MRS is used to identify metabolites and monitor the absolute

and relative concentrations of metabolites; thereby providing a non-invasive measure of

physiology and pathology in body tissues.

3.5.1 Contrasting MRI and MRS

The main difference between MRI and MRS is that MRS does not result in images with

spatial information, but rather results in a set of spectral peaks. The frequency encoding gradient









(read out) during MRI serves as a giant chemical shift in spatially discriminating proton spins

along the conventional x-axis (Boesch 1999). In MRS, chemical shift is produced by the inherent

nature of metabolites. Such inherent nature of the metabolites such as water and fat represent an

artifact and spatial mis-registration in MRI. For example: artifacts at organ fat interface in the

abdomen, vertebral body and intervertebral disc interface, bone muscle interface etc. Secondly,

MRI uses the protons in the body's water molecules that have a concentration of 110M to obtain

information about anatomy and pathology (Garlick and Maisey 1992; Boesch 1999). The sample

to be imaged is placed in a magnetic field gradient and depending upon the frequency of protons,

spatial information of the structure under is obtained. In MRS, proton nuclei and a variety of

magnetic nuclei present at concentrations of 2-10mM are used to obtain information about tissue

biochemistry. Precise localization to small volumes of body tissue are achieved using

surface/volume coils in combination with single volume or multiple volume spectroscopic

techniques (Alger 1994). Accordingly, MRS can quantify smaller concentrations of muscle

tissue (example lipid) that might not be feasible with imaging techniques (Schick, Machann et al.

2002).

3.5.2 Nuclei Studied with MRS

Hydrogen, phosphorus and carbon are widely used in MRS because these nuclei produce

strong MR signal and are of biomedical interest. Hydrogen is used to study hydrogen compounds

such as lipids. Phosphorus is used for phosphate compounds such as ATP and PCr that play a

key role in the bioenergetics of resting and exercising muscle states. Carbon is especially used

for studying the metabolic fate of glycogen and metabolites of the tricarboxylic acid, which play

a major role in carbohydrate metabolism (Boesch 1999). For detection by MRS, the nuclei need

to be in adequate concentrations as well as fairly mobile. For example, phosphorus nuclei at

370C in a 1.5T magnet have 1,000,026 nuclei in the low energy state and 1,000, 000 nuclei in the









high-energy state. The MR signal therefore comes from 0.0001% of the total nuclei (Garlick and

Maisey 1992). In most experiments, a threshold concentration of 0.5mmol of metabolite per kg

of wet weight is required for detection with MRS (Heerschap, Houtman et al. 1999). Metabolites

present in a concentration lower than milli molars (mM) are not detectable by MRS. Also,

molecules that are in a bound state are not detectable. For example: ADP is a phosphate

compound not detectable by 31P-MRS because most of the ADP exists in the bound form.

3.5.3 Spectral Components of MRS

Identifying different nuclei in the form of different peak resonances is an outstanding

feature of MRS. Each spectral peak is defined by a specific resonance frequency, height and

width. The height of the spectral peak or the area under it yields relative measurements of

metabolite concentrations. The area under a fully relaxed spectral peak is directly proportional to

the concentration of nuclei that make up the peak. The spectral width at half its height is called

the line width and gives relaxation time information because it is proportional to 1/7T2 (Garlick

and Maisey 1992). Spectral width is influenced by magnetic field inhomogeneties. The position

of the spectral peak with a specific resonant frequency on the plot is expressed as parts per

million. Therefore, the peaks have a specific positional relation with respect to each other in the

spectra (see below for detailed description of chemical shift). Thus, the chemical shift difference

between fat and water peaks will always remain 3.5 ppm in all strength fields. Accordingly,

metabolites can be identified from the position of spectral peaks. Figure 3-10 shows a typical

proton spectrum with its principle components.

3.5.4 Chemical Shift in MRS

The distinct peaks seen in spectroscopy can be attributed to the inherent "chemical shift" of

nuclei that are tested. Even in a perfectly homogenous magnetic field, not all protons resonate at

the same frequency. Depending upon the chemical environment of the nucleus in different









biochemical compounds, the same nuclei are subject to very small variations in frequency. For

example: hydrogen protons in water and fat have different chemical environments and therefore

their precessional/resonant frequencies are also different. In fact, protons at different sites within

the same complex molecule will have different precessional frequencies. Precessional frequency

of phosphorus nuclei (31P) at IT is approximately 17.18 MHz and that of hydrogen nucleus at IT

is approx 42.6MHz.

As seen in Figure 3-10, the two resonance signals of fat and water peaks are distinct. In

different magnetic field strengths, these resonance frequencies will vary and therefore the

numerical locator representing the two peaks will change. Therefore, in order to characterize and

specify the location of MR signal irrespective of magnetic field strength, an alternative method is

necessary. One method of solving this problem is to report the location of the MR signal in a

spectrum relative to a reference signal. Tetramethylsilane, (CH3)4Si, usually referred to as

TMS, has become the reference compound of choice for proton and carbon MR1. Furthermore,

to correct these frequency differences for their field dependence, they are divided by the

spectrometer frequency (example: 63.9 MHz for protons in a 1.5 T magnet). This difference in

the resonance frequencies from a reference frequency is called chemical shift (6). The resulting

number would be very small, since Hz is divided by MHz, and is measured in parts per million

(ppm), which is a dimensionless quantity independent of field strength. Chemical shift equation

is given by:

Chemical shift () = 106 [Frequency (sample)-Frequency (reference)]

(3-1)

Frequency (operating)


1 hIp \ "\ \ .cem.msu.edu/-reusch/VirtualText/Spectrpy/nmr/nmrl.htm









Note that frequency (sample) is the resonant frequency of the sample signal (example fat),

frequency (reference) is the frequency of the reference signal (TMS) and frequency (operating) is the

frequency of the spectrometer (magnet). When chemical shift between tissues is expressed in

absolute frequency, it is proportional to the magnetic field (Ray H Hashemi 1997).

3.5.5 Correction of Saturation Effects

Absolute or relative quantification of metabolites using MRS is best obtained after

correction of T1 and T2 saturation effects. This is because spectroscopic sequences employed in

obtaining metabolite concentrations from spectral peaks typically use relatively longer echo

times and shorter repetition times (less than 6T1). While the long TE permits T2 decay and phase

modulation; the shorter TR times allow for incomplete saturation of longitudinal magnetization.

Collectively, there is a net loss of the acquired MRS echoes that in turn underestimate metabolite

concentration. In addition, because of different T1 and T2 relaxation times of metabolites under

study, simple metabolite ratios at any one given TE and TR are inaccurate representations of

relative metabolite proportions. Therefore, signal correction is achieved by calculation of precise

values of relaxation times and calculating for saturation factors for each metabolite. Final

metabolite concentrations are obtained after correction with saturation factors.

3.5.6 Proton Spectroscopy ('H-MRS)

Hydrogen protons have been used traditionally for spectroscopy because of their natural

abundance in organic structures and high nuclear magnetic sensitivity. 1H-MRS has been a

sensitive and precise tool to quantify gross tissue fat content and derangements in lipid

metabolism. Figure 3-10 represents components of a typical proton spectroscopy spectrum from

a human skeletal muscle. Muscle creatine contains contributions from several metabolites

including creatine phosphate. It serves as a reserve for high energy phosphates and a buffer for

ATP/ADP reservoir (Castillo, Kwock et al. 1996). Interestingly, the lipid peak has contributions









from a variety of lipid components and these components can be resolved in a spectrum.

Decomposition of the fat signal into components (Figure 3-11) gives specific information

regarding intramyocellular lipids (IMCL) and extramyocellular lipids (EMCL). The IMCL is

suggested to correspond to lipids within the myocytes that serve as an important source of energy

supply during long duration endurance activities. The EMCL corresponds to the

extramyocellular lipid pool that constitutes the long term storage depot for lipids (Szczepaniak,

Babcock et al. 1999).

Intense signals from tissue water and fat swamps the less conspicuous signals from other

metabolites of biomedical interest (muscle metabolites such as creatine, choline, etc) that are

present in much lower concentrations. Successful signals from these metabolites can be obtained

by using water and fat suppression techniques during acquisition of spectra or by mathematical

elimination of the water peaks from the FIDs during post-processing of raw data (Hope and

Moorcraft 1991).

3.5.7 Phosphorus Spectroscopy (31P-MRS)

Magnetic properties of the phosphorus nucleus have gained momentum because of the

pivotal role of phosphorus containing compounds in energy metabolism of skeletal muscle.

Hoult et al published the first observations of metabolites from isolated rat skeletal muscle using

31P-MRS in 1974 (Hoult, Busby et al. 1974). Since then, 31P-MRS has made considerable impact

in studying the bioenergetics of normal and pathological neuromuscular tissues and used

expansively to quantify metabolic costs of various physiological processes in skeletal muscle.

Almost all muscle metabolic processes involve phosphates and visualizing energy metabolism of

skeletal muscle with 31P-MRS (both at rest and during exercise) has proven to be valuable. One

of the most outstanding features of 31P-MRS is its ability to continuously obtain time dependent

metabolic information from living tissues. The time resolution of 3P-MRS is around Ims and









this makes it possible to evaluate and quantify muscle oxidative and glycogenolytic energetic

in-vivo (Argov and Arnold 2000).

In general, seven peaks can be identified in a phosphorus spectrum acquired from a

skeletal muscle (Figure 3-12). The major peaks correspond to inorganic phosphate (Pi),

phosphocreatine (PCr), and three phosphate groups of adenosine triphosphate (ATP). In addition,

peaks of phosphomonosterase (PME) and phosphodiesterase (PDE) can be identified with

greater spectral resolution.

Metabolite concentrations are commonly quantified from relative spectral amplitudes using

ATP peaks as an internal standard. Normal Pi concentration is 3-5mM. Pi/PCr ratio is closely

related to the phosphorylation potential and reflects the energy state of the muscle (Veech,

Lawson et al. 1979; Chance 1984). At rest, PCr/Pi ratios range from 6-12 in healthy human

muscles, depending upon the muscle studied. The single Pi peak is actually a combined peak of

two molecules (HP042- and H2P04-) that are in fast exchange with each other. These acidic and

basic molecules resonate at two different positions in the spectrum. Position of the Pi peak

depends upon the relative concentrations of the two molecules and based on frequency shifts of

the Pi peak, intracellular pH can be determined. Intracellular pH calculated based on the

chemical shift difference between PCr and Pi (d) is rather accurate (up to 0.02pH units) and

calculated by the following equation:

Intracellular pH = 6.75 + log [(3.27-d)/(d-5.69)] (3-2)

This equation is derived from the more general Henderson-Hesselbach equation that

defines the pH value of a sample via a titration curve (pH as a function of resonance shift);

base concentration

Intracellular pH = pKa + log acid concentration (3-3)











Where, pKa is the dissociation constant of Pi (6.9), generally defined as the ability of an

acid to give away its H+ protons. Lastly, it is noteworthy that otherwise undetectable metabolites

including adenosinediphosphate (ADP) and ionic concentration such as Magnesium (Mg+2) can

also be indirectly estimated using 31P-MRS based on measurements of creatine, phosphocreatine,

pH and the equilibrium constants.

As discussed below, variations in phosphate metabolite concentrations from rest to

exercise and during recovery have been used to illustrate muscle energetic in-vivo.

3.6 Application of Magnetic Resonance Spectroscopy in Skeletal Muscle

Depending upon the nucleus studied, MRS allows observation of high-energy phosphates

(31P-MRS), glycogen (13C-MRS) or intramyocellular fat (1H-MRS). In 1955, skeletal muscle was

the first biological tissue that was studied using MRS (Odeblad and Lindstrom 1955). While

initial application of the technique was restricted to cell cultures and biological systems in situ,

MRS is now widely used for a range of in-vivo metabolite measurements in animal and human

muscle. These include a) studying different sites of the same muscle and a number of muscles

simultaneously, b) yielding metabolite data from deep muscles that are difficult to access by

biopsy, c) directly quantifying muscle metabolites and scrutinizing cellular components of

mitochondrial metabolism instead of relying on global and/or invasive measures of oxidative

phosphorylation (for example VO2max measures) d) determining real time in-vivo biological

processes with a temporal resolution on the order of seconds, e) obtaining longitudinal

measurements of skeletal muscle bioenergetics thereby permitting the study of disease

progression over time and reliably quantifying effects of therapeutic interventions.









3.6.1 Role of H-MRS : Quantify Intramuscular Fat

1H-MRS has the unique ability to measure intramuscular lipid content free from muscle

tissue, thereby overcoming limitations of partial volume filling (seen with MRI). In addition to

the total lipid content, 1H-MRS can separately quantify lipid muscle tissue into its intracellular

and extra cellular components. Specifically, 1H-MRS is the only currently available non-invasive

tool to quantify intramyocellular (IMCL) and extramyocellular lipid (EMCL) within skeletal

muscle. As discussed in chapter 1, intramyocellular lipid (IMCL) content has the potential to

serve as a non-invasive marker of insulin resistance, especially in sedentary individuals. Non-

invasive measures of IMCL via H-MRS are found to correlate well with IMCL quantified by

electron microscopy and histochemistry using Oil Red O staining (Hinderling VB 2006).

Sensitivity of 1H-MRS to changes in lipid measurements is high and 1H-MRS has proven to be a

more valid technique for measuring IMCL than morphometry and histochemical analysis of

IMCL (Schrauwen-Hinderling, Hesselink et al. 2006). The accuracy and sensitivity of 1H-MRS

in measuring IMCL concentrations is also sufficient to measure changes in IMCL (depletion and

recovery) after exercise (Boesch, Decombaz et al. 1999). As a result, a variety of studies have

utilized spectroscopy to quantify whole muscle fat and its components and their possible

association with insulin resistance (Schick, Eismann et al. 1993; Machann, Haring et al. 2004;

Boesch 2007).

3.6.2 Role of 'H-MRS: Assess Muscle T2 Characteristics

Chemical shift differences between protons bound to muscle and protons bound to fat

yield separate water and lipid peaks; thereby allowing measurements of T2 relaxation properties

of muscle and fat tissues separately. Accordingly, muscle tissue composition including muscle

damage and edema is more reliably measured using 1H-MRS measures. Investigators are using

this ability of 1H-MRS to decipher between fatty and damaged muscle (Walter, Cordier et al.









2005) and to identify muscle degeneration in a variety of muscle disorders (Bongers, Schick et

al. 1992).

3.6.3 Role of 31P-MRS: Quantify Resting Muscle Metabolites

At rest, most of the muscle energy supplies come from the formation of ATP from ADP

and inorganic phosphate (Pi) that takes place in the electron transport chain of the mitochondrion

(mitochondrial oxidative phosphorylation). Metabolism at rest occurs at a low rate. Accordingly,

it is easy to obtain baseline 31P-MRS spectrum. Studies report 31P-MRS measured PCr/Pi ratios

as reliable markers of phosphorylation potential (Chance 1984; McCully, Kent et al. 1988).

Phosphorylation potential is the energetic potential of the cell. The variables [ADP][Pi]/[ATP]

describe the effects of energy demand and potential for ATP supply by oxidative

phosphorylation. Alterations in resting metabolite content, metabolite ratios and change in pH

reflect disturbances in the metabolic pathways and/or structural dysfunctions of mitochondria or

a diseased state of the muscle as a whole (Heerschap, Houtman et al. 1999; Mattei, Bendahan et

al. 2004). Increase in resting Pi content and Pi/PCr ratios of skeletal muscle obtained by 31P-MRS

have been observed in a variety of disorders including primary mitochondrial diseases,

myopathies, muscle injury, disuse and denervation (McCully, Kent et al. 1988; Argov and

Arnold 2000; Tartaglia, Chen et al. 2000). In the lower limb muscle disuse model, Pathare et al

have reported an increase in basal Pi along with concurrent decrease in the PCr/Pi ratios in the

plantarflexor muscles of individuals following cast immobilization secondary to ankle fracture

(Pathare, Walter et al. 2005; Pathare, Vandenborne et al. 2007). Moreover, the elevated Pi

content and Pi/PCr ratios were found to significantly impact the force generating capacity of the

skeletal muscle. Elevated Pi/PCr ratios are also demonstrated during muscle injury (McCully,

Kent et al. 1988; Pathare, Vandenborne et al. 2007). Furthermore, right shift of the Pi peak









relative to the PCr peak in a 31P spectrum implies elevated H+ ions produced by lactate and

hence an acidic state of the muscle.

Information provided by resting spectra reflects metabolic state of the muscle at rest.

Abnormalities in muscle bioenergetics however, are better characterized in dynamic experiments

performing 31P-MRS during exercise and recovery. The subsequent section describes one of the

most popular applications of 31P-MRS measurement of metabolic oxidative capacity of the

skeletal muscle.

3.6.4 Role of 31P-MRS: Identify Fiber-type and Muscle Fatigue

Three individual Pi peaks have been identified in the 31P-MRS spectrum of the human

gastrocnemius muscles following brief periods of muscle contraction (Vandenborne, McCully et

al. 1991). Based upon distinct positions of the three peaks in the MRS spectrum that correspond

to heterogeneity in pH, the authors hypothesized that the three Pi spectral peaks correspond to

slow oxidative, fast twitch oxidative and fast twitch glycolytic fibers. Similarly, split in the Pi

peak has been reported by Kutsuzawa et al in patients with chronic respiratory impairment

during moderate exercise intensity of the forearm muscles; implying the contribution of different

fiber types to muscle work. Interpretation of muscle fiber type is also based upon the ratios of

phosphate metabolites (Kutsuzawa, Shioya et al. 1992). Resting Pi/PCr ratios are higher in red

muscle as compared to white muscles because of baseline higher levels of Pi in red muscle and

higher PCr levels in white muscle (Meyer, Brown et al. 1985; Kushmerick, Moerland et al.

1992).

Amongst various other sources of peripheral muscle fatigue, altered levels of muscle

metabolites (example excessive Pi levels, H and H2P04-) are purported as a potential source of

skeletal muscle fatigue at the myocyte level (Kevin K McCully 1994). Fatigue is described as the

decreased force generating capacity of the muscle. Metabolic aspects of muscle fatigue can be









evaluated by monitoring concentration of oxidative metabolites and pH of exercising muscle. For

example: A strong relationship has been observed in the time frame of fatigue in the dorsiflexor

muscles of healthy subjects and the accumulation of H2P04- (Kent-Braun, Ng et al. 2002).

3.6.5 Role of 31P-MRS: Measure Muscle Oxidative Capacity

One of the most valuable contributions of 31P-MRS is the non-invasive measurement of

the oxidative capacity of skeletal muscle (McCully, Fielding et al. 1993; Kemp, Sanderson et al.

1996; Argov and Arnold 2000). Measuring oxidative capacity via 31P-MRS has gained

tremendous attention because of participation of energy rich phosphates in muscle bioenergetics.

One of the most reliable measures of oxidative capacity using 31P-MRS is the rate of PCr

resynthesis. PCr recovery rates have been extensively used in both healthy and diseased muscles

as estimates of muscle oxidative capacity (Meyer 1988; Paganini, Foley et al. 1997; McCully,

Mancini et al. 1999; Argov and Arnold 2000; Kent-Braun and Ng 2000; Pathare, Vandenborne et

al. 2007).

Energy released from ATP hydrolysis forms the principal energy source for muscle

metabolism during rest and work. The immediate source of ATP at onset of exercise is provided

by hydrolysis of PCr catalyzed by the enzyme creatine kinase.

MgADP- + PCr + H 4 N MgATP2- + Creatine (3-4)

Creatine Kinase

The resultant ATP is quickly hydrolyzed into inorganic phosphate (Foley, Jayaraman et

al.) and ADP while releasing energy enough to meet energy demands of the cell.

PCr + H+ _Creatine + Inorganic phosphate (3-5)

Noteworthy, homeostatic mechanisms exist within the myocyte that couple overall ATP

utilization with ATP synthesis; thereby maintaining nearly steady concentrations of ATP

(Erecinska and Wilson 1982; Kushmerick 1995). In this respect, one of the most important









contributions is the temporal and spatial buffer role of creatine kinase reaction in maintaining

energy homeostasis. High PCr content and creatine kinase enzyme activity in skeletal muscle

makes the creatine kinase equilibrium reaction an extremely efficient enzymatic system that is

capable of buffering transient changes in ATP (Kushmerick and Meyer 1985). A closer look at

bioenergetics' data during moderate exercise reveals that while ATP is held constant, the PCr

levels continue to deplete (PCr hydrolysis). The following steps (Bessman and Geiger 1981)

briefly describe the creatine kinase buffer phenomena (Figure 13) : a) During beginning of

exercise, cystolic PCr hydrolysis produces ATP b) Subsequent cystolic respiration (aerobic and

anaerobic) is controlled through kinetic control of ADP (respiratory control of ADP) (Dudley,

Tullson et al. 1987) while the reverse creatine kinase continues to maintain ATP levels (temporal

buffer role of the creatine kinase reaction by myofibrillar creatine kinase) c) the creatine-kinase

reaction at sites of ATPase activity also triggers the creatine-phosphocreatine shuttle mechanism

such that creatine released from sites of contraction diffuses (shuttled) into the mitochondria d)

ATP produced by oxidative phosphorylation and glycogenolysis is used by mitochondrial

creatine kinase isoenzyme to resynthesize PCr. This PCr is then shuttled back for participation in

the CK reaction to release ATP at the sites of muscle contraction. Thus, the mitochondrial

creatine kinase serves as an intermediate in the transfer of high energy phosphate from sites of

ATP production (mitochondria and glycolytic loci) to ATP consuming locations in the myocyte

(Kushmerick 1995; Heerschap, Houtman et al. 1999). Therefore, while muscle creatine kinase

controls the backward reaction (PCr + ADP + H+ Creatine + ATP) outside the mitochondria;

the forward reaction is controlled by mitochondrial creatine kinase isoenzyme (Sahlin, Harris et

al. 1979). In this regard, the creatine kinase reaction acts as a spatial buffer and maintains cell

ATP homeostasis (Figure 3-13) e) However, the rate of ATP release from oxidative









phosphorylation and glycogenolysis is not as fast as the rate of ATPase reaction' thereby

accounting for depletion of PCr; which is in proportion to ATP turnover until the end of the

workloads. Large declines in PCr are therefore accompanied by small or no changes in ATP and

ADP. At steady workloads, the oxidative phosphorylation reaches a steady state of ATP release,

thereby leveling off PCr levels as long as no further demand for ATP arises. [Note that the

shuttle mechanism explained above is only one theory that describes PCr replenishment by ATP

in the skeletal muscle. This metabolite homeostasis can also occur by simple diffusion processes

(the buffer and spatial roles); which are the alternate and more modern explanations to describe

this homeostatic mechanism (Kushmerick 1995, Kushmerick and Meyer 1985)].

3.6.6 Relationship between PCr Recovery Rates and Muscle Oxidative Capacity

During recovery from exercise, ATP breakdown is minimal and PCr levels return to

baseline values. Studies have well established that during recovery from exercise, glycolysis

ceases and PCr in the muscle cell is replenished at the expense of ATP produced via

mitochondrial oxidative phosphorylation (Taylor, Bore et al. 1983; Meyer 1988; Quistorff,

Johansen et al. 1993; Kemp, Roberts et al. 2001; Mattei, Bendahan et al. 2004). Indeed, for

steady state, low intensity exercise, where no change in intracellular pH (Phillips, Wiseman et

al.) is expected, a constant positive relationship exists in the time scales and rates of oxygen

consumption in the mitochondria following exercise and PCr recovery rates (Piiper and Spiller

1970; Meyer 1988; McCully, lotti et al. 1994; Thompson, Kemp et al. 1995). These results

confer that PCr resynthesis after exercise is mediated via oxidative phosphorylation.

Accordingly, rate constant of PCr recovery (PCr recovery rates) is conventionally been

recognized as an index of mitochondrial oxidative capacity.

PCr resynthesis follows a pseudo first order kinetics and the rate constant is described by

a monoexponential curve as long as PCr is depleted by a sufficient amount (-50%) and pH does









not decline below 6.75 units (Taylor, Bore et al. 1983; McCully, lotti et al. 1994; Boesch 2007).

In addition, for the rate constants of PCr recovery to correctly reflect oxidative capacity, several

assumptions are made: a) the creatine kinase reaction is in equilibrium b) presence of an intact

vascular (hence oxygen) supply to the muscle during recovery c) the PCr resynthesis is

predominantly due to ATP released by oxidative processes (thus anaerobic ATP production is

negligible) (Meyer 1988; Paganini, Foley et al. 1997). Furthermore, PCr resynthesis following

depletion necessitates the presence of intact blood supply and oxygen availability (Sahlin, Harris

et al. 1979; Quistorff, Johansen et al. 1993).

Importantly, while PC recovery rates can be used as indices of oxidative capacity,

absolute estimations of metabolic capacity from PCr recovery data is possible using rigorous

theoretical framework (Paganini, Foley et al. 1997). For practical purposes, the rate constant of

PCr recovery (k) that reflects muscle oxidative capacity is calculated using the following

equation (Hartkopp, Harridge et al. 2003; Kitahara, Hamaoka et al. 2003):

PCr (t) = PCro + APCr (1-e kt) (3-6)

Where, PCr is the concentration of PCr at a given time t during post exercise recovery;

PCro is the PCr concentration at end of exercise and APCr is the change in PCr concentration

after recovery from exercise. Note that the rate constant (k) is influenced by change in pH such

that lower pH decreases the rate constants of PCr recovery (Bendahan, Confort-Gouny et al.

1990; McCully, lotti et al. 1994; Paganini, Foley et al. 1997; Heerschap, Houtman et al. 1999).

Alterations in pH apparently alter the equilibrium state of the creatine kinase reaction and/or

mitochondrial ATP yield (Bendahan, Confort-Gouny et al. 1990; McCully, lotti et al. 1994).

Accordingly, measurements of oxidative capacity typically accompanying pH decrease do not

correctly reflect oxidative capacity. The inverse of the rate constant kPCr is called the time









constant of PCr recovery (' PCr,) and is related to peak oxygen uptake (V02) and maximal

oxygen consumption (V02 max) (Thompson, Kemp et al. 1995). The T PCr is reported to be

independent of exercise intensity and end exercise levels of PCr.

Another reliable measure, initial post exercise PCr resynthesis rate (Vmeas), is also

demonstrated to reflect mitochondrial oxidative capacity and is less affected by the end exercise

PCr levels and pH (Meyer 1988; Kemp, Thompson et al. 1994; Lodi, Kemp et al. 1997). This

initial rate of PCr resynthesis during the first few seconds is estimated from the first two-three

data points in recovery and is quantified by:

Vmeas = (d[PCr]/dt) (3-7)

Also, a more complete estimation of maximum mitochondrial capacity (Qmax) has also

been used (Kemp, Thompson et al. 1994);

Qmax = k [basal PCr] (3-8)

where k is the rate constant of PCr recovery and A [PCr] is the basal PCr levels.

As mentioned above, increase in ADP levels that follow exercise, tend to return to baseline

levels soon after exercise. Faster the recovery of ADP levels, faster is ATP formed and therefore

this implies an increased efficiency of mitochondria. Indeed, ADP concentrations in myocytes

are known to regulate mitochondrial ATP synthesis. As ATP is used up during exercise, the

[ADP] increases and return of [ADP] levels back to normal are indicative of in vivo

mitochondrial function or oxidative capacity (Argov, De Stefano et al. 1996). Few studies have

in fact proven that the rate of decline in ADP is a more sensitive measure of oxidative capacity

than PCr recovery rates because of its robustness to changes in intracellular pH (Kemp and

Radda 1994; Argov and Arnold 2000). However, since ADP levels are not directly measurable, it

is less commonly used. Also, owing to its relatively lower concentration; [ADP] cannot be









directly measured from a normal 31P spectrum; but can be estimated using 31P-MRS assuming a

constant magnesium ion concentration (Kemp, Sanderson et al. 1996; Durozard, Gabrielle et al.

2000).

3.7 Summary

This chapter reviews the fundamental concepts of MR with main focus on MRI and MRS

applications in skeletal muscle. MR has become the investigative tool of choice because of its

non-invasiveness, ability to perform longitudinal studies and obtain a wealth of anatomical and

biochemical information of skeletal muscle. While MRI more clearly assesses muscle

morphology, MRS estimates physiological metabolic processes in real time. Various applications

of MRI in skeletal muscle include assessment of muscle size, identification of muscle damage

and quantification of fatty tissue infiltration. The most common nuclei used in MRS include

proton and phosphorus. Through its unique ability to decipher between protons in water and fat

peak in muscles, 1H-MRS has gained widespread application in characterizing tissue T2

relaxation properties and separately quantifying IMCL and EMCL fat components. 31P-MRS has

been utilized to estimate muscle pH and quantifying energy rich phosphates that participate in

basic physiological processes. The high time resolution and the ability to quantify metabolites in

real time has made 31P-MRS a unique non-invasive tool to reliably measure muscle oxidative

capacity in-vivo.










Table 3-1. Longitudinal (T ) relaxation times (in milliseconds) of water and lipid components
obtained from human skeletal muscle at different magnetic field strengths.

Reference Muscle Magnetic Water Fat IMCL EMCL
field strength
Bruhn 1991 Plantar flexors 1.5T n/a 300 n/a n/a
Bongers 1992 Plantar flexors 1.5T 1100-1400 280-330 n/a n/a
Schick 1993 Soleus 1.5T 1100-1500 270-280 n/a n/a
Sinha2002 Soleus 2.1T 1220-1370 n/a 306-378 221-461
Hwang Jong Hee Soleus 4T 1300-2300 n/a 340-440 340-440
2001


Table 3-2. Transverse (T2) relaxation times (in milliseconds) of water and lipid components
obtained from human skeletal muscle at different magnetic field strengths.

Reference Muscle Magnetic Water Fat IMCL EMCL
field strength
Bongers 1992 Plantar flexors 1.5T n/a 75-100 n/a n/a
Bruhn 1991 Plantar flexors 1.5T 30 90 n/a n/a
Szczepaniak 1999 Soleus 1.5T 37-43 n/a 82-90 66-76
Schick F, 1993 Soleus 1.5T 50 70-85 n/a n/a
Sinha R, 2002 Soleus 2.1T 29-34 n/a 70-83 76-86
Hwang Jong Soleus 4T 21-31 n/a 68-82 58-78
Hee 2001









Bo


prwf!io V.


dipole
moment


nucleus
I jj 13 C 31lp


/


iar gnet


Figure 3-1. Schematic representation of a nucleus and its behavior as a bar magnet in an external
magnetic field Bo



Bo






@ Iif -

l z


Figure 3-2. Application of a 900 RF pulse.











a7%


I



Ti 2T1




Figure 3-3. T1 relaxation time


Mvz


TI relExation
("Ciymy~3


1 .eexpt[ ~


3Ti


T2V relation



S I

Time


Figure 3-4. T2 relaxation time


















r Fourier Traisfrni.





Time
Figure 3-5. Fourier transform of the FID signal.


Mz


Fa -


FreqIeucy


.


Timh


Figure 3-6. Schematic representation of two tissues with different T1 relaxation times. A) Faster
Tl relaxation times of fat tissue and relatively slower T1 relaxation times of skeletal
muscle tissue. B) Representative T1 weighted trans-axial image of a healthy human
calf muscle obtained 3T.



















for PD'w image -for T- wejbt inm~e


A B
Figure 3-7. Schematic representation of two tissues with different T2 relaxation times. A) Faster
T2 relaxation times of muscle tissue and relatively slower T2 relaxation times of fat
tissue. B) Representative T2 weighted trans-axial image of a healthy human calf
muscle obtained at 3T.


At pieulse wab -L Widw brfe A
kmpmcy- iand a biPsdwift off-beq~aW


AbppLckai~ooofiradteu
a8~oqzaxi30zz


ELcitation otslice with a specific
heee kns s a secfc wdth of slie h in thair e s
Figure 3-8. Bandwidth of frequencies excites a specific width of slice in the sample.










II
II


/'

II


/
fiaB


'I


Application ofvadieult i y-direcioi


I


r


/

'IB


Appicatio of adieut in x-dirfcLion


Figure 3-9. Frequency and phase encoding gradient effects on spins. A) Spins (arrows depict spin
phases) in all the rows of the grid are aligned in the same direction of the main
magnetic field before application of a phase encoding gradient (Gy) B) After
application of Gy, spins in the upper row experience a higher magnetic field and spins
in the lowest row experience the least magnetic field. C) After application of
frequency encoding gradient, spins in the upper and lower rows change phases while
those in the center row experience no change in magnetic field.


Water peak


Muscle metabolites


f at peak

4.7 1.2 (prpm)


Figure 3-10. Representative 1H-MR spectrum of a healthy human soleus muscle at 1.5Tesla.




104


t
t
II
II


1'
fi
'I


f

1'










EMCL
(CH, in


IMCL
(CH,i)n


CIholine ( t'n. )


Cr (C H,1) |



5 3.7 2.5 1.25 0 (ppm)
Figure 3-11. Representative 1H-MR spectrum of a human skeletal muscle at 1.5Tesla showing
decomposition of the lipid peak into its IMCL and EMCL components after water
suppression (at ppm = 0). The creatine (Cr) and choline peaks represent muscle
metabolites.


PCr













yATP aATr


BATP


PME P A_ _ADH_

4.8 0 -2.5 -7.5 -16.0 (ppm)
Figure 3-12. Representative typical 31p-MR spectrum of a rat calf muscle at 11Tesla (NADH
nicotinamide dehydrogenase; PME= phosphomonoesterase)









I myofibdillar m~m
---- ---- -- --- ---- ----


Cr + ADP
A


U


'iliI


ceatime Lwinfs


ATI


+ Cr


\ bys ATT awe/
il*"lli ll l

/

lillllll iuuiuuumuuuuuuu ri U 11 Ei lluuuuuuum mumuul lllliu


a yf I LWA j


F"""""'


iLLrT IA


oxidaive


'I mitcoodia. I
Figure 3-13. Schematic representation of the creatine-phosphocreatine shuttle and buffer role of
the creatine-kinase reaction muscle.


.,_++









CHAPTER 4
OUTLINE OF EXPERIMENTS

The overall objective of this dissertation was to investigate the lower extremity skeletal

muscle adaptations after incomplete spinal cord injury. An outline of aims and hypotheses

related to experiments is given below.

4.1 Experiment One

4.1.1 Specific aim

a) To quantify lower extremity skeletal muscle size in persons with chronic incomplete

spinal cord injury (SCI) and in age-matched healthy individuals b) To determine atrophic

response in anti-gravity versus the non anti-gravity muscles c) To examine the impact of

ambulatory ability on muscle size.

Maximum cross sectional areas (CSA) of the thigh and calf muscles were quantified using

high-resolution magnetic resonance imaging (MRI).

4.1.2 Hypotheses

a) Chronic incomplete SCI leads to a decline in lower extremity muscle CSA.

b) Greater decreases are observed in the lower extremity anti-gravity muscles as compared

to the non anti-gravity muscles.

c) Persons with incomplete SCI who use a wheelchair as primary means of mobility show

a larger atrophic response than persons with SCI who do not use a wheelchair for ambulation.

4.2. Experiment Two

4.2.1 Specific Aim

a) To characterize muscle characteristics via T2 relaxation times of lower extremity

muscles in persons with incomplete SCI and compare that with age-matched controls. b) To









quantify the intramuscular lipid content along with intramyocellular and extramyocellular lipid

composition of the soleus muscle after incomplete SCI in humans.

Skeletal muscle T2 relaxation properties and soleus muscle lipid content are determined

using combinations of MRI and proton spectroscopy (1H-MRS).

4.2.2 Hypotheses

a) Persons with incomplete SCI show alterations in the T2 relaxation times of the lower

extremity muscles as compared to healthy individuals.

b) Persons with incomplete SCI show elevated soleus muscle lipid content along with

elevations in both the intramyocellular (IMCL) and extramyocellular lipid (EMCL) components

as compared to healthy individuals.

4.3 Experiment Three

4.3.1 Specific Aim

To assess the impact of two and nine weeks of locomotor training on markers of muscle

damage in persons with incomplete SCI. Lower leg skeletal muscle T2 relaxation properties are

determined using combinations of MRI and spectroscopy (1H-MRS) measures.

4.3.2 Hypothesis

Two and nine weeks of locomotor training alter the T2 relaxation properties of lower leg

muscles of persons with incomplete SCI.

4.4. Experiment Four

4.4.1 Specific Aim

a) To assess the impact of nine weeks of locomotor training on the lower extremity muscle

size of persons with incomplete SCI. b) To assess the impact of nine weeks of locomotor training

on the soleus muscle composition of persons with incomplete SCI.









Skeletal muscle CSA and soleus muscle lipid content are determined using combinations

of MRI and spectroscopy (1H-MRS).

4.4.2 Hypotheses

a) Locomotor training attenuates the atrophic response and increases the muscle cross-

sectional area in lower extremity skeletal muscle size of persons with incomplete SCI.

b) Locomotor training alters the lipid content of soleus muscle in persons with incomplete

SCI, with changes seen both in the IMCL and EMCL.

4.5. Experiment Five

4.5.1 Specific Aim

a) To determine the impact of acute spinal contusion (one week) on the content of resting

phosphates, and hence the phosphorylation potential of rat calf muscle after spinal cord

contusion. b) To longitudinally monitor alterations in the phosphorylation potential of the calf

muscles.

Phosphorus magnetic resonance spectroscopy (31P-MRS) at a high magnetic field strength

(11T) was used to monitor basal phosphate metabolites for three weeks after spinal cord

contusion. Intracellular quantification of phosphate metabolites was achieved using biochemical

assays.

4.5.2 Hypotheses

a) Immediately after spinal cord contusion (one week), there is an alteration in the resting

muscle phosphate content; and hence a change in the phosphorylation potential of the paralyzed

hind limb muscle.

b) Muscle phosphate levels approach control values by three weeks after spinal cord

contusion.









4.6. Experiment Six


4.6.1 Specific Aim:

a) To determine the impact of acute spinal contusion (one week) on oxidative capacity of

rat hind limb muscle after spinal cord contusion. b) To longitudinally monitor alterations in the

skeletal muscle oxidative capacity of the hind limb muscles.

Phosphorus magnetic resonance spectroscopy (31P-MRS) was performed on the animal

hind limb muscle using an electrical stimulation protocol to quantify in-vivo muscle

bioenergetics in real time. Measurements were obtained once weekly for three weeks starting at

one week post injury.

4.6.2 Hypotheses

a) Immediately after spinal cord contusion (one week), there is a decrease in the oxidative

capacity of the paralyzed hind limb muscle.

b) Muscle oxidative capacity approach control values after three weeks of spinal cord

contusion.









CHAPTER 5
METHODOLOGY

In this dissertation, experiments one through four are focused on studying muscle

adaptations in humans with incomplete spinal cord injury. Experiments five and six are

conducted in a rat model of spinal contusion injury. While non-invasive MR techniques are

employed as the key tool for all experiments; combinations of biochemical assays, histology and

pap smears have been employed as additional methodologies to supplement MR findings in the

animal studies. The following sections discuss relevant experimental protocols and data analyses

relevant to both human and animal studies.

5.1 Human Studies

5.1.1 Subjects

Persons with incomplete SCI and age-matched controls were recruited for this study. Prior

to participation in the study, all subjects were informed of the study purpose and all provided

written informed consent as approved by the Institutional Review Boards at the University of

Florida.

Able-bodied controls: Age, weight, height and gender matched individuals from the local

University of Florida setting were recruited as healthy volunteers for the study. Control subjects

were recreationally active, but not engaged in any rigorous exercise program. Controls were

screened for MR compatibility before participation.

Individuals with incomplete spinal cord injury: Individuals with SCI were recruited

from the local community at the University of Florida in Gainesville, FL or at the University of

Georgia, Athens, GA. The inclusion criteria for participants were 1) diagnosis of traumatic SCI

at cervical or thoracic levels (C4-T12) resulting in upper motor neuron lesions in the lower

extremity, 2) history of SCI as defined by the American Spinal Injury Association (ASIA)









Impairment Scale categories C or D, and 3) a medically stable condition at the time of testing 4)

persons who were MR compatible. Participants had varied ambulatory capabilities and

accordingly, used a wheelchair, cane, crutches and/or ankle foot orthosis for assistance in

mobility.

5.1.2 Clinical Assessments

American Spinal cord Injury Association (ASIA) scores for neurological classification

of spinal cord injury: The ASIA impairment scale for neurological classification of spinal cord

injury is a nominal measure for measuring completeness of injury (ASIA 2001). Injury to the

spinal cord is considered complete if there is "absence of sensory and motor functions in the

lowest sacral segments" and incomplete if there is "preservation of sensory or motor function

below the level of injury, including the lowest sacral segments". Impairments in muscle strength

and sensory function are graded by the ASIA impairment scale (ASIA 2001) as follows:

ASIA A Complete: No sensory or motor function is preserved in sacral segments S4-S5.

ASIA B Incomplete: Sensory, but not motor, function is preserved below the neurological

level and extends through sacral segments S4-S5.

ASIA C Incomplete: Motor function is preserved below the neurological level, and most

key muscles below the neurological level have muscle grade less than 3.

ASIA D Incomplete: Motor function is preserved below the neurological level, and most

key muscles below the neurological level have muscle grade greater than or equal to 3.

ASIA E Normal: Sensory and motor functions are normal.

The sacral fibers are located more at the periphery of the cord making them least

susceptible to injury after a spinal cord traumatic event. Accordingly, after a SCI, sacral-sparing

is evidence of the physiologic continuity of spinal cord long tract fibers. "Indication of the

presence of sacral fibers is of significance in defining the completeness of the injury and the









potential for some motor recovery. This finding tends to be repeated and better defined after the

period of spinal shock".

Lower Extremities Motor Score (LEMS): This is a commonly used measure to define

the motor capabilities after SCI. This score uses the manual muscle testing scores of the ASIA

key muscles in both lower extremities with a total possible score of 50 (i.e., maximum score of 5

for each key muscle per extremity). Accordingly, a LEMS score of 20 or less indicates that

individuals with incomplete SCI are less likely to be community ambulators. In contrast, a

LEMS of 30 or more suggests a better likelihood for community ambulation (ASIA 2001).

Walking Index for Spinal Cord Injury (WISCI): Originally developed by the

international study group, the Walking Index for Spinal Cord Injury (WISCI) score is a scale that

indicates the ability to walk after SCI. It is a reliable and valid tool that has been found to

correlate well with ASIA scores (Burns, Golding et al.). Researchers and clinicians have widely

used the WISCI score to assess the walking ability after SCI. The testing incorporates assessing

the ability to walk for 10meters with the help of assistive aids, crutch and physical assistance of

one or two persons. Accordingly, the severity of walking impairment is graded from most to

least severe on a scale of 0 to 20. A score of zero implies inability to stand and/or walk at all with

assistance and a score of 20 implies the ability to ambulate independently for 10m (Ditunno,

Ditunno et al. 2000). Note worthily, the scale is not reflective of the functional ambulatory status

of the individual in the community.

5.1.3 Locomotor Training

Locomotor training (LT) consisted of 9 weeks of step training (30 minutes, 5days/week)

on the treadmill with body weight support and manual assistance followed by over ground

training (20 minutes). Bodyweight support, initially set to 40% of the subjects' body weight was

adjusted to maintain proper limb kinetics while also maximizing bilateral limb loading. If









necessary, manual assistance was provided by trainers to assist in correct stepping. In addition,

assistance was provided in maintaining an upright trunk by stabilizing the pelvis. Subjects were

encouraged to swing their arms voluntarily or with the aid of poles. Verbal encouragement by

trainers along with visual cues provided by a mirror placed in front of them served as additional

sensory cues to facilitate a near-normal walking pattern. Speed of treadmill stepping was kept in

a range consistent with normal walking, optimized for each subject (2.0-2.8 miles/hr). Training

sessions were interspersed with adequate rest periods. During each rest break, the participant

stood with minimal BWS required to maintain balance with minimal assistance from the trainers.

Initially, treadmill sessions required up to 60 minutes to achieve 30 minutes of stepping, but the

amount decreased with improved walking ability and endurance such that stepping could be

completed in 45 minutes or less. Progression of training was achieved by decreasing BWS,

altering speed, increasing trunk control, decreasing manual assistance for limb control and

increasing the stepping time on the treadmill per bout. All participants received the same number

of sessions and spend approximately the same amount of time involved in training, although

progression of training parameters was individualized. Immediately following step training on

the treadmill, each participant engaged in 20 minutes of over ground training. Over ground

training incorporated the use of assistive devices, but participants were otherwise bearing full

weight on their lower extremities. A more detailed description of the training principles and

parameters has been provided (Behrman and Harkema 2000; Behrman, Lawless-Dixon et al.

2005).

5.1.4 Proton Magnetic Resonance Imaging (1H-MRI)

Proton magnetic resonance imaging (1H-MRI) was used to determine a) maximum muscle

CSA and b) T2 relaxation properties of the lower extremity muscles.









A 1.5 Tesla super conducting magnet scanner (Signa; General Electric)2 was used to

collect trans-axial images of the leg and thigh. The self-reported more involved leg of the SCI

group and the right leg of control subjects was scanned. A standard (20cm long) lower extremity

quadrature coil or body coil was employed for imaging. The extremity coil covered the length of

the leg starting from above the lateral malleolus and extending to -3 cm centimeters proximal to

the superior patella. TI weighted image of the leg with a pulse sequence of TR=300ms, TE=

minimum, matrix = 128*128 and FOV = 16-30cm served as a coronal localizer for axial imaging

(Figure 5-1). Trans-axial images for measurements of CSA and T2 relaxation properties were

obtained using distinct NMR sequences.

5.1.4.1 Muscle cross sectional area: data collection and analysis

Spin echo or fat suppressed 3D SPGR imaging sequence was utilized with the following

imaging parameters: acquisition matrix size, 256x256 to 256x192; field of view of 16cm to 32cm

for the leg and 22cm to 40cm for the thigh; pulse repetition time, 51ms-300 ms; echo time,

10ms-27ms; slice thickness of 5-7mm; slice gap, 0-5mm.

The images were transferred to a silicon graphics UNIX workstation and fat-free maximal

muscle CSA of lower extremity muscles was determined using a custom-designed interactive

computer program as previously described (Elliott, Walter et al. 1997). Briefly, multiple slices

(8-20) of each muscle were outlined taking care to avoid fascia and blood vessels, manually

thresholded to include pixels of similar signal intensity that represent muscle tissue and then

segmented to determine the slice with maximum CSA. A unique feature of this software was its

calculations for partial volume effects thereby yielding an accurate measure of muscle CSA.

Maximum CSA of the quadriceps femoris (QF) and hamstring (HAMS) muscle groups in the


2 GE Medical Systems global headquarters: Waukesha, Wisconsin









thigh, the soleus (SOL), medial gastrocnemius, lateral gastrocnemius (LG), tibialis anterior (TA)

muscles in the lower leg and maximum CSA of the entire posterior compartment (PC) of the

lower leg (that includes the tibialis posterior muscle) were calculated (Figure 5-2). In addition,

maximum CSA of the ankle anti-gravity muscles [plantar flexors (PF)] was considered at the

level of the lower leg where the SOL, MG and LG taken together resulted in the largest CSA.

5.1.4.2 T2 relaxation times: data collection and analysis

T2 weighted imaging using multiple slice spin echo sequence was performed with

following imaging parameters TR = 2000ms; TE = 26, 52, 78 and 108ms; FOV = 16cm, slice

thickness = 7mm, matrix of 256x128.

The T2 weighted images were transferred to a UNIX workstation and characteristic T2

relaxation time of the muscle and bone marrow were calculated using a custom designed

software assuming a singe exponential decay with respect to the four imaging echo times. The

T2 relaxation times were represented on resultant images called T2 maps (Figure 5-3). Specific

regions of interests (ROI) that avoid visible blood vessels in the SOL, MG, LG and TA muscles

were identified in 8-10 T2 map slices and the average T2 of each muscle was subsequently

calculated. T2 times of bone marrow were measured as internal references to assess

reproducibility of T2 values.

5.1.5 Proton Magnetic Resonance Spectroscopy ('H-MRS)

Localized unsuppressed spectra were acquired from the soleus muscle to measure a)

muscle lipid content b) T2 relaxation characteristics of muscle independent of fat.

For precise localization of volume of interest, a voxel (35mm thickness) was prescribed

over the localized trans-axial image of the soleus muscle avoiding visible blood vessels and

muscle fascia (Figure 5-4). In order to ascertain voxel position over the muscle without signal

contamination from non-selected surrounding tissue, a phantom experiment was performed to









obtain an image of the prescribed voxel. Figure 5-5 shows a raw image of the voxel acquired at

the 1.5T from a CuSO4 phantom (TR =2000ms, TE = 18, FOV =16cm). Note that the boundaries

of the voxel are sharp and its dimensions the same as the prescribed voxel dimensions. An

unsuppressed water stimulation acquisition mode spectroscopic sequence STEAM (Bruhn,

Frahm et al. 1991) sequence was used with four different echo times (13ms, 30ms, 60ms and

120ms), a TR of 6000ms, 4 scans, 512 points and 2500 Hz spectral width. In addition, a 32scan

average was performed at the 120ms TE to estimate total lipid content and individual lipid

components; intramyocellular (IMCL) and extramyocellular lipids (EMCL).

5.1.5.1 Muscle lipid: data analysis

A zero order phase correction was performed on the 32 scan raw spectrum and water and

lipid spectral peaks were quantified using an Advanced Magnetic Resonance (AMARES) time-

domain-fitting algorithm using jMRUI (Naressi, Couturier et al. 2001). Prior knowledge values

were constructed from healthy controls for measuring water and lipid amplitudes. Whole lipid

peak and water peak were identified at 1.5ppm and 4.7ppm (Figure 5-4). Once amplitudes from

the water and whole lipid peak were calculated, the water peak was manually suppressed during

data analysis. Lipid resonance peak was subsequently deconvoluted to estimate IMCL and

EMCL at approximately 1.25ppm and 1.4ppm using the AMARES method (Figure 5-6).

Thereafter, IMCL and EMCL lipid amplitudes were calculated from their spectral peaks and

corrected for T2 relaxation effects using T2 relaxation times of 85ms for IMCL and 75ms for

EMCL. T2 relaxation times of IMCL and EMCL in the soleus muscle used in this study match

with lipid T2 relaxation values reported in literature (Bruhn, Frahm et al. 1991; Szczepaniak,

Babcock et al. 1999). In concurrence with other studies, the overall lipid, IMCL and EMCL

content in our study were expressed as a ratio using the spectral water peak as an internal

reference (Sinha, Dufour et al. 2002; White, Ferguson et al. 2006).









Uniqueness of this method to analyze fat was that it exclusively represents intramuscular

fat. Studies in the past have quantified fat within muscle using imaging techniques. However,

this method is plagued by the inclusion of inter-muscular fat (Elder, Apple et al. 2004) and

requires a good amount of fat to adequately quantify it (Huang, Majumdar et al. 1994). Proton

spectroscopy on the other hand, can yield a good lipid peak along with fat components from

within the same muscles.

5.1.5.2 Muscle T2 relaxation times: data analysis

The T2 relaxation time of soleus muscle independent of fat was calculated assuming a

single exponential decay with respect to the four spectral echo times.

Mono-exponential decay versus multi-exponential decay curves: In this study, only

four echo times were used to acquire both imaging and spectroscopy data, which consequently

required assumption of a single exponential decay to calculate T2 relaxation times from each

muscle. This might pose a potential limitation to data collection in our present study. Chances

prevail that the single exponential decay curves might in fact be multi-exponential, with

contributions from multiple T2 components. Various algorithms including the non-negative least

squares (NNLS) are commonly used to decompose multiple component curves into its distinct

components. However, for decomposition of the signal into its components using NNLS, several

TEs are necessitated during data collection.

5.2 Animal Studies

5.2.1 Animals

Sixteen adult Sprague Dawley female rats (16 weeks of age, 228-260g; Charles River, NJ)

either underwent a spinal cord contusion (n=8) or served as controls for wet lab procedures

relevant to the study. Briefly, experimental rats were tested for outcome measures before injury

and were followed up longitudinally for three weeks after injury. Animals were housed in a









temperature controlled room at 21 oC with a 12:12 hour light: dark cycle and were provided

rodent chow and water ad libitum. All experimental procedures were performed in accordance

with the U.S. Government Principle for the Utilization and Care of Vertebrate Animals by

approval of the Institutional Animal Care & Use Committee at the University of Florida. At the

end of the study, all animals were euthanized and hind limb muscles were extracted and

processed for biochemical assays.

5.2.2 Gender Differences in Animal Models of SCI

Most studies pertaining to spinal cord injury research have used the female rat model of

spinal injury. While studying both male and female models for muscle adaptations will assist in

teasing out any gender differences in muscle adaptations, we have chosen to utilize female rats in

the present work. One of the main reasons for our choice vests in remaining consistent with

reports in literature and taking advantage of valid comparisons with published work by other

investigators.

5.2.3 Spinal Cord Contusion Injury

Spinal cord contusion injury was produced using a NYU (New York University) impactor

device. A 10g weight was dropped from a 2.5-cm height onto the T8 segment of the spinal cord

exposed by laminectomy under sterile conditions. Animals received two doses of Ampicillin per

day for 5 days, starting at the day of surgery. Procedures were performed under ketamine

(100mg/kg)-xylazine (6.7mg/kg) anesthesia (Reier, Anderson et al. 1992; Thompson, Reier et al.

1992). Subcutaneous lactated Ringer's solution (5 ml) and antibiotic spray were administered

after completion of the surgery. The animals were kept under vigilant postoperative care,

including daily examination for signs of distress, weight loss, dehydration, and bladder

dysfunction. Manual expression of bladders was performed 2-3 times daily, as required, and

animals monitored for the possibility of urinary tract infection. Animals were housed









individually following surgery. At post-operative day 7, open field locomotion was assessed

using the 21 Basso-Beattie-Bresnahan (BBB) locomotor scale (Basso, Beattie et al. 1995) and

animals that did not fall within a preset range (0-7) were excluded from the study. After baseline

measurements before injury, rats underwent once weekly MRS measurements for three weeks

starting at week one after injury.

5.2.4 Experimental Electrical Stimulation Protocol

An electrical muscle stimulation protocol was adopted to determine the mitochondrial

oxidative capacity of the rat hind limb muscle in-vivo. Animals were anesthesized using gaseous

isoflurane in oxygen (3% box induction), and maintained at 0.5%-2.5% during the MR

procedures. After shaving the limb and cleaning it with alcohol, an oval surface coil tuned to 31P

(190.5 MHz) was placed over the belly of the gastrocnemius muscles. A 1H surface coil was

placed underneath the hind limb to perform swimming. Two needle electrodes were placed

subcutaneously one over the region of the third lumbar vertebrae and the other land marked

over the greater trochanter to stimulate the hind limb plantarflexor muscles via stimulation of

the sciatic nerve (Figure 5-9). Electrical stimulation was carried out for four to six minutes to

deplete PCr by 30 to 40%. A Grass Stimulator (Quincy MA) with a Grass Model SIU8T

stimulation isolation unit (Grass Instruments, West Warwick, RI) was used to deliver a

monophasic, rectangular pulse with a Ims pulse duration, 1Hz frequency and 10V. Following the

electrical stimulation, the muscle was allowed to recover for twenty minutes. Conceptually,

electrical stimulation depletes phosphocreatine (PCr) from the contracting muscle and the rate of

PCr recovery is measured to determine skeletal muscle oxidative capacity. During the entire

duration of stimulation and recovery, spectra were collected. No attempts were made to

synchronize the radiofrequency pulse with the muscle stimulation. The FIDs were multiplied by









an exponential corresponding to a 25Hz line broadening. Vital signs of the animal were

monitored throughout the experimental procedure.

5.2.5 Phosphorus Magnetic Resonance Spectroscopy (31P-MRS): Data Collection

All data was collected using a high magnetic field strength Bruker 11Tesla/470 MHz

spectrometer. A 1.5 x 1.7 cm oval surface coil tuned to 31P (190.5 MHz) was placed over the

belly of the gastrocnemius muscle. A 3-cm standard 1H surface coil was placed underneath the

hind limb to perform swimming and the animal's hind limb was extended such that the calf

muscles were centered over the surface coil. For the baseline 31P MRS spectra data, spectra were

acquired with a 50 |.s square pulse, a TR of 2s, spectral width of 10,000 Hz, 150 averages and

8000 complex data points. For the 31P kinetic data, spectra were averaged into 20s bins and

acquired at rest (5 min), electrical stimulation (4-6 min), and recovery (20 min); thus amounting

to a total of 93 fids. The partially relaxed spectra were then calibrated by comparison with fully

relaxed spectra (acquired at TR of 15seconds) to determine correction factors (CF).

5.2.5.1 31P-MRS spectral analysis at rest

Resting spectra yields measures of basal Pi and PCr concentration from the resting

gastrocnemius rat hind limb muscles. The spectra were manually phased, and the areas of the P

ATP, Pi, and PCr peaks determined by area integration. The enzymatically determined ATP

concentration in frozen muscle tissue was equated with the integral of the 3-ATP. Equating

tissue measurements of ATP under the 3-ATP peak is established as valid for skeletal muscle

(Hitchins, Cieslar et al. 2001). Pi and PCr concentrations were determined by using 3-ATP as an

internal standard and after accounting for correction factors (CF) as follows:









[Metabolite] (tlmol/g wet wt) = CFMetabolite x IntegralMetabolite x [ATP] ([tmol/g wet wt)

Integral P-ATP

(5-1)

ATP concentration in gastrocnemius muscle obtained by ATP assay was 4.6 0.3 tlmol/g

wet wt and is similar to ATP concentrations in rat gastrocnemius muscle reported in literature

(Authier, Albrand et al. 1987; Hitchins, Cieslar et al. 2001; Gigli and Bussmann 2002).

Similarly, our correction factors for Pi and PCr were 1.69 and 1.40 respectively and match well

with published values from rat gastrocnemius muscle (Mizobata, Prechek et al. 1995).

Intracellular pH were calculated from the chemical shift of the Pi peak relative to PCr using the

equation, pH = 6.75 + log [(6 3.27) / (5.69 6)], where 6 is the chemical shift of the Pi peak in

ppm. The cystolic phosphorylation potential was calculated in reciprocal form as

[Pi][ADP]/[ATP] since the phosphorylation potential itself is not normally distributed. Free

cystolic ADP was calculated from the creatine kinase equilibrium reaction as previously

described (Mizobata, Prechek et al. 1995; Thompson, Kemp et al. 1995; Pathare, Vandenborne et

al. 2007):

[ADP] = {[free creatine] [ATP] }/[PCr] [H+] [Keq] (5-2)

where the free creatine was quantified by subtracting the PCr content obtained by 31P-MRS

from the total creatine content (42.2mM) determined biochemically. Intracellular Mg

concentration and equilibrium constant (Keq) of the creatine kinase reaction were assumed as

ImM and 1.66 x 109 respectively for mammalian skeletal muscle (Veech, Lawson et al. 1979).

5.2.5.2 31P-MRS spectral analysis of electrical stimulation protocol

The electrical stimulation protocol data yields kinetic measurements of PCr. Dynamic

changes in PCr levels were measured using complex principal component analysis (Elliott,









Walter et al. 1999). Recovery data were fitted to a single exponential curve, and the pseudo first-

order rate constant for PCr recovery (kPCr) was determined (Meyer 1988). The maximal rate of

PCr resynthesis, a measure of mitochondrial oxidative capacity (Vmax-lin) was calculated based on

kPCr and PCr rest (Vmax-lin = kPCr. [PCr] rest) (Walter, Vandenborne et al. 1997). Initial rates of PCr

recovery (Vmeas mM/min, a direct measure of mitochondrial ATP synthesis) was determined from

the first three to four data points in recovery; depending upon the best linear curve fit. The initial

rate of PCr resynthesis was also extrapolated from the product of kPCr and amount of PCr

depletion (APCr). Thus, Vex (mM/min) = kPCr. APCr (Walter, Vandenborne et al. 1997).

Rates of PCr depletion at onset of stimulation (Vdep mM/min, a measure of ATP demand)

were determined from the first three to nine data points (first through three minutes) of PCr

declines during stimulation.

The maximum oxidative ATP synthesis rate (Qmax), which is a function of intrinsic

mitochondrial content and enzyme activity, oxygen and substrate supply to the mitochondrion,

and cytosolic redox state (Kemp, Sanderson et al. 1996), was calculated from the known

hyperbolic relationship between PCr resynthesis and cytosolic free [ADP] and from PCr

resynthesis and [ADP][Pi]/[ATP].

Qmax-ADP = Vmeas (1 + Km/[ADP]) mM/min, where Km is the Michaelis constant and is

assumed as 50 [tM for rat leg muscle (Thompson, Kemp et al. 1995). (5-3)

Qmax-[ADP][Pi]/[ATP] = Vmeas (1 + Km/[ADP][Pi]/[ATP]) mM/min, where Km is assumed as

0.11 mM. (5-4)

5.2.6 Biochemical Assays

Animals were sacrificed at the end of the experiments and gastrocnemius muscle excised

and snap frozen at -800C for subsequent biochemical quantification.









5.2.6.1 ATP measurements

ATP was measured as previously described (Hitchins, Cieslar et al. 2001; Gigli and

Bussmann 2002; Pathare, Vandenbome et al. 2007). Frozen gastrocnemius muscle was ground to

fine powder using a mortar and pestle under dry-ice. 100mg of the tissue was homogenized for

30seconds with a Mini-bead-beater in a plastic Eppendorf tube containing beads and ice-cold

0.9% perchloric acid (5v/w). The sample was then centrifuged at 9000 g for 15 minutes at 40C

(-4000rpm). The supernatant was extracted and added to 4M KOH (1.125v/w) and centrifuged

again for 5 minutes at 40C and 9000 g. The supernatant was frozen at -800C and processed for

ATP measurements with an ATP assay kit (Sigma) using a luminometer (Biotek Instruments,

CA).

5.2.6.2 Total creatine measurements

Total muscle creatine content was determined using the diacetyl/a-napthtol assay ((De

Saedeleer and Marechal 1984; Vandenberghe, Gillis et al. 1996; Tarnopolsky and Parise 1999).

Approximately 10-15 mg (average 11.55 2.25 mg) of muscle tissue was cut and placed in a

microfuge tube, and then placed in a vacuum centrifuge (Savant ISS110 SpeedVacTM

Concentrator, Thermo Scientific, Milford, MA) to be spun for 18-24 hours. After sufficient

muscle drying, the samples were then placed in an ultra-low freezer at -800C. Dried muscle was

powdered by grinding on a porcelain plate with a pestle. Connective tissue was removed and

discarded, whereas powdered muscle was placed into pre-weighed microfuge tubes and dry

weight determined. Powdered muscle (average 2.27 0.44 mg) was extracted in a 0.5 M

perchloric acid/i mM EDTA solution at a relative ratio of 800 [l per 10 mg powdered muscle on

ice for 15-minutes, while periodically vortexing. Samples were then spun at 15,000 rpm at 4C

for 5-minutes. The supernatant was transferred into a microfuge tube and neutralized with 2.1 M

KHCO3 / 0.3 M MOPS solution at a ratio of 1:5 and then centrifuged again at 15,000 rpm for 5-









minutes. The resulting supernatant was then stored at -80C for future use. In order to determine

muscle total creatine concentration, 40 ul of the supernatant from the above reaction was

combined with 140 ul ddH20 and 20 ul 0.4 N HC1 and heated at 65C for 10-minutes to

hydrolyze phosphate groups. The solution was then neutralized with 40 ul of 2.0 N NaOH and

analyzed as described above.

TCr and ATP levels were determined in [tmol (g wet weight)- and mmol (1 tissue)-',

assuming a muscle density of 1.06 gml- These were converted to mmol 1-1 intracellular water

assuming a cellular water fraction of 0.83 in the rat gastrocnemius muscle (Veech, Lawson et al.

1979; Cieslar, Huang et al. 1998).


































Figure 5-1. Representative 1H-MRI coronal image of the calf muscles from a healthy control at
1.5T. Trans-axial images were obtained along the length of the coronal image
(represented by horizontal green lines)


Figure 5-2. Representative 'H-MRI trans-axial image of the calf muscles from a healthy control
at 1.5T. A 3D fast gradient echo imaging sequence was used in a 1.5Tesla magnet.
(A= anterior; P=posterior; M= medial; L=lateral)




























e"p OEMT2)


14 TL(


TZMap


Figure 5-3. A) Representative 1H-MRI T2 weighed images of the calf muscles obtained at four
different echo times. B) Individual pixel signal intensities were fitted to an
exponentially decaying curve along four different echo times (TE) C) Representative
T2 map of the T2 weighted images acquired in A.


Water peak









Muscle mrtabolites

Fat peak

4.7 1.2 (ppm)

Figure 5-4. Representative trans-axial image presents a voxel prescribed over the soleus muscle
and a resultant 1H-MR spectrum with water and fat peaks.











EMCL
'7H, )n


DICL
(CH-)n


ChotweCe CH



CT f


S3.7 Z. 12i5 0 (pPLm)

Figure 5-5. Decomposition of a lipid peak obtained from a healthy soleus muscle into its
intramyocellular and extramyocellular components using jMRUI.


Figure 5-9. Experimental-setup for electrical stimulation protocol during 31P-MRS data
acquisition.


i/j
21


r
rl









CHAPTER 6
EXPERIMENT ONE LOWER EXTREMITY MUSCLE CROSS-SECTIONAL AREA
AFTER INCOMPLETE SPINAL CORD INJURY

6.1 Summary

The purpose of this study was to: a) To quantify skeletal muscle size in lower extremity

muscles in persons after incomplete-SCI, b) to assess differences in muscle size between

involved lower limbs, c) to determine the impact of ambulatory status (using wheelchair for

community mobility versus not using a wheelchair for community mobility) on muscle size after

incomplete-SCI, d) to determine if differential atrophy occurs among individual muscles after

incomplete-SCI. Seventeen persons with incomplete-SCI and 17 age, gender, weight and height

matched non-injured controls participated from the university research setting. Maximum cross

sectional area (CSA) of individual lower extremity muscles soleuss, medial gastrocnemius,

lateral gastrocnemius, tibialis anterior, quadriceps femoris and hamstrings] was assessed by

Magnetic Resonance Imaging. Overall, subjects with incomplete-SCI demonstrated significantly

smaller (24% 31%) average muscle CSA in affected lower extremity muscles as compared to

control subjects (P<0.05). Mean differences were highest in the thigh muscles (-31%) compared

to the lower leg muscles (-25%). No differences were noted between the self-reported more- and

less-involved limbs within the incomplete-SCI group. Dichotomizing the incomplete-SCI group

revealed significantly lower muscle CSA values in both the wheelchair [range = 21% 39%] and

non-wheelchair groups [range = 24% 38%]. In addition, the wheelchair group exhibited

significantly greater plantar flexor muscle atrophy compared to the dorsi-flexors, with maximum

atrophy in the medial gastrocnemius muscle (39%). Our results suggest marked and differential

atrophic response of the affected lower extremity muscles that is seemingly impacted by

ambulatory status in persons with incomplete-SCI.









6.2 Introduction

An emerging trend in the care and treatment of persons after spinal cord injury (SCI) is the

increased proportion of persons diagnosed with incomplete injuries. Persons with incomplete-

SCI exhibit variable paralysis and paresis of affected muscles, typically resulting in impaired

motor performance and varying degrees of functional limitations (Subbarao 1991; Tang, Tuel et

al. 1994; Bums, Golding et al. 1997). Interestingly, although incomplete-SCI constitutes -51%

of all new spinal injuries, the majority of human and animal research related to physiological and

morphological adaptations following SCI has focused on subjects with complete injuries. As

such, a large body of literature exists that describes skeletal muscle adaptations after this type of

injury (Baldi, Jackson et al. 1998; Hopman, Dueck et al. 1998) with few data describing

adaptations within affected skeletal muscle after incomplete injuries. Similar to persons with

complete-SCI, persons with incomplete-SCI exhibit a variety of clinically relevant motor and

functional deficits, including local muscle fatigue, weakness of affected muscles (Sloan,

Bremner et al. 1994; Johnston, Finson et al. 2003) and diminished capacity to ambulate(Waters,

Adkins et al. 1994; Ulkar, Yavuzer et al. 2003). We recently demonstrated that after chronic

upper motor lesions and incomplete-SCI, both knee extensor and plantar flexor skeletal muscles

generate -70% less peak torque (Jayaraman, Gregory et al. 2006). Other studies have shown a

significant reduction in ambulatory capacity, with a reduced gait speed, step frequency and stride

length. Despite such obvious motor dysfunction, no studies have documented the extent of

muscle atrophy in paralyzed skeletal muscle following incomplete-SCI in humans. Given that

muscle atrophy relates strongly to compromised muscle strength (Berg, Dudley et al. 1991;

Ploutz-Snyder, Tesch et al. 1995; Vandenborne, Elliott et al. 1998; Stevens, Walter et al. 2004)

as well as locomotor ability,(Visser, Kritchevsky et al. 2002; Visser, Goodpaster et al. 2005) an









in-depth understanding of the extent of impairment in this population would be valuable to the

field of rehabilitation research.

Persons with incomplete-SCI constitute an extremely heterogeneous group. For example,

people after this type of injury exhibit a continuum of ambulatory abilities ranging from being

completely wheelchair dependent to nearly normal walking without the use of assistive devices.

Consequently, the mechanical loading and activation of the affected lower extremity muscles is

extremely variable (Melis, Torres-Moreno et al. 1999). Given that forced inactivity of lower

extremity muscles (i.e. immobilization, limb suspension) results in differential skeletal muscle

atrophy (Adams, Hather et al. 1994; Adams 2002; Alkner and Tesch 2004) one might expect

variable patterns of muscle adaptations in persons after incomplete-SCI. Accordingly, we sought

to examine the morphological characteristics of lower extremity skeletal muscles in persons with

incomplete-SCI. Specifically, the purpose of our study was four-fold: 1) to compare lower

extremity muscle maximum cross sectional area (CSA) in persons with incomplete-SCI to a

group of age, gender, height and weight matched controls 2) to make comparisons of maximum

muscle CSA between the self-reported more and less involved limbs within a group of persons

with incomplete-SCI 3) to evaluate whether ambulatory status (i.e. using wheelchair for

community mobility versus not using a wheelchair for community mobility) influences lower

extremity skeletal muscle CSA after incomplete SCI 4) to compare the magnitude of atrophic

response a) between the flexor and extensor muscles about the knee and ankle, b) between

proximal and distal anti-gravity extensor muscles, and c) among individual ankle plantar flexor

muscles.









6.3 Methods

We performed a case-control study in which the lower extremity maximum muscle CSA of

persons with incomplete-SCI was compared with maximum muscle CSA of age, gender, weight

and height matched non-injured controls. To address the impact of ambulatory status on skeletal

muscle size, we dichotomized our incomplete-SCI subjects into those who did not have upright

mobility in the community, but demonstrated ambulatory ability through use of a wheelchair

(W/C group) and those who did not use a wheelchair for community mobility (non-W/C group).

6.3.1 Subjects

Persons with incomplete-SCI: A convenient sample of seventeen persons (2 women, 15

men; 139 months post-injury) with incomplete-SCI (3915 yr, 7613 kg, 17810 cm)

volunteered to participate in the study. Of these, 10 subjects were studied at the University of

Florida, Gainesville, Florida; and 7 subjects were studied at the University of Georgia, Athens,

Georgia. All participants 1) had a diagnosis of traumatic SCI at cervical or thoracic levels (C4-

T12) resulting in upper motor neuron lesions in the lower extremity, 2) had a history of SCI as

defined by the American Spinal Injury Association (ASIA) Impairment Scale categories C or D,

and 3) had a medically stable condition at the time of testing. Seven of the persons with

incomplete-SCI used a wheelchair, two subjects used forearm crutches, and six used a single-

point cane for community ambulation (Table 6-1).

Controls: Seventeen persons (2 women, 15 men; 39 12 yr, 78 12 kg, 1788 cm)

volunteered to serve as control subjects. These subjects were matched to incomplete-SCI

subjects on the basis of age, gender, and height and body mass. Though large demographic

variability existed among subjects in both the incomplete-SCI and control groups, each control

person was closely matched (age 7 yr, height 10 cm, and body mass 8 kg) to the

corresponding person with incomplete-SCI. Control subjects were recreationally active, but not









engaged in any rigorous exercise program, and were recruited from the Gainesville, FL

community.

6.3.2 Maximum Muscle Cross-sectional Area

Proton magnetic resonance imaging (MRI) was used to determine maximum muscle CSA

of the lower extremity. MRI for all subjects was performed specifically for the study. Details of

the MRI procedures are described in Chapter 5 (section 5.1.4.1). Figure 6-1 illustrates

representative trans-axial proton magnetic resonance images of patient and control subjects

obtained at 1.5Tesla magnetic field.

6.3.3 Data Analysis

Independent samples t-tests were employed to determine if differences existed in the

demographic characteristics (age, height and body weight) and to compare the maximum muscle

CSA of the muscles of interest in the pooled incomplete-SCI (n=17) and control groups (n=17).

For skewed data, distribution-free Mann Whitney tests were used to compare muscle CSA

between controls and persons with incomplete-SCI. The self reported more- and less-involved

limbs of persons with incomplete-SCI (n=7) were compared using a two-related sample

Wilcoxon test. To determine the impact of ambulatory status on maximum muscle CSA we

compared the mean CSA of the above-mentioned muscles in the W/C (n=7) and non-W/C groups

(n=10) with their corresponding matched controls using Mann Whitney test. In all analyses

involving comparisons for incomplete-SCI with controls, a unidirectional hypothesis

(incomplete-SCI CSA < control CSA) was tested. Further, relative differences between the

extensor and flexor muscles about the knee and ankle were determined by intra-compartment

(PF:TA, QF:HAMS) ratios. In addition, relative differences between proximal and distal anti-

gravity muscles were compared using inter-compartment (QF:PF) ratios. Lastly, differential

atrophy among the specific plantar flexor muscles was determined by normalizing each









individual plantar flexor muscle to the maximum total posterior compartment (PC) muscle CSA

(SOL:PC, MG:PC, LG:PC). The analyses for relative atrophy between muscles were done both

on the pooled incomplete-SCI groups versus controls and the wheelchair versus non-wheelchair

groups. After obtaining the proportions, a proportionality test was run (Ho: HnI=2) (Alan Agresti

1997) to determine if statistical differences existed between the groups. SPSS for Windows

(Version 11.0.1)3 was utilized for all statistical analyses. Alpha level was set at 0.05 and Dunn-

Bonferroni corrections for multiple comparisons were made where appropriate). Percent

differences in maximum muscle CSAs between groups were calculated by taking the average of

the individual percent differences between persons with incomplete-SCI and their corresponding

control subjects.

6.4 Results

Demographic data: No differences existed between the incomplete-SCI and control group

with respect to age, height or weight (p>0.562).

Maximum muscle CSA after incomplete-SCI: Lower extremity muscle size in the

pooled incomplete-SCI subjects was significantly smaller than the control group for all of the

tested muscles p<0.004). Mean differences in muscle CSA ranged from 24% (TA) to 31% (QF)

in the incomplete-SCI group relative to controls. Muscle specific CSA data are presented in

Figures 6-2A and 6-2B.

Bilateral differences in maximum muscle CSA after incomplete-SCI: No significant

differences in maximum muscle CSA were found between the self-reported more-involved

versus the less-involved limbs within the incomplete-SCI subjects for any of the muscles tested

(p>0.473).



3 SPSS Inc, 233 S. Wacker Drive, 11th floor, Chicago, Illinois 60606









Impact of ambulatory status on maximum muscle CSA after incomplete-SCI: Our

results showed that persons with incomplete-SCI in both the W/C and non-W/C group showed a

lower skeletal muscle size as compared to controls. As shown in Table 2, subjects in the W/C

group had significantly smaller muscle CSA values for all of the anti-gravity muscles (i.e. SOL,

MG, LG, QF) relative to their corresponding control group (p<0.048). Mean differences in lower

extremity muscle CSA in the W/C group ranged from 21% (QF, TA) to 39%. Interestingly,

neither TA (p=0.064) nor the HAMS (p=0.120) were significantly different in the W/C group

when compared to matched controls. The non-W/C i-SCI group also showed significant

differences in muscle CSA values relative to control subjects. With the exception of the MG, all

of their lower extremity muscle CSA values were significantly smaller than those measured in

the control group (p<0.021). As shown in Table 2, mean differences in muscle CSA in the non-

W/C group ranged between 24% (SOL) and 38% (QF) relative to the control group.

Differential atrophy after incomplete-SCI: Comparisons in the magnitude of atrophy

across affected lower extremity muscles revealed no differential atrophy between extensor and

flexor muscle groups about the ankle (PF:TA, p = 0.433) or knee (QF:HAMS, p = 0.769) in the

pooled incomplete-SCI group and control groups (Table 3). However, as shown in Figure 6-3,

similar comparisons suggest greater relative atrophy of the anti-gravity muscles in the leg in the

W/C group compared to the non-W/C group (PF: TA, p = 0.043). When examined separately,

the proportion of the overall plantar flexor CSA occupied by SOL or the LG was not different

between the pooled incomplete-SCI and control groups (Table 3). However, the proportion of

overall CSA occupied by the MG was significantly smaller in the W/C group relative to the non-

W/C group (MG:PC, p = 0.002) (Figure 6-4). Lastly, the relative ratios between proximal and

distal anti-gravity muscle CSA (QF:PF) were similar for all comparisons made (p>0.273).









6.5 Discussion

The findings of our study indicate marked atrophy of lower extremity muscles following

incomplete-SCI. Overall, subjects with incomplete-SCI demonstrate a 24% 31% smaller

average muscle CSA in affected lower extremity muscles as compared to control subjects. Mean

differences were highest in the thigh muscles (-31%) compared to the leg muscles (-25%), with

no differences noted between the self-reported more- and less-involved limbs within the

incomplete-SCI group. Dichotomizing the incomplete-SCI group into a W/C and non-W/C users

revealed that both the W/C and non-W/C groups had a significantly lower muscle CSA than their

respective control groups. In addition, the W/C group exhibited greater atrophy in their ankle

plantar flexor muscles compared to the dorsi-flexors, suggesting a preferential atrophic response

in the anti-gravity muscles.

The magnitude of atrophy in our subjects after incomplete-SCI is much less than that

reported in persons following complete-SCI. An overall 46% decline in average CSA of the

lower extremity muscles in persons after complete-SCI has been reported 24 weeks after injury;

with decreases in the SOL (68%), gastrocnemius (54%), TA (20%), QF (42%) and HAMS (44%)

muscles reported relative to controls (Castro, Apple et al. 1999). These values (except for the TA

muscle) are approximately twice that seen in the present study. One of the main reasons is likely

related to the partial sparing of voluntary motor control following motor incomplete-SCI.

Skeletal muscle atrophy following SCI is a result of primary injury to motor neurons in the spinal

cord and concurrent inactivation of affected skeletal muscle along with subsequent changes in

muscle length and mechanical loading conditions (Gordon and Mao 1994). Fractional presence

of neural inputs to the affected muscle allows for variable activation of lower extremity

musculature after incomplete-SCI. In fact, research subjects in our study were typically able to

load their lower extremities during transfers and/or ambulate with crutches or a cane.









An interesting finding in the present study was the significant difference in lower extremity

muscle CSA in both the W/C and the non-W/C incomplete-SCI groups. When compared to the

control group, the W/C group showed atrophy in all the anti-gravity muscles. The non-W/C

subjects demonstrated a similar pattern, with the exception that no differences were seen in the

MG. One likely explanation for the atrophy seen in both the incomplete-SCI groups is that

persons who use assistive aids (cane, crutches, etc) for ambulation transfer much of the weight

bearing demands of daily activities to their upper extremities (Lee and McMahon 2002; Mulroy,

Farrokhi et al. 2004). Consequently, weight bearing through the affected lower extremities is

reduced. This reduced use and activation of the affected lower extremity muscles triggers a cycle

of added muscle atrophy and further dependence on assistive devices. In fact, in a recent study,

Clark et al. demonstrated reductions in leg muscular activity after use of a crutch or walker for

ambulation in able-bodied subjects (Clark, Manini et al. 2004). The authors found that the knee

extensor (vastus lateralis) and ankle plantar flexor muscles soleuss) incurred maximum decreases

in muscle activity thereby suggesting a predisposition of the anti-gravity muscles to dysfunction

following unloading. This finding is consistent with both animal and human models of

unweighting (Thomason and Booth 1990; Adams, Caiozzo et al. 2003).

Unlike anti-gravity muscles, the proximal and distal flexors in the W/C and non-W/C

groups showed varied findings. The ankle dorsi-flexor muscles showed significant differences in

CSA in the non-W/C (-26%), but not in the W/C group (-21%). This finding seems counter

intuitive given the perceived differences in the pattern of muscle activation between W/C and

non-W/C users. However, it has been reported that anti-gravity extensor muscles contribute

much more than the flexors toward an efficient gait (Saunders JBD 1953; Sutherland, Cooper et

al. 1980; Basmajian JV 1988). Therefore, the discrepancy noted in the ankle and knee flexor









muscle response between the two groups is probably not related to activation patterns during

gait. Interestingly, unlike the W/C group (1/7), a greater proportion of the subjects in the non-

W/C group (5/10) were fitted with ankle-foot orthosis for daily use (Table 6-1). This

characteristic may serve to explain the greater magnitude of atrophic response in the non-W/C

group given the limited dorsi-flexion ROM allowed with the orthosis. However, the use of an

orthosis did not necessarily correspond with lower extremity motor scores (LEMS, Table 6-1) in

our incomplete-SCI groups. As such, we cannot fully explain the dorsi-flexor atrophic response

in the non-W/C group.

When the pooled subject data were examined, the proportion of individual plantar flexor

muscles relative to the compartments they occupy was consistent between the incomplete-SCI

and control groups. This finding suggests that all the muscles studied underwent similar relative

amounts of atrophy, independent of mobility status. However, when the W/C and non-W/C

groups were examined individually, the MG muscle relative to the posterior compartment in the

W/C group showed a differential atrophy compared to the other groups. Similarly, though no

differences existed between the PF to TA ratios in the pooled incomplete-SCI and control

groups, significantly lower PF to TA ratios are noted in the W/C group versus the non-W/C

group suggesting more overall atrophy of the plantar flexors in the W/C group (-32% versus

21% in the W/C versus non-W/C, respectively). The most intuitive explanation for this finding

would be the greater relative loading imposed on this muscle group during an upright (non-W/C)

versus a seated posture (W/C). Previous studies have documented that the ankle plantar flexors

are critical during locomotion and generate a majority of the propulsive forces necessary for

efficient walking (Sutherland, Cooper et al. 1980; Basmajian JV 1988). Since walking is

comparatively more compromised in persons who use a wheelchair versus a cane or crutch for









ambulation, mechanical loading via weight bearing on the paralyzed muscles is less. In addition

to differential loading, a prolonged flexed position of the knee during use of a wheelchair might

shorten the gastrocnemius muscle at the knee joint resulting in greater plantar flexor muscle

atrophy relative to the non-W/C group.

In contrast to differences in leg muscle atrophy, differences in the thigh muscle CSA in the

W/C and the non-W/C group were not significant. The QF to HAMS ratio was similar in both the

i-SCI groups, implying relatively similar atrophy of the thigh musculature independent of

ambulatory status. Lastly, the degree of atrophy between the anti-gravity proximal and distal

extensor muscles (QF:PF) was also similar. Collectively, our results suggest that although using

a cane or crutch for ambulation might attenuate the atrophic response of the ankle plantar flexor

muscles (primarily the MG), yet marked adaptations are seen throughout the entire lower

extremity after incomplete-SCI.

The atrophic response in persons after incomplete-SCI as described in this study most

likely has dramatic functional implications. Previous studies have shown that decreases in

muscle CSA are strongly related to impaired muscle strength (Ploutz-Snyder, Tesch et al. 1995;

Vandenborne, Elliott et al. 1998; Stevens, Walter et al. 2004). In addition, muscle strength after

incomplete-SCI plays an important role in functional walking performance (Kim, Eng et al.

2004). Also, gains in skeletal muscle size have been associated with improvements in motor

function. For example, in a study assessing motor and sensory recovery following incomplete-

SCI, hypertrophy of the partially innervated skeletal muscles was suggested as one of the factors

that could account for the motor recovery following rehabilitation (Waters, Adkins et al. 1994).

As such, if specific deficits associated with incomplete-SCI can be discerned, efficient strategies

can be designed for functional rehabilitation.









Lastly, there are some shortcomings in our study. Subjects with i-SCI were, on an average,

13 months post injury (range, 5-37 months). Motor recovery following ISCI is a continuous

process that reaches a plateau around one year post-injury, with a significantly slower rate of

recovery in the second half year interval following injury (Waters, Adkins et al. 1994). As a

result, subjects with i-SCI in this study were at slightly different stages of motor recovery and

not necessarily in a steady condition (Waters, Adkins et al. 1994). Furthermore, our subjects

underwent routine rehabilitation treatment prior to participation in the study. Therefore, it is

possible that the true values of actual atrophy might have been underestimated. In addition, we

cannot definitely confirm the subjects' pre-injury CSA values to be similar to the control group.

However, our control subjects were matched to gender, age height and weight and have

comparable CSA values to controls in other human studies (Castro, Apple et al. 1999). Despite

these limitations, this study provides unique findings regarding the impact of ambulatory status

on muscle CSA in individuals with incomplete-SCI.

In conclusion, this study demonstrates that incomplete-SCI is associated with significant

muscle atrophy in the affected lower extremity that is uniform between limbs and somewhat

influenced by mobility status. Interestingly, the majority of therapeutic approaches for improving

locomotor performance in subjects with incomplete-SCI are compensatory rather than

physiologically based. As such, persons after incomplete-SCI are often left with significant

motor deficits despite long-term therapeutic intervention. With the increasing prevalence of

persons living with incomplete-SCI, there is an urgent need to develop appropriate therapeutic

techniques with the goal of maximizing motor recovery thereby reducing disability. When

developing therapeutic interventions to enhance functional recovery after SCI, an understanding

of the underlying physiology of muscular responses that occur after this type of injury may









promote different intervention strategies that rely less heavily on compensatory rehabilitation.

This study will provide a foundation from which the relationship between muscle size and

function in this population can be further explored. Future research efforts can be directed

towards understanding relationships between physiological deficits in skeletal muscle following

SCI and parameters of functional ability like walking balance, speed and muscle strength.










Table 6-1. Characteristics of subjects after incomplete-SCI.
Months Level ASIA Mobility
after injury of injury classification status Orthosis LEMS
S1 5 C5 D W/C NA 35
S2 10 C5 D Forearm crutch AFO 40
S3 13 C5 D Single point cane AFO 43
S4 15 C6 C W/C NA 35
S5 16 C5 C W/C Bil-AFO 33
S6 20 T1 D Single point cane NA 44
S7 37 T1 D Bilateral forearm crutches AFO 35
S8 15 T1 D W/C NA 45
S9 11 C4 C W/C NA 17
S10 18 C6 D Single point cane NA NT
S11 7 C6 C W/C NA 26
S12 13 T4 C W/C NA 20
S13 7 C6 D Single point cane AFO 39
S14 7 C3 D Single point cane AFO 49
S15 7 T7 D Single point cane NA 43
S16 12 T12 D No assistive aid NA 45
S17 12 C5 D No assistive aid NA 49
Abbreviations: W/C: using wheelchair for community ambulation; AFO=ankle foot orthosis; Bil=bilateral; LEMS:
Lower extremity muscle score (normal is 50/50) as assessed by the American Spinal cord Injury Association (ASIA)
motor impairment scale (2001), NT = not tested, NA= Not applicable

Table 6-2. Percentage differences between the lower extremity maximum muscle CSA of
wheelchair (W/C) and non-wheelchair (non-W/C) groups relative to corresponding
controls. Data are expressed as differences in percentage means (% difference) +
standard error of percentage (s.e %).
W/C group (n=7) non-W/C group (n= 10)
Muscle Groups Individual muscles % difference s.e%
SOL 27.5+7.5* 23.88.7t
Plantar flexors MG 38.8+5.3* 13.7+11.8
LG 30.1+9.8* 24.710.3t
Dorsi flexors TA 20.7+6.1 26.1+8.3t
Knee extensors QF 21.4+3.7* (n=6) 37.59.3t (n=8)
Knee flexors HAMS 23.1+5.1 (n=6) 34.9+6.8t (n=8)
*Significant difference between the W/C and control groups (p<0.05). ? Significant difference between the non-
W/C and control groups. Abbreviations : SOL=soleus; MG=medial gastrocnemius; LG=lateral gastrocnemius;
TA=tibialis anterior; QF = quadriceps femoris; HAMS=hamstrings










Table 6-3. Relative proportions of muscles in the pooled i-SCI and control groups.
Description Proportions Incomplete-SCI Controls
Individual plantar flexors relative to posterior SOL:PC 0.47 0.47
compartment
MG:PC 0.27 0.27

LG:PC 0.16 0.17

PF:TA 5.08 5.53
Intra-compartment ratios:
QF:HAMS 1.94 1.99
Inter-compartment ratios [] QF:PF 1.74 1.70

SOL/PC = soleus/posterior compartment, MG/PC = medial gastrocnemius/posterior compartment, LG/PC = lateral
gastrocnemius/posterior compartment; Flexors and extensor ratios of leg and thigh: PF/TA= plantar flexor/Tibialis
Anterior, QF/HAMS = Quadriceps/Hamstrings; []Proximal-distal antigravity muscle ratios: QF/PF =
quadriceps/plantarflexor.









SRemememativeincoete SCIsujet


Figure 6-1. Representative trans-axial proton magnetic resonance images obtained at 1.5Tesla
magnetic field. Leg (I, II) and thigh (III, IV) in a subject with incomplete-SCI (right)
and corresponding age-matched control (left). [A= anterior; P = posterior; M=medial;
L=lateral; MG=medial gastrocnemius; LG= lateral gastrocnemius; SOL=soleus,
TP=tibialis posterior; TA=tibialis anterior, QF=quadriceps; HAMS=hamstrings].


keperemam8tive Cm-rol










25

is

1.1


80L


MG


30

20


*Inc1DmJ3W SCI
ContraIm


i-I--I


aircomn pt SCI
controls


HAMS


Figure 6-2. Muscle CSA in the pooled incomplete-SCI and control groups. A) leg muscles [SOL
soleuss), MG (medial gastrocnemius), LG (lateral gastrocnemius) and TA tibialiss
anterior)] and B) thigh muscles [QF (quadriceps) and HAMS (hamstrings)]. Values
are means + sem. denotes significant differences between incomplete SCI and
control subjects.













6


S.

?.4
2
0.
I3
2I-


5.50


PF:DF


*VWC group

non-WiC group


QF:HAMS


QF:PF


Figure 6-3. Proportion ratios of muscle groups within the leg and thigh. (PF/TA= plantar
flexors/tibialis Anterior; QF/HAMS = quadriceps femoris/Hamstrings) and proximal
and distal antigravity muscles (QF/PF = quadriceps femoris/plantar flexors) between
the W/C and non-W/C group. denotes significant differences between W/C and
non-W/C incomplete SCI subjects.


m WIC group
0.6 o non -WIC group


Ef,
S0.4
o
0 .3

I .2


S0.1

0.0


0,46


0.16


SOL-PC


MGAS:PC


LGAS:PC


Figure 6-4. Ratio of individual plantar flexor muscles to the max CSA of the posterior
compartment of the leg. (SOL/PC = soleus/posterior compartment, MG/PC = medial
gastrocnemius/posterior compartment, LG/PC = lateral gastrocnemius/posterior
compartment) in the W/C and the non-W/C group. denotes significant differences
between W/C and non-W/C incomplete SCI subjects.









CHAPTER 7
EXPERIMENT TWO NON-INVASIVE ASSESSMENT OF LOWER EXTREMITY
MUSCLE COMPOSITION AFTER INCOMPLETE SPINAL CORD INJURY

7.1 Summary

A sedentary lifestyle makes persons with SCI extremely vulnerable to cardiovascular

complications. Glucose intolerance and insulin resistance is purported as one of the major

metabolic risk factors in the development of cardiovascular complications after SCI. Several

studies have established a positive correlation between the intramyocellular lipid (IMCL)

concentration and insulin resistance in sedentary individuals and a variety of patient populations.

Persons with complete spinal cord injury (SCI) show elevated intramuscular lipid levels that are

well correlated with development of insulin resistance. The purpose of this study was to assess

lower extremity muscle composition of persons with incomplete SCI. Combinations of T2

weighted magnetic resonance imaging from the calf muscles and localized unsuppressed proton

spectroscopy from the soleus muscle were acquired in persons with chronic incomplete SCI and

age-matched controls. Results of the study show elevated T2 relaxation times of the soleus and

gastrocnemius muscle in persons with incomplete SCI. In addition, estimates of both the

intramyocellular lipid (IMCL) and extramyocellular lipid (EMCL) content of the soleus muscle

in persons with incomplete SCI were 3-5 times higher compared to that in controls. Higher

IMCL concentrations in skeletal muscle may indicate that persons with incomplete SCI are

predisposed to the development of peripheral insulin resistance and have a relatively greater risk

to develop Type 2 diabetes.

7.2 Background

Amongst the 10,000 spinal cord injuries (SCI) that occur annually in the United States,

almost 55% are classified as incomplete (NSCISC 2006). Similar to complete SCI (Castro,

Apple et al. 1999; Shields 2002), individuals with incomplete SCI display a variety of skeletal









muscle adaptations including a decrease in muscle cross sectional area (Shah, Stevens et al.

2006), and decrement in voluntary force production and muscle activation (Jayaraman, Gregory

et al. 2006). Collectively, these musculoskeletal deficits limit the overall functional capabilities

of this patient population (Waters, Adkins et al. 1994; Ulkar, Yavuzer et al. 2003; Kim, Eng et

al. 2004). Furthermore, a relatively sedentary lifestyle following the neurological injury makes

persons with incomplete SCI extremely vulnerable to metabolic risk factors. Glucose intolerance

and insulin resistance is purported as one of the major metabolic risk factors in the development

of cardiovascular complications after SCI (Dallmeijer, van der Woude et al. 1999; Bravo,

Guizar-Sahagun et al. 2004; Jacobs and Nash 2004; Manns, McCubbin et al. 2005).

Strong evidence in the literature suggests obvious positive correlations between

intramyocellular lipid (IMCL) and insulin resistance in individuals with a relatively sedentary

lifestyle including healthy individuals (Krssak, Falk Petersen et al. 1999; White, Ferguson et al.

2006), elderly (Cree, Newcomer et al. 2004) and in persons with obesity (Sinha, Dufour et al.

2002; Weiss and Caprio 2006). Accordingly, IMCL depots in skeletal muscle may provide an

indirect measure of insulin resistance. Given that the higher reported incidence of insulin

resistance in persons with incomplete SCI (Bauman, Spungen et al. 1999; Bauman and Spungen

2001) and the relatively inactive lifestyle (Subbarao 1991; Ditunno, Bums et al. 2005), we

hypothesized that the IMCL content would be elevated in the lower extremity skeletal muscles of

these individuals. Though studies have demonstrated elevated lipid depots in the lower extremity

muscles of persons with complete and incomplete SCI using magnetic resonance imaging (Elder,

Apple et al. 2004; Gorgey and Dudley 2006), we are not aware of any studies that have

specifically reported alterations in the IMCL content in persons with motor incomplete SCI.









Therefore, the overall objective of this study was to investigate alterations in the skeletal

muscle composition of persons with motor incomplete SCI using a combination of non-invasive

magnetic resonance imaging (MRI) and spectroscopic (MRS) measures. Specifically, we

measured the T2 relaxation times of lower leg muscles and quantified the intramyocellular lipid

(IMCL) and extramyocellular lipid (EMCL) content of the soleus muscle in persons with

incomplete SCI.

7.3 Methods

General design: We performed a case-control study in which lower extremity muscle

characteristics were compared between individuals with incomplete-SCI and age, gender, weight

and height matched, non-injured healthy persons. For this purpose, we used a combination of

proton imaging and spectroscopy techniques.

Subjects: Eight individuals (2 women) with chronic (17+9 months post injury) motor

incomplete SCI (C4-T12; ASIA C or D) participated in this study. Four subjects used a cane or

forearm crutches, while four used a powered wheelchair as their primary means of mobility

(Table 7-1). In addition, eight able-bodied persons matched in age, weight, height and gender

volunteered to serve as control subjects (mean SD; 42+11 yrs old, 7313kg, 17410cm).

Control subjects were recreationally active, but not engaged in any rigorous exercise program,

and were recruited from the Gainesville, FL community.

Prior to participating in the study, written informed consent was obtained from all subjects,

as approved by the Institutional Review Board at the University of Florida, Gainesville. The

conduct of all investigation conformed to the protocol and the ethical and humane principles of

research.

Magnetic resonance measurements: A total of eight persons with incomplete SCI

participated in the study. However, for logistical reasons, magnetic resonance imaging was









performed on seven individuals and proton magnetic resonance spectroscopy was performed on

six participants.

Details of T2 weighted Magnetic Resonance Imaging (MRI section 5.1.4) and Proton

Magnetic Resonance Spectroscopy (MRS section 5.1.5) measures and data analysis are provided

in chapter 5.

Statistical analysis: Levene's test showed unequal variance between the SCI and control

groups for both T2 and lipid measures. Accordingly, non-parametric Mann Whitney tests were

used to compare the T2 relaxation times and baseline lipid measures between the SCI and control

groups. SPSS for Windows (Version 11.0.1)4 was utilized for all statistical analyses. Alpha level

was set at 0.05.

7.4 Results

Muscle T2 relaxation times: Representative T2 weighted axial images of the lower leg of

healthy controls, ambulatory and non-ambulatory incomplete SCI subjects are provided in Figure

7-1. Overall, persons with incomplete SCI showed 11% -26% higher T2 relaxation values in the

tested lower leg muscles (Table 7-2). Significant differences were observed in the T2 relaxation

times of the SOL (p= 0. 011, 11%) and MG (p=0.005; 26%) muscles. Interestingly, all persons

with incomplete SCI showed higher T2 values of the MG and SOL muscles obtained by MRI. In

contrast, the T2 relaxation time of bone marrow was consistent in the SCI and control subjects

(1% difference).

Calculations of soleus muscle T2 relaxation times by spectroscopy coincided with T2

measurements from MRI. Patients with an incomplete SCI showed a significant (14%, p=0.017)

increase in the baseline T2 relaxation times of the soleus muscle as compared to the control group

[median (minimum-maximum) 35.8ms (30.8 41.5ms) versus 31.1ms (29.1-31.4ms)]

4 SPSS Inc, 233 S. Wacker Drive, 11th floor, Chicago, Illinois 60606









Intramuscular lipid: Persons with incomplete SCI demonstrated significantly higher total

lipid, IMCL and EMCL levels in the soleus muscle as compared to healthy controls. Spectra

acquired from the soleus muscle of healthy controls, ambulatory and non-ambulatory incomplete

SCI subjects are provided in Figure 7-2. As shown in Figure 7-3, the total lipid content

normalized to water was on average 3.2 times higher in the subjects with incomplete SCI as

compared to controls. IMCL and EMCL values were 3.3 and 4.5 times higher in the incomplete

SCI subjects. Note that incomplete SCI subjects also showed a much wider distribution of values

for all lipid ratios as compared to the controls (Figure 7-3). Finally, individual data revealed that

every person, ambulatory and non-ambulatory, with incomplete SCI showed an elevation in the

overall lipid, IMCL and EMCL measures as compared to their corresponding controls (Figures

7-4 and 7-5).

7.5 Discussion

This study utilized T2 weighted imaging and localized proton spectroscopy to characterize

skeletal muscle in persons with chronic, motor incomplete SCI. Our results show marked

elevations in the T2 relaxation times of the locomotor muscles soleus and medial

gastrocnemius, as compared to controls, with maximum relative changes in the medial

gastrocnemius muscle. In addition, estimates of both the intramyocellular lipid (IMCL) and

extramyocellular lipid (EMCL) content of the soleus muscle in persons with incomplete SCI

were 3-5 times higher compared to that in controls. Higher IMCL concentrations in skeletal

muscle may indicate that persons with incomplete SCI are predisposed to the development of

peripheral insulin resistance and have a relatively greater risk to develop Type 2 diabetes.

Using image-guided volume localized proton spectroscopy we quantified intramuscular

lipid in the predominantly slow twitch soleus muscle. Interestingly, we found a marked increase

in the intramuscular lipid content of all individuals with SCI compared to controls. This









phenomenon of elevated muscle lipid has been reported in a number of clinical populations

including individuals with stroke (Ryan 2002) as well as both complete (Elder, Apple et al.

2004) and incomplete SCI (Gorgey and Dudley 2006). Elder et al demonstrated that persons

with complete SCI display an almost 4-fold increase in intramuscular fat relative to controls, and

that these values correlate with plasma glucose levels during an oral glucose tolerance test. In

another study, a three fold elevation in intramuscular fat was observed in persons with chronic

motor incomplete SCI (Gorgey and Dudley 2006). Previous studies in SCI patients however, did

not distinguish between IMCL and EMCL, but instead used MRI to estimate total intramuscular

lipid content. An advantage of the present study is that using localized H-spectroscopy we were

able to individually quantify both IMCL and EMCL.

Our findings demonstrate significant elevations in the ratios of IMCL and EMCL to water

following incomplete SCI. Moreover, the IMCL and EMCL content measured in the soleus

muscle (relative to the water peak) of control subjects in our study fell within the range of similar

measures reported in the literature (Krssak, Falk Petersen et al. 1999). Though IMCL is

purported to be influenced by diet and exercise, studies also report strong correlations between

IMCL and insulin resistance irrespective of diet, age, weight, activity level and gender (for

reviews see (Goodpaster and Wolf 2004; Boesch, Machann et al. 2006). Nevertheless, in our

study, while gender, body weight, physical activity and age were well controlled, elevations in

myocellular lipids are far greater than those attributable to dietary influences (0.32 times

elevation in previous dietary studies) (Stettler, Ith et al. 2005). The highly elevated IMCL levels

in the incomplete SCI group raises concern because of their reported association with insulin

resistance in sedentary individuals with normal body weights (Goodpaster and Wolf 2004;

Boesch, Machann et al. 2006) as well as obese individuals (Visser, Kritchevsky et al. 2002;









Goodpaster and Wolf 2004). Moreover, skeletal muscle is the major depot (-80%) for blood

glucose and marked muscle atrophy has been shown to be a secondary risk factor (Goodpaster

and Kelley 1998; Goodpaster and Brown 2005). The combination of muscle atrophy (Shah,

Stevens et al. 2006), relative inactivity (Subbarao 1991; Ditunno, Burns et al. 2005) and high

IMCL levels in persons with incomplete SCI renders this patient population particularly

vulnerable to altered glucose homeostasis.

In addition to measuring the intramuscular lipid content, we also studied the T2 relaxation

characteristics of the lower extremity muscles in persons with chronic SCI. We found 11-26%

higher T2 values in the lower extremity muscles of persons with incomplete SCI, with maximum

relative changes in the medial gastrocnemius muscle. Because of partial volume filling estimates

of muscle, T2 relaxation time using MRI reflect properties of both water and lipid protons within

the muscle. Lipid has an inherently longer T2 relaxation time (85-90ms) than muscle (T2 equal

to 31-33ms) at 1.5T) (Bruhn, Frahm et al. 1991) and hence it is not surprising that persons with

incomplete SCI, which show higher amounts of intramuscular lipid, would display increased T2

relaxation times on MRI. However, using proton spectroscopy we also assessed the T2 relaxation

time of the soleus muscle independent of contributions of lipid, and found that the muscle T2 in

persons with incomplete SCI remained elevated. Increased skeletal muscle T2 values have been

reported in a number of conditions, including edema(Ploutz-Snyder, Nyren et al. 1997; Ababneh,

Beloeil et al. 2005), peripheral denervation (Koltzenburg and Bendszus 2004; Wessig,

Koltzenburg et al. 2004) and exercise induced muscle damage (Walter, Cordier et al. 2005).

Persons with motor incomplete SCI have vascular disturbances including venous vascular

dysfunction (Hopman, Nommensen et al. 1994) and a continuous dependent position of their

lower extremities may lead to accumulation of myocellular water in the postural leg muscles.









Increases in intramyocellular and extramyocellular water content can cause elevations in proton

density and a subsequent increase in T2 relaxation times of muscle (Ploutz-Snyder, Nyren et al.

1997; Ababneh, Beloeil et al. 2005). Interestingly, we observed a pattern consistent with higher

elevations in non-ambulatory subjects, that is, in persons who used a wheelchair for mobility

(S5, S6, S7, S8). Furthermore, we found relatively greater T2 enhancements in the soleus and

medial gastrocnemius as versus the tibialis anterior muscle. Concurrent to our findings, increases

in muscle T2 relaxation times have also been reported after spinal contusion in rats. Liu et al

have shown significant elevations in the T2 relaxation times of rat hind limb muscles after one

week of spinal contusion (Liu, Bose et al. 2006). Interestingly, T2 elevations in the rat soleus

muscle did not recover until three months after injury in contrast to the tibialis anterior muscle

that showed normal T2 values by four weeks. The authors attributed the elevated T2 relaxation

times to increase in extramyocellular fluid as indicated by an enhancement in extracellular space

during concomitant qualitative histological assessment of the involved muscle (Liu, Bose et al.

2006). Similarly, elevated muscle T2 times after peripheral denervation in both humans (Uetani,

Hayashi et al. 1993; Koltzenburg and Bendszus 2004) and animals (Wessig, Koltzenburg et al.

2004) have been attributed to capillary enlargement and increased muscular blood volume.

T2 weighted MRI has also been successfully used to monitor muscle damage during

reambulation following models of disuse such as cast immobilization (Frimel, Walter et al.

2005). Based on increase in muscle T2 after isometric contractions, previous studies suggest an

increased susceptibility to muscle injury after complete spinal cord injury in humans (Bickel,

Slade et al. 2004). Factors including unloading and inactivation of affected muscles, and skeletal

muscle atrophy seemingly contribute to this phenomenon. Although persons with incomplete

SCI have partial sparing of the spinal cord, relative unloading and inactivation of their lower









limb muscles might predispose skeletal muscle to injury. As such, the elevated baseline T2 values

from our data might be reflective of damaged muscle. However, we believe that muscle damage

is a less likely factor in the present study because neither locomotor training in humans

(submission under review) or animal (Liu, Bose et al. 2006) has shown to enhance muscle T2

after incomplete SCI. Using MR imaging and spectroscopy measures, we recently demonstrated

that muscle loading and activation during locomotor training does not result in lower limb

muscle damage after incomplete SCI in humans (submission under review). In fact, locomotor

training in spinal contused rats has shown to accelerate the normalization in elevated muscle T2;

with no link between muscle damage and T2 (Liu, Bose et al. 2006). Future studies using

advanced imaging and spectroscopic measurements will be needed to identify the cause of

elevated T2 relaxation times.

We recognize potential limitations of our study. First, the sample size of our incomplete

SCI group limits conclusions concerning the impact of injury level, duration and/or severity of

injury on myocellular lipid levels. We observed a much larger variation in all lipid measures of

our SCI group as compared to variation in a similar sized control group (see Figure 7-3). Since

incomplete SCI reflects a very heterogeneous population with varying ambulatory status, future

studies on a larger sample size with inclusion criteria based on severity of injury or duration after

injury might prove worthwhile. Such studies can quantify skeletal muscle lipid composition,

especially if these ratios could serve as non-invasive biomarkers of insulin resistance. Secondly,

we did not test glucose tolerance or insulin sensitivity of our subjects; thereby limiting our ability

to directly associate the myocellular lipids with the development of insulin resistance. Future

studies on persons with incomplete SCI are warranted to confirm the association of IMCL

accumulation and development of insulin resistance. Nevertheless, based on extensive literature









that correlates IMCL content with development of insulin resistance, we believe our data yield

valuable information concerning potential risks associated with this type of injury and can

provide a foundation for future studies.

In conclusion, we found an increase in T2 relaxation properties of leg muscles and elevated

fatty depots within the soleus muscle of persons with chronic incomplete SCI. Our results

suggest altered muscle composition in this patient cohort. Future studies are warranted to identify

the relationships between IMCL and insulin resistance along with the functional and

physiological impacts of increased IMCL in this population.










Table 7-1. Characteristics of individuals with incomplete-SCI

Subject Age (years) BMI Months post Injury ASIA Type of WISCI-II
/Gender (kg/m2) injury level Grade assistive aid
S1 46/F 18.6 10 C5 D Forearm crutch 15
S2 22/M 21.5 13 C5 D NAD 20
Single point
S3 23/M 19.9 8 T1 D cane 19
S4 48/M 20.3 37 T1 D Forearm crutch 12
S5 59/M 18.2 15 C6 C Wheelchair 8
S6 42/F 28.3 16 C5 C Wheelchair 6
S7 37/M 25.1 15 T1 D Wheelchair 13
S8 58/M 27.4 11 C4 C Wheelchair 8
Abbreviations: NAD= No assistive aid; WISCI-II = Walking Index for spinal cord injury (normal is 20/20).

Table 7-2. Percent differences between the T2 relaxation times of lower extremity muscles in
persons with incomplete SCI and corresponding controls (n=7). Data are expressed as
median, minimum-maximum values (min-max) and percentage change in mean T2
relaxation times.
Incomplete SCI Control subjects % difference in T2 p value
median (min max) of T2 values by MRI
SOL 31.7 (30.8 41.5) 30.5 (29.1 31.4) 11% 0.011*
MG 35.8 (30.1 45.7) 29.5 (27.8 32.4) 26% 0.009*
LG 33.0 (26.7 39.0) 30.9 (27.9 33.0) 11% 0.132
TA 28.8 (24.8 38.9) 27.0 (26.4 29.4) 12% 0.209
BM 49.2 (46.8 51.6) 47.9 (45.7 51.6) 1% 0.667
*Significant T2 differences between the incomplete SCI and control groups (p<0.05). Abbreviations: SOL=soleus;
MG=medial gastrocnemius; LG=lateral gastrocnemius; TA=tibialis anterior; BM = Bone marrow.


















gure 7-1. Representative T2 weighted trans-axial proton magnetic resonance images of the
lower leg. Ambulatory individual with incomplete SCI (S1) ; non-ambulatory
individual with incomplete SCI (S8); able-bodied control subject (C8). [A= anterior;
P = posterior; M=medial; L=lateral; MG=medial gastrocnemius; LG= lateral
gastrocnemius; SOL=soleus, TA=tibialis anterior]















[Ipidi



prto sp ecr) 3soi 1g 13 (1ePr

Water 1




Lipid(



4,7 [p5 p 4 = c at3







lipid


4.7 1-5pp 3,310 1. 3 (pp1)

Figure 7- 2. Representative proton magnetic resonance spectra obtained from the soleus muscle.
Ambulatory individual with incomplete SCI (S1); non-ambulatory individuals with
incomplete SCI (S8) ; able-bodied control subject (C8). The left column represents
proton spectra showing water (4.7ppm) and lipid (1.5ppm) peak. The right column
illustrates lipid components after water suppression [1= intramyocellular lipid
(IMCL) at 1.3ppm; 2 = extramyocellular lipid (EMCL) at 1.5ppm; 3 = creatine (CH3)
at 3.0ppm; 4 = choline at 3.2ppm]









.140



S,10








EMCL IMCL Total Lipid
(0).
s"J


,04J


.020 Control

0.00 Incomplete SC
N= 6 6 6 6 6
EMCL IMCL Total Lipid
Figure 7-3. Box-plot depicting variability in EMCL/water, IMCL/water and total soleus muscle
lipid/water ratios. Note the large variability in the SCI (hatched bars) versus control
group for all ratios. The EMCL/water ratio shows an extreme value from subject S6
(0).









* Incomrpiete SCI Control


0.09

0.07
0.06
0.05
0o.04
O.DS

0.03
0.02
0.01
0.00
C.O2


Figure 7-4. Individual data comparisons of IMCL to water ratio (IMCL/water)


* Incomplete SCI


S1


Figure 7-5. Individual data comparisons of EMCL to water ratio (EMCL/water)


I 5
S .54


5
95


I
5E


I7
S7,


Control


0.09

alas




0.02
0.01
0.03
G-.011


I
S6


S5









CHAPTER 8
EXPERIMENT THREE MAGNETIC RESONANCE ASSESSMENT OF MUSCLE
DAMAGE DURING LOCOMOTOR TRAINING IN PERSONS WITH INCOMPLETE
SPINAL CORD INJURY

8.1 Summary

The purpose of this study was to assess the impact of locomotor training (LT) on muscle

T2, an in vivo marker of muscle damage before, at 2 weeks and 9 weeks of training in persons

with incomplete spinal cord injury (SCI). Persons with chronic incomplete SCI underwent nine

weeks of LT (45 sessions) that involved step training (30 minutes) on the treadmill with body

weight support (BWS) and manual assistance followed by over ground walking (20 minutes). T2

relaxation time was measured in lower extremity muscles soleuss, medial gastrocnemius, lateral

gastrocnemius and tibialis anterior] before LT and after 2 and 9 weeks of LT using Magnetic

Resonance Imaging (MRI) and Spectroscopy (MRS). Overall, no significant differences were

detected in the T2 relaxation times of lower extremity muscles of individuals with chronic i-SCI

during LT using either MRI or MRS measures. Our pilot data suggest that muscle loading and

activation during LT as provided in the present study does not result in significant muscle

damage in the lower limb, following i-SCI as determined by MRI and MRS.

8.2 Introduction

Secondary to chronic alterations in loading and inactivity, persons with complete spinal

cord injury experience extensive muscle atrophy (45-80%) and an increased susceptibility to

muscle damage (Bickel, Slade et al. 2004). With recent advances in the management of acute

SCI, most injuries are now classified as incomplete (i-SCI). Individuals with i-SCI demonstrate a

24-31% decrease in lower extremity muscle size, considerable loss of muscle strength and an

increased dependence on the use of assistive devices for ambulation (Melis, Torres-Moreno et al.

1999; Shah, Stevens et al. 2006). However, the degree of functional impairment and reduction in









loading/activity after i-SCI is variable and the muscle's susceptibility to damage has not been

studied.

Recently, rehabilitation of persons with i-SCI has focused on the use of treadmill

locomotor training (LT) to facilitate recovery of walking (Wemig, Nanassy et al. 1998;

Behrman, Lawless-Dixon et al. 2005). LT takes advantage of the phasic, peripheral sensory

information and loading associated with stepping to promote neural plasticity and maximize

residual function. However, a variety of animal studies have shown that reambulation following

a period of unloading and inactivity can cause muscle damage in postural muscles such as the

soleus (Kasper, White et al. 1990; Frimel, Walter et al. 2005). Consequently, the potential for

muscle damage to occur during LT especially at the onset of training is a concern. Therefore the

objective of this study was to determine the effect of two (2wk-LT) and nine weeks of LT (9wk-

LT) on in-vivo markers of muscle damage in the lower limb muscles of persons with chronic i-

SCI using magnetic resonance techniques.

8.3 Methods

Subjects: Seven subjects (489 yr; 70+14 kg; 17513 cm) with chronic (17+10 months)

motor i-SCI (C4-T12; ASIA C or D) participated in this study. Three subjects used a cane or

forearm crutches, while four used a powered wheelchair as their primary means of mobility.

Locomotor training: LT consisted of 9 weeks (45 sessions) of step training (30 minutes)

on the treadmill with body weight support and manual assistance followed by over ground

training (20 minutes). A detailed description of the training principles, parameters and

progression has been described in Chapter 5 (5.1.3) of this work.

Magnetic Resonance Imaging (MRI) and Spectroscopy (MRS): Details of T2 weighted

Magnetic Resonance Imaging (MRI section 5.1.4) and Proton Magnetic Resonance









Spectroscopy (MRS section 5.1.5) to measures T2 relaxation times of muscles and data

analysis are provided in chapter 5.

Statistical analysis: A two-way repeated measures ANOVA was used to test for

differences in T2 relaxation times. SPSS for Windows (vl 1.0.1)5 was utilized for all statistical

analyses (. = 0.05).

8.4 Results

No significant differences were found in the T2 relaxation times of lower limb muscles in

persons with chronic i-SCI following LT using either MRI or MRS. Muscle T2 relaxation times

measured using MRI and MRS in the tested muscles are provided in Table 8-1. The T2

relaxation times of bone marrow did not change (variance = 4%) with LT.

8.5 Discussion

Pilot data presented in this study demonstrate that during 2 weeks and 9 weeks of LT, T2

relaxation times of lower extremity muscles do not significantly change in persons with chronic

i-SCI. These results indicate that body weight supported LT does not induce muscle damage in

affected lower extremity muscles of persons with chronic i-SCI, despite significant muscle

atrophy and weakness.

Several studies have demonstrated that chronic unloading and inactivity renders skeletal

muscles more susceptible to muscle damage. Using MRI, Bickel et demonstrated that persons

with complete SCI experience muscle damage even after a single bout of isometric contractions

(Bickel, Slade et al. 2004). Alterations in the MR T2 relaxation properties of skeletal muscle,

indicative of muscle damage, have also been reported during reambulation following cast

immobilization (Frimel, Walter et al. 2005). MRI has been used extensively to monitor muscle

damage in a variety of conditions. It has the distinct advantage to provide spatial information and

5 SPSS Inc, 233 S. Wacker Drive, 11th floor, Chicago, Illinois 60606









allows the investigation of multiple muscles simultaneously. However, similar to other imaging

techniques, T2 weighted MRI suffers from partial volume filling, making it challenging to

reliably assess the inherent T2 of skeletal muscle in the presence of increased amounts of

intramuscular lipid. MRS on the other hand, while providing limited spatial information, offers

the opportunity to monitor changes in muscle T2 without contamination from lipid signal

(Walter, Cordier et al. 2005). In this study, we found no significant change in the T2 relaxation

times of lower extremity muscles during LT using either method.

Of note, our data demonstrate relatively higher baseline (pre-LT) T2 values in the i-SCI

group relative to published control values at the same magnetic field strength (Akima, Ushiyama

et al. 2003). Seemingly, some baseline alterations in muscle composition may be present in

skeletal muscle of persons with i-SCI, independent of ambulatory status, which warrants further

investigation.

8.6 Conclusion

In conclusion, these pilot data suggest that bodyweight supported treadmill LT does not

induce muscle damage in the lower-limb muscles of persons with chronic i-SCI.









Table 8-I. MR measures of T2 relaxation times of lower extremity muscles before and after LT.
The soleus (SOL), medial gastrocnemius (MGAS) and lateral gastrocnemius (LGAS)
and tibialis anterior (TA) muscles prior to LT (pre-LT), after two weeks (2wk-LT)
and nine weeks (9wk-LT) of locomotor training in persons with incomplete SCI.
T2 Relaxation times Muscle Pre-LT 2wk-LT 9wk-LT
[Mean T2 (ms) + standard error]
SR SOL 33.71.5 37.11.9 36.02.0
Magnetic Resonance
Imaging MGAS 36.9+2.7 38.8+2.4 38.8+2.6
LGAS 33.51.8 37.23.2 38.43.3
TA 30.91.9 33.51.4 32.61.9
Bone marrow 49.70.6 49.90.5 50.10.3

Magnetic Resonance
Spectroscopy SOL 34.51.5 37.1+0.7 37.21.2









CHAPTER 9
EXPERIMENT FOUR IMPACT OF LOCOMOTOR TRAINING ON MUSCLE SIZE AND
INTRAMUSCULAR FAT AFTER SPINAL CORD INJURY

9.1 Summary

The use of repetitive locomotor training with body weight support to improve motor

recovery and ambulation following incomplete-SCI has gained considerable momentum in the

last two decades. The purpose of the present study was to investigate if LT impacted muscle

physiology; especially lower extremity whole muscle size and fat content in persons with

incomplete SCI. Nine persons with incomplete SCI underwent 45 sessions of locomotor training

protocol that consisted of treadmill stepping and over ground walking (LT). Magnetic resonance

imaging and proton spectroscopic measures were utilized to measure lower extremity muscle

size and soleus muscle composition after nine weeks of the training. LT effect on skeletal muscle

was variable. Our findings show significant increases (8-10%) in the plantarflexor muscle cross

sectional area of persons with incomplete SCI after the LT; with no marked changes in the thigh

muscles. In addition, LT did not cause any alterations in the soleus muscle lipid composition in

our patient cohort.

9.2 Introduction

Atrophy of paralyzed muscles is one of the most obvious muscular adaptations following

SCI. Depending upon the severity of injury; individuals with SCI demonstrate a noticeable 25-

80% decline in average cross-sectional area (CSA) of their lower leg and thigh muscles (Castro,

Apple et al. 1999; Shah, Stevens et al. 2006). Consequent to this atrophy are a battery of

disabling motor impairments including decreases in absolute muscle force production, muscle

torque and power (Lieber 1986; Shah, Stevens et al. 2006). Functionally, atrophy significantly

diminishes muscle strength, motor performance, and locomotor capabilities and makes the

paralyzed muscle susceptible to injury (Bickel, Slade et al. 2004; van Hedel, Wirth et al. 2005).









Recently, atrophy following SCI has also been linked to substitution of the paralyzed muscle by

fatty tissue infiltration (Elder, Apple et al. 2004; Gorgey and Dudley 2006). Though direct

functional implications of the presence of fat in skeletal muscle remain unidentified at this time,

wide arrays of studies have reported strong associations between intramuscular fat, specifically

intramyocellular lipid (IMCL) and development of insulin resistance (Perseghin, Scifo et al.

1999; Furler, Poynten et al. 2001). Furthermore, persons with SCI are predisposed to

development of insulin resistance (Bauman, Spungen et al. 1999; Bauman and Spungen 2001)

that shows a significant association with muscle fat (Elder, Apple et al. 2004).

Several studies have aimed at attenuating atrophy after SCI to optimize limb function and

improve the locomotor capabilities of this patient cohort (Bremner, Sloan et al. 1992; Dudley,

Castro et al. 1999; Creasey, Ho et al. 2004). An emerging trend in promoting motor recovery and

ambulation following incomplete-SCI is the use of repetitive locomotor training with body

weight support (Edgerton, Tillakaratne et al. 2004; Behrman, Bowden et al. 2006). Locomotor

training has been suggested to have a positive impact on the walking ability, functional

independence and subjective well being of persons with chronic incomplete SCI (Phillips,

Stewart et al. 2004; Behrman, Bowden et al. 2006; Hannold, Young et al. 2006). Moreover,

studies have shown that locomotor training involves sufficient mechanical loading to increase

muscle fiber size and muscle glucose tolerance, while simultaneously enhancing locomotor

capacities (Phillips, Stewart et al. 2004; Stewart, Tamopolsky et al. 2004; Adams, Ditor et al.

2006). However, to our knowledge, no study has investigated the effects of locomotor training

on lower extremity whole muscle size and fat content of skeletal muscle.

Therefore, the main purpose of this study was to assess the effect of locomotor training on

the muscle size and fat content of lower extremity muscles after incomplete SCI. Specifically,









we measured the cross-sectional area of the lower leg and thigh muscles and quantified fat

content (along with intramyocellular lipid and extramyocellular lipid) of the soleus muscle after

9 weeks of locomotor training in persons with incomplete SCI.

9.3 Methods

Subjects: Nine individuals (2 women) with chronic (17+9 months post injury) motor

incomplete SCI participated in this study. Details of subject selection are provided in Chapter 5.

Five subjects used a cane or forearm crutches, while four used a powered wheelchair as their

primary means of mobility.

Locomotor training protocol: Described in chapter 5

Magnetic resonance measurements: For logistical reasons, magnetic resonance imaging

for cross sectional area measurements was performed on nine individuals and proton magnetic

resonance spectroscopy for estimation of lipid content was performed on five participants.

Details of the procedures and data analysis are provided in chapter 5.

Statistical analysis: Paired t-tests were used to compare CSA of the plantar flexors and

thigh muscles before and after nine weeks of LT. In addition, non-parametric Mann Whitney

tests were used to determine the effect of LT on TA muscle CSA and the lipid content. SPSS for

Windows (Version 11.0.1)6 was utilized for all statistical analyses. Alpha level was set at 0.05.

9.4 Results

Nine weeks of LT produced selective hypertrophy of lower extremity muscles in persons

with incomplete SCI (Figures 9-1). Significant changes were found in the CSA of the individual

plantarflexor muscles after nine weeks of LT. SOL muscle CSA increased by 8% (p=0.040) and





6 SPSS Inc, 233 S. Wacker Drive, 11th floor, Chicago, Illinois 60606









the MG (p=0.046) and LG (p=0.045) increased by 10%. Increases in CSA of the TA (4%), QF

(4%) and HAMS (6%) were not significant after LT (Figure 9-2).

No effect of LT was seen in the total lipid, IMCL and EMCL contents of the soleus muscle

(Figure 9-3).

9.5 Discussion

Findings of our present study reveal selective hypertrophy of the plantar flexors and no

change in the dorsiflexors (TA) and thigh muscle size after nine weeks of LT in persons with

incomplete SCI. In addition, LT does not alter the lipid content of the soleus muscle in our

patient group.

Our results are in concurrence with animal models of spinal transaction, where maximum

effect of LT is seen in the slow extensor muscles with minimal or no effect on the fast extensor

or flexor muscles (Roy and Acosta 1986; Roy, Talmadge et al. 1998). In addition, a 23%

increase in the soleus muscle fiber cross-sectional area has been shown after one week of

locomotor training in spinal contused rats (Stevens, Liu et al. 2006). To our knowledge, no

studies in humans have determined the impact of LT on whole skeletal muscle size. Previous

studies in humans have shown that around 65 sessions of locomotor training with manual

assistance from therapists increases vastus lateralis mean fiber area by almost 25% in persons

with incomplete SCI (Stewart, Tarnopolsky et al. 2004). While our study demonstrates

significant hypertrophy of the plantarflexor muscles, we see an insignificant 4% increase in the

overall size of the quadriceps muscles after LT. In the study by Stewart et al, the greater number

of training sessions might have probably produced the quadriceps hypertrophic response. In

contrast to LT, comparatively lesser training frequencies (as less as 30 sessions) of strength

training interventions such as electrical stimulation has shown relatively greater hypertrophy of









the treated musculature. In persons with incomplete SCI, Gregory et al have reported an increase

in quadriceps muscle CSA by 8% and of the plantar flexors by 14% after 30 sessions of electrical

muscle stimulation accompanied with plyometric training (Gregory, Bowden et al. 2007). Sloan

et al have shown an increase in the quadriceps muscle CSA of chronic incomplete SCI

individuals by 9% after similar sessions of electrical stimulation induced cycling training (Sloan,

Bremner et al. 1994). These morphological improvements in muscle size have also been

associated with simultaneous gains in muscle strength and subjective reports of functional

activities of daily living. Despite relatively lesser training sessions, hypertrophy observed in

these studies is probably because of the hypertrophic stimulus of strength training that is

achieved because of specific muscle activity against adequate resistance. In contrast, the intensity

of LT used at every session in the current study was just enough to generate an alternating slow

stepping pattern that simulates near-normal walking. Though subjects in our study gradually

progressed to lesser body weight support, the speeds and overall duration of training might have

deemed insufficient to elicit a major hypertrophic response. Perhaps, a much stronger stimulus is

necessitated to elicit specific quadriceps hypertrophy. On the other hand, the gain in CSA of the

plantarflexor muscles after LT is probably because of their selective activation during the

training. LT protocol in our study placed constant emphasis on adequate foot placement and push

off that largely involves activity in the plantar flexor muscles. Deitz et al show have shown that

four-five weeks of LT drastically increases the EMG activity of gastrocnemius muscle in

individuals with incomplete SCI as compared to the dorsiflexors or thigh muscles (Dietz,

Colombo et al. 1995). Of note, our sample size (n=6) for the thigh muscle CSA data limits us

from making any conclusive remarks about LT effect on thigh muscle size. Nevertheless, our

findings suggest that LT has the potential to selectively enhance skeletal muscle size.









In response to LT in our patient group, we did not find any significant alterations in the

soleus muscle fat content after 9 weeks. The phenomenon of elevated muscle lipid has been

reported in a number of clinical populations including individuals with stroke (Ryan 2002),

multiple sclerosis (White 2007) as well as both complete (Elder, Apple et al. 2004) and

incomplete SCI (Gorgey and Dudley 2006). A common precipitating factor for increased fatty

tissue infiltration in these conditions is the presence of a sedentary lifestyle. Evidence from

literature demonstrates an improvement in glucose uptake in skeletal muscle (hence a decrease in

insulin resistance) after LT in persons with incomplete SCI (Phillips, Stewart et al. 2004). Given

the strong associations of IMCL with insulin resistance (Jacob, Machann et al. 1999; Krssak,

Falk Petersen et al. 1999; Schrauwen-Hinderling, Hesselink et al. 2006), we hypothesized that

increased physical activity through LT would decrease the accumulated muscle fat (and hence

IMCL) in our patient cohort. Using non-invasive methods of MRS we have been able to quantify

the lipid components in the soleus muscle. However, we did not encounter any alterations in the

lipid content in our present study. Counterintuitive as it might seem, but studies have

unequivocally shown an increase in the IMCL content after both short and long term endurance

training (such as running and cycling) in healthy individuals (VanLoon 2004). Apparently,

despite an increase in the IMCL content, endurance trained individuals (specifically athletes),

have markedly greater insulin sensitivity. Studies suggest that unlike in sedentary individuals,

excess IMCL in endurance athletes actually contributes positively by playing an integral role in

mitochondrial oxidation and increasing the oxidative capacity of the muscle (Schrauwen-

Hinderling, Hesselink et al. 2006). We are unsure however, if the intensity of LT used in this

study makes a difference in altering muscle lipid content. Phillip et al have shown an increase in

the glucose uptake after LT in persons with chronic incomplete SCI; thereby suggesting an









increase in sensitivity to insulin resistance in this patient cohort. However, patients in their study

underwent 68 sessions of LT as versus 45 sessions in the present study. Nevertheless, based on

our sample size and non-availability of direct measures of insulin resistance, interpretations on

insulin sensitivity and any potential of relationship of IMCL with oxidative capacity etc are

difficult to make.

As a potential limitation, the sample size of our incomplete SCI group limits conclusions

concerning the impact of high intensity of LT on atrophy and myocellular lipid levels.

Furthermore, we simply studied the soleus muscle to assess lipid composition and different

muscles might respond to therapy differently. Future studies are probably needed to identify

effect of different doses (duration and intensity) and types of exercise (endurance versus strength

training on atrophy and lipid content in persons with incomplete SCI. Concurrent measurements

of oxidative capacity and standard measurements of insulin sensitivity will help verify the direct

effects of these training on the paralyzed skeletal muscle.












SPreLT PostLT


* T


SOL


MG


LG


Figure 9-1. Cross sectional area of lower leg muscles after nine weeks of locomotor training
(9wk-LT). *Significant change in selective plantar-flexor muscles after LT).


I Pre LT Post LT


50.0

S40.0

5 30.0

20.0

10.0

0.0



Figure 9-2. C
L


HAMS


ross sectional area of thigh muscles after nine weeks of locomotor training (9wk-
F). LT did not change CSA of the thigh muscles in persons with incomplete SCI.


30.0


25.0

20.0
rt
S15.0

U 100L

5.0

&0


70.0

60.0


....,















*Pre LT Post LT


Lipidl


[MCL


EMCL


Figure 9-3. Estimates of soleus muscle lipid before (Pre LT) and after (Post LT) nine weeks of
locomotor training (LT) in persons with incomplete SCI. LT did not produce any
significant change in muscle lipid composition.


0.1200


u0.000







0.020



0l.0000









CHAPTER 10
EXPERIMENT FIVE MONITORING ALTERATIONS IN INORGANIC PHOSPHATE OF
HINDLIMB MUSCLE AFTER SPINAL CORD CONTUSION IN RATS

10.1 Summary

The overall objective of the present study was to determine the inorganic phosphate

content [Pi] and phosphorylation potential of the rat hindlimb muscle after moderate spinal cord

contusion in rats. Eight young adult female rats were moderately injured at the T8-T10 thoracic

spinal cord. 31P MRS measurements were performed at weekly intervals for assessments of

phosphorylation potential of the rat hindlimb muscle for three weeks. Spectra were acquired in a

Bruker 11T/470 MHz spectrometer using a 31P (190.5 MHz) surface coil, placed over the belly

of the gastrocnemius muscles. Resting spectra were manually phased, and the areas of the y-

ATP, Pi, and PCr peaks were determined using area integration. Absolute concentrations of the

metabolite ratio were obtained after accounting for correction factors and through biochemical

determination of total creatine [TCr] and [ATP] content in gastrocnemius muscle. Our data

revealed marginally significant elevation in [Pi]/[PCr] ratios, significantly elevated

[ADP][Pi]/[ATP] ratios and marked decreases in [PCr]. These data suggests an imbalance of the

intracellular energy buffer system of the paralyzed hindlimb muscle that recovers by three weeks

after injury.



10.2 Introduction

Relative inactivity and a sedentary lifestyle following incomplete SCI leads to unloading

and disuse of the paralyzed lower extremity muscles (Melis, Torres-Moreno et al. 1999). We

recently reported marked skeletal muscle atrophy and an overall decrease in the ability of lower

extremity skeletal muscles to generate muscle peak torque in persons with incomplete SCI (Shah,

Stevens et al. 2006).









Studies of muscle disuse show evidence that forced inactivity of lower extremity muscles

(i.e. immobilization, limb suspension and denervation) result in metabolic alterations of involved

musculature that may ultimately hinder muscle function (Lai, Jaweed et al. 1992; Vandenborne,

Elliott et al. 1998; Yoshida, Ikata et al. 2001; Pathare, Walter et al. 2005). Specifically, studies

have shown that cast immobilization is accompanied by marked elevations in the basal inorganic

phosphate (Pi) concentration and the Pi-to-PCr ratio. Pathare et al demonstrated elevated levels

of resting Pi in the immobilized skeletal muscles of patients with an orthopedic injury (Pathare,

Walter et al. 2005) and also in a mouse model of cast immobilization (Pathare, Vandenborne et

al. 2007). Pi/PCr ratio is closely related to the phosphorylation potential and reflects the energy

state of the muscle (Veech, Lawson et al. 1979; Chance 1984). Though the exact mechanisms of

elevated Pi levels secondary to disuse are not well established, pathophysiological processes

accompanying disuse itself have been hypothesized as potential contributions (Lai, Jaweed et al.

1992; Pathare, Vandenborne et al. 2007). Notably, elevated Pi levels in the above-mentioned

studies have been shown to correlate well with declines in muscle force. In-vitro skinned muscle

fiber studies have unanimously reported that inorganic phosphate suppresses skeletal muscle

force production (Kentish 1986; Chase and Kushmerick 1988; Martyn and Gordon 1992).

Inhibitory effects of Pi on force development are shown to result from alterations in Ca+2

sensitivity of myofibrils that subsequently impacts the actomyosin cross-bridge force-Ca+2

relationship (Kentish 1986).

Increases in basal Pi concentration have also been noted following peripheral denervation.

Lai et al showed that following crush denervation of the sciatic nerve, the Pi/PCr ratios increased

in the denervated gastrocsoleus muscle (Lai, Jaweed et al. 1992). These ratios returned to normal

values following reinnervation. Interestingly, metabolic recovery of the muscle showed similar









patterns of recovery as parameters of nerve conduction (Lai, Jaweed et al. 1992). In addition,

spinal transaction studies in the animal model have shown tendencies of an altered

phosphorylation potential after SCI (Durozard, Gabrielle et al. 2000). However, these studies on

metabolic adaptations following neurological injuries have been conducted at relatively lower

magnetic field strengths and suffer from low signal to noise ratios.

The main aim of the present study was to characterize metabolic adaptations; specifically

the quantification of phosphate metabolites (and hence the phosphorylation potential) of the

resting rat hind limb muscle following spinal cord contusion. We used non-invasive phosphorus

magnetic resonance spectroscopy (31p_ MRS) at magnetic field strengths of 11Tesla to monitor

the phosphate metabolite content in the contused rat hind limb muscles over a course of three

weeks.

10.3 Specific Aims and Hypothesis

10.3.1 Specific Aim

a) To determine the impact of acute spinal contusion (one week) on the resting phosphate

metabolite content, and hence the phosphorylation potential of rat calf muscle after spinal cord

contusion. b) To longitudinally monitor alterations in the phosphorylation potential of the calf

muscles.

Phosphorus magnetic resonance spectroscopy (31P-MRS) at a high magnetic field strength

(11T) was used to monitor basal phosphate metabolites for three weeks after spinal cord

contusion. Skeletal muscle ATP and total creatine content (TCr) were quantified by biochemical

assays.

10.3.2 Hypotheses

a) Immediately after spinal cord contusion (one week), there is an alteration in the resting

phosphate metabolite content and hence a change in the phosphorylation potential of the









paralyzed hind limb muscle. b) Muscle phosphate metabolites begin to recover at three weeks

after spinal cord contusion.

10.4 Methods

10.4.1 Experimental Design

Two groups of randomly distributed rats were studied. Animals either underwent a spinal

cord contusion (n=8) or served as controls for wet lab procedures relevant to the study (see

below). The spinal injured group underwent 31P magnetic resonance spectroscopic (31P-MRS)

measurements prior to injury and at weekly intervals for three weeks after injury.

10.4.2 Animals

Sixteen adult Sprague Dawley female rats (12 week, 228-260g; Charles River, NJ) were

housed in a temperature controlled room at 210C with a 12:12 hours light: dark cycle and

provided with rodent chow and water ad libitum. Of these, 8 rats were moderately injured at the

T8-T10 thoracic spinal cord levels and 8 rats served as controls in providing healthy muscle

tissue to run biochemical assays for the quantification of phosphate compounds. The control

group also provided reproducibility results of our 31P data. All experimental procedures were

performed in accordance with the U.S. Government Principle for the Utilization and Care of

Vertebrate Animals by approval of the Institutional Animal Care & Use Committee at the

University of Florida.

Details about spinal cord contusion operation procedures have been described in detail in

chapter 5 (Section 5.2.2)

10.4.3 Magnetic Resonance Spectroscopy: Data Collection and Analysis

Phosphate metabolites including Pi, PCr and ATP were quantified from the hind limb rat

muscle at rest using a high magnetic field strength Bruker 11Tesla/470 MHz spectrometer. A 1.5

x 1.7 cm oval coil, tuned to the frequency of 31P nuclei at the 1 1T (190.5 MHz) was placed over









the belly of the gastrocnemius muscle. A 3-cm standard 1H surface coil was placed underneath

the hind limb to perform swimming and the animal's hind limb was extended such that the calf

muscles were centered over the surface coil. Spectra were acquired with a 50 gts square pulse, a

TR of 2s, spectral width of 10,000 Hz, 150 averages and 8000 complex data points. In order to

account for the differences in the rate of longitudinal relaxation times of phosphate compounds,

fully relaxed spectra with a TR of 15 seconds were acquired from non-stimulated rat hind limb

muscle. Subsequently, empirical calculations of correction factors (CF) were made by comparing

the amplitudes of phosphate spectral peaks at TR of 2s with those at 15s. In concurrence with CF

from the rat gastrocnemius muscle in literature (Mizobata, Prechek et al. 1995), the CF for PCr

and Pi in our study were 1.40 and 1.69 respectively.

10.4.3.1 3P-MRS spectral analysis at rest

The spectra were manually phased, and the areas of the 0 ATP, Pi, and PCr peaks

determined by area integration. The enzymatically determined ATP concentration in frozen

muscle tissue was taken as the standard to estimate absolute concentration of phosphate

metabolites. Absolute Pi and PCr concentrations were determined by using 3-ATP as an internal

standard and after accounting for correction factors (CF) as follows:

[Metabolite] (jtmol/g wet wt) = CFMetabolite X IntegralMetabolite X [ATP] (jtmol/g wet wt)

Integral P-ATP

(10-1)

Intracellular pH was calculated from the chemical shift of the Pi peak relative to PCr using

the equation, pH = 6.75 + log [(6 3.27) / (5.69 6)], where 6 is the chemical shift of the Pi peak

in ppm. The cystolic phosphorylation potential was calculated in reciprocal form as

[Pi][ADP]/[ATP] since the phosphorylation potential itself is not normally distributed. Free









cystolic ADP was calculated from the creatine kinase equilibrium reaction as previously

described (Mizobata, Prechek et al. 1995; Thompson, Kemp et al. 1995; Pathare, Vandenborne et

al. 2007):

[ADP] = {[free creatine] [ATP] }/[PCr] [H+] [Keq] (10-2)

Where, the free creatine was quantified by subtracting the PCr content obtained by 31p_

MRS from the total creatine content determined biochemically. Intracellular Mg concentration

and equilibrium constant (Keq) of the creatine kinase reaction were assumed as ImM and 1.66 x

109 respectively for mammalian skeletal muscle (Veech, Lawson et al. 1979).

10.4.4 Biochemical Assays

Details of ATP analyses are described in chapter 5 (Section 5.2.5.1).

10.4.5 Data Analysis

Repeated measures ANOVA were used to statistically compare the metabolite

concentrations, ratios ([Pi]/PCr) and phosphorylation ratios [ADP][Pi]/[ATP] of the hind limb

muscle prior to and over three weeks of spinal cord contusion. All hypotheses were tested at an

alpha level of 0.05 and post-hoc Bonferroni corrections were used for multiple comparisons.

Analyses were performed using SPSS for Windows, Version 13.3.

10.5 Results

Our data reveal that after one week of spinal contusion, there is marginal elevation in the

[Pi]/[PCr] ratios that did not reach statistical significance (p=0.067) (Figure 10-1). A significant

elevation in the phosphorylation ratio (1.5fold, p = 0.002) of the paralyzed gastrocnemius

muscles was observed at one week after contusion (Figure 10-2). This increase in the ratio after

spinal contusion was observed in all, but one animal (Figure 10.3). Specifically, the elevated

phosphorylation ratios were accompanied by significant elevations in [ADP] (-52%, p = 0.01)

and significant decreases in [PCr] content (13%, p = 0.035) (Table 1, Figure 10-4, Figure 10-5).









Figure 10.3 shows 31P spectra from the gastrocnemius muscle before and after spinal contusion.

Note the decrease in the amplitude of PCr peak after spinal contusion in contrast to before injury.

No significant change in baseline pH was observed between time points (Table 1). Biochemical

analyses revealed no differences in the total creatine and ATP content of gastrocnemius muscle

before and after injury. The TCr content (meanSEM) from gastrocnemius muscle assays was

44.22.6 mM and 44.23.9 mM for the control and injured group respectively. The ATP content

was 4.620.44 imol/g wet weight and 4.630.77 imol/g wet weight from the control and

injured gastrocnemius muscles respectively.

Longitudinal follow up of our data show that the phosphorylation ratio at two weeks

recovers to 1.38 fold of basal values that recover to control values (1.03times) at three weeks

after contusion (p > 0.01). The resting [PCr] and [ADP] levels recovered towards normal along

the course of three weeks (Table 1).

10.6 Discussion

The main objective of the present study was to quantify the [Pi]/[PCr] ratios in the

paralyzed rat gastrocnemius muscle at rest after moderate spinal contusion injury. Results of our

study show that there are elevations in the [Pi]/[PCr] ratios, albeit marginal. Additionally, we

found drastic elevations in the resting phosphorylation ratios ([ADP][Pi]/[ATP]) of the rat

gastrocnemius muscle following one week of moderate spinal contusion. These metabolic

alterations recover to baseline values by three weeks after spinal contusion injury.

The [Pi]/[PCr] ratio is a measure of bioenergetic potential of the muscle and has significant

bearing to muscle fatigue. In-vitro skinned muscle fiber studies have unanimously reported that

elevated inorganic phosphate suppresses skeletal muscle force production (Kentish 1986; Chase

and Kushmerick 1988; Martyn and Gordon 1992). Given that the paralyzed skeletal muscle is

extremely fatigable and accompanies marked muscle atrophy (Roy, Talmadge et al. 1998; Kjaer,









Mohr et al. 2001; Shields 2002) we expected that the [Pi]/[PCr] ratios in the paralyzed hind limb

would increase dramatically. As expected, we found an elevation in the [Pi]/[PCr] ratio (-18%)

after one week of moderate spinal cord contusion. This, however, was not statistically significant

(p=0.067). Nevertheless, based upon the 6.7% chance that the decrease in the ratios is

insignificant, we believe that the low statistical power (0.6) of this data might have probably

accounted for this insignificance. In fact, following complete spinal cord transaction in rats,

modest trends towards an increase in the resting Pi/PCr ratios have been reported after one week

of injury (Durozard, Gabrielle et al. 2000).

Despite the elevation in Pi/PCr, to our surprise, the increase in the [Pi]/[PCr] ratio

observed in our study are largely due to decreases in [PCr] content as versus elevations in [Pi].

Published data from our lab demonstrate a marked elevation in the [Pi]/[PCr] ratio (-75%) of the

plantarflexor muscles of mice that are immobilized at the hind limb for two weeks (Pathare,

Vandenborne et al. 2007). In animals models of muscle disuse due to denervation induced by

crush injuries, Lai et al report an almost 54% increase in the Pi/PCr ratios of the rat

gastrocnemius muscle at two weeks after denervation (Lai, Jaweed et al. 1992). In fact, studies

have shown that the severity of muscle denervation following the crush injuries dictates the

increases in Pi/PCr ratio, with greater increases in the ratios seen after severe injuries (Zochodne,

Thompson et al. 1988). Lastly, disuse due to damage to the motor neurons in the spinal cord, as

in poliomyelitis, is also associated with an increase in Pi/PCr ratios of the denervated muscle that

can get as large as 4fold high (Barany, Siegel et al. 1989). In these studies, elevations are not

only significantly higher than that seen in the spinal cord injury models, but the greater elevation

in the ratio is accounted predominantly by elevations in [Pi] (Pathare, Vandenborne et al. 2007)

or by combinations of elevations in [Pi] and decreases in [PCr] (Barany, Siegel et al. 1989). In









contrast, based on the significant depletion in resting [PCr] content (-13%) as opposed to the less

obvious elevations in the [Pi] content (-3% increases) of the paralyzed gastrocnemius muscle,

our results support the finding that the [Pi]/[PCr] ratio after spinal contusion is largely influenced

by declines in [PCr]. We are unsure why the [Pi] in our study remain unaltered after injury.

Declines in resting [PCr] have also been reported after complete spinal cord transaction in

the rat gastrocnemius muscle (Durozard, Gabrielle et al. 2000). A decrease in resting PCr

content is also observed in patients with muscular dystrophies including severe myotonic,

Duchene's and Becker's muscular dystrophies (Taylor, Kemp et al. 1993), mitochondrial

myopathy (Taylor, Kemp et al. 1993), hypothyroidism (Taylor, Rajagopalan et al. 1992),

hyperthyroidism (Erkintalo, Bendahan et al. 1998) and in chronic fatigue syndrome (Wong,

Lopaschuk et al. 1992). Though authors of the above-mentioned studies have not been able to

pinpoint the exact cause of depletions in the resting [PCr] in skeletal muscle, the association of

the depleted stores in [PCr] to a drastic intolerance to exercise has been well recognized. The

authors suggest that the decrease in [PCr] at rest indicates uncoupling of the phosphorylation

potential such that the energy available for muscle contraction and other cellular work is

decreased (Taylor, Kemp et al. 1993). Similar to muscular dystrophies, SCI is accompanied by

marked muscle weakness, atrophy and extreme intolerance to activity (Gerrits, De Haan et al.

1999; Hutchinson, Linderman et al. 2001; van der Salm, Nene et al. 2005).

Irrespective of the mechanism, we observed striking increases in the phosphorylation

potential of the paralyzed hind limb muscle after one week of spinal cord contusion. In order to

maintain steady state metabolic conditions of a skeletal muscle, increased flux through functional

ATPases must be met by an equal increase in ATP synthesis. The driving force, or the link,

between cytosolic ATP demand and supply is the phosphorylation ratio [ADP][Pi]/[ATP]. The









free energy for ATP hydrolysis depends on the mass action term of [ADP][Pi]/[ATP].

Accordingly, this ratio is used as a measure of bioenergetic reserve and oxidative metabolism.

Small changes in [ATP], [ADP] or [Pi] provide sensitive parameters through which

mitochondrial control can be exerted (Klingenberg 1969; Holian, Owen et al. 1977; Veech,

Lawson et al. 1979). Mitochondrial respiration is directly regulated by [ADP] or by the

phosphorylation ratio [ADP/[Pi]/[ATP] and the total creatine content only functions to buffer

changes in ATP/ADP (Blei, Conley et al. 1993; Kemp, Thompson et al. 1994). An increase in

the phosphorylation ratio is more typical of energy demanding states such as exercises.

Specifically, during exercises, elevations in [ADP] stimulate ADP entry into the mitochondria to

drive mitochondrial respiration (Dudley, Tullson et al. 1987; Houston 2006). The high

[ADP][Pi]/[ATP] ratios at rest, as observed in the present study, suggests an uncoupling of the

intracellular energy buffer system of the paralyzed hind limb muscle after one week of moderate

spinal contusion. As such, the elevated basal [ADP] is reflective of a heightened driving force

that most probably triggers respiration at rest after a contusion injury.

The [Pi]/[PCr] phosphorylation ratios are most altered at one week after injury and return

to baseline by two weeks of the injury. This reversal of metabolite adaptations of the paralyzed

rat gastrocnemius muscle to control values by two weeks after SCI is remarkable suggestive of

the extreme plasticity of the skeletal muscle in response to neural input; but not surprising.

Others and we have previously reported an acute response of the skeletal muscle to loss of

descending neural input and recovery from this loss in moderate spinal cord injury models

(Hutchinson, Linderman et al. 2001; Liu, Bose et al. 2006; Stevens, Liu et al. 2006; Liu, Bose et

al. 2008). These studies demonstrate marked atrophy of the paralyzed hind limb muscles as early

as one week after spinal contusion; that starts to recover by three weeks. Similarly, Liu et al have









shown acute response in MR relaxation properties as early as one weeks that recovers by three

weeks after injury (Liu, Bose et al. 2006). We are not aware of studies that have studied

mechanisms related to the reversal of skeletal muscle adaptations after spinal contusion injuries.

Nevertheless, given that moderate SCI involves partial loss of descending neural drive,

spontaneous motor and physiological recovery after injury is expected. In fact, the return of our

animal group to functional levels as evident by normal BBB scores at three weeks after injury,

support our speculations.

In conclusion, the cellular capacity for the phosphorylation potential in the paralyzed hind

limb muscle, as reflected in an uncoupling of the phosphorylation ratio at rest, is significantly

reduced until two weeks of moderate spinal contusion injury in rats. Whether this interferes with

the oxidative capacity of the muscle needs further investigation. We explore this inquiry in the

next experiment (Chapter 11).









Table 10-1. Absolute phosphate metabolite concentrations before and at various time points (one
week, two week, three weeks) after spinal cord contusion in rats. Data are expressed
as mean standard error.
Pre SCI Iwk SCI 2wk SCI 3wk SCI
[Pi](mM) 2.46+0.16 2.51+0.18 2.55+0.11 2.21+0.18
[PCr](mM) 26.95+0.84 23.41+1.13* 24.36+0.82 25.81+0.63
Pi/PCr 0.09+0.01 0.11+0.01 0.10+0.004 0.09+0.01
pH 7.14+0.01 7.18+0.01 7.17+0.01 7.17+0.01
[ADP] (tM) 38.10+3.53 57.85+4.81* 51.25+4.08 44.47+2.59
[ADP][Pi]/[ATP] x 106M 13.95+1.55 21.31+2.0* 19.26+1.32 14.44+1.14
*Statistically significant differences (p<0.05) between pre injury and lwk post SCI. Abbreviations: [Pi] = inorganic
phosphate; [PCr] = phosphocreatine; [ADP] = free cystolic adenosine triphosphate; [ADP] [Pi]/[ATP] =
phosphorylation ratio.





















340 -

10-
S1.71





0 1XXXX).

PI (mM) PCr (rM) PI/PCR Creatine (mM) ADP (mM) PP


-13.11
-20-


Figure 10-1. Percent change in resting phosphate metabolites of the rat hind limb muscle one
week after spinal cord contusion.



25.00 -



2 0.00-






10.00
2S.










Pre SCI 1wk SO 2A SCI 3wk SCI



Figure 10-2. Change in phosphorylation ratios [ADP][Pi]/[ATP] before and after spinal cord
contusion.


52.69


51.56


























Figure 10-3. Representative 31P-spectra before and after one week SCI obtained at 1 IT.


19.00 -

18.00
270-
17.00-
1a.oo -

15.00 -



B4.00 -

13.0 -

22.00 -

11.00


Pre S l wk SI


Sb


3wk SCI


2wk SOe


Figure 10-4. Change in [PCr] before and after spinal cord contusion. *Statistically significant
difference.


- mmmmm .....................














EPreSCI DlwkSCi
JD -


is-

E


u 2
C.



I0




1 2 a 4 S 6 7 a



Figure 10-5. [PCr] in individual rat hind limb muscle after one week of spinal cord contusion.









CHAPTER 11
EXPERIMENT SIX IN-VIVO ASSESSMENT OF SKELETAL MUSCLE BIOENERGETICS
AFTER SPINAL CORD CONTUSION IN RATS

11.1 Summary

Declines in skeletal muscle oxidative capacity following spinal cord injury (SCI) have the

potential to decrease exercise capacity and negatively impact muscle fatigability. Though altered

oxidative capacity after SCI appears to be well documented, most investigators have utilized in

vitro measurement techniques in their studies. 31P MRS offers a unique non-invasive alternative

of measuring oxidative capacity of skeletal muscle and is especially suitable for longitudinal

investigations. The purpose of this study was to determine the impact of spinal cord contusion on

the oxidative capacity of the rat hindlimb using 31P MRS. Eight young adult female rats were

moderately injured at the T8-T10 thoracic spinal cord. 31P MRS measurements were performed

at weekly intervals for assessments of oxidative capacity of the rat hindlimb muscle for three

weeks. Spectra were acquired in a Bruker 11T/470 MHz spectrometer using a 31P (190.5 MHz)

surface coil, placed over the belly of the gastrocnemius muscles. The sciatic nerve was

electrically stimulated by subcutaneous needle electrodes with a frequency of 1Hz and a Ims

duration. Kinetic data were collected before, during and after EMS. The PCr area during

recovery was fit to a single exponential curve, and the psuedo-first-order rate constant for PCr

recovery (kpcr) was determined. As compared to control rats, spinal cord injured rats at one week

showed markedly faster PCr depletions rates. Additionally, PCr recovery rates were significantly

declined after one week of SCI and regained within two weeks after the injury.



11.2 Introduction

Paralysis or paresis of lower extremity muscles renders persons with SCI to early muscle

fatigue and increased energy demands for simple functional activities (Hopman, Dueck et al.









1998; Ulkar, Yavuzer et al. 2003). Skeletal muscle alterations following SCI have the potential

to significantly impact daily functional motor performance and locomotor capabilities of persons

with SCI that ultimately culminates into long-term disability (Yakura, Waters et al. 1990;

Gordon and Mao 1994; Wang, Hiatt et al. 1999). Specifically, declines in skeletal muscle

oxidative capacity following the injury have the potential to decrease exercise capacity and

negatively impact muscle fatigability (Wang, Hiatt et al. 1999; Bhambhani, Tuchak et al. 2000).

Muscle oxidative capacity is defined as the ability of muscle mitochondria to synthesize ATP

and is a function of the intrinsic mitochondrial volume, mitochondrial enzyme activation,

cystolic redox carriers in mitochondria and vascular oxygen delivery and substrate to the

mitochondria (Kemp, Sanderson et al. 1996; McCully, Mancini et al. 1999; Kemp, Roberts et al.

2001). A variety of studies have shown drastic declines in muscle enzyme activity of paralyzed

skeletal muscles after complete SCI in humans (Kjaer, Mohr et al. 2001) and in spinalized

animal models (Jiang, Roy et al. 1990; Gregory, Vandenborne et al. 2003). In addition, an

overall decrease in the mitochondrial DNA content, capillary density and blood flow of the

paralyzed muscle, along with declines in skeletal muscle oxygen uptake following exercise are

reported following chronic SCI (Scelsi, Marchetti et al. 1982; Barstow, Scremin et al. 1995;

Wang, Hiatt et al. 1999; Bhambhani, Tuchak et al. 2000). Unanimously, these alterations are

observed in relatively faster muscles such as the gastrocnemius and vastus lateralis, with

relatively no metabolic alteration in the slow soleus muscle (Roy, Talmadge et al. 1998; Otis,

Roy et al. 2004).

Though altered oxidative capacity after SCI appears to be well documented, most

investigators have utilized in vitro measurement techniques in their studies. These assessments

are not only invasive, but suffer from their inability to yield bio-energetic data from a









functioning muscle in real time. As a result, kinetic changes in muscle metabolism are not best

represented using invasive techniques. Moreover, longitudinal follow-up of assessments from the

same tissue becomes practically infeasible. Though some investigators have used non-invasive

measures such as maximum oxygen consumption (VO2max) as markers of oxidative capacity

(McCully, Fielding et al. 1993; Wang, Hiatt et al. 1999), VO2max measures are more global and

do not characterize specific muscle metabolic states. Given that skeletal muscle is largely

effective in maintaining adequate oxidative ATP formation under most conditions (of rest and

exercise), directly studying the cellular events that provide the substrates for ATP synthesis in

muscle can prove invaluable.

Magnetic resonance spectroscopy (MRS) seemingly overcomes the limitations that are

posed by invasive techniques. In particular, the high spectral resolution (around Is) enables

kinetic assessment of metabolites in real time. In this respect, phosphorus MRS (31P-MRS) has

gained tremendous momentum in measuring in-vivo muscle oxidative capacity. One of the most

reliable measures of oxidative capacity using 31P-MRS is the rate of PCr recovery after exercise.

Studies have established well that the time scale and rates of PCr recovery after exercise

intimately match with the time scales and rates of oxygen consumption in the mitochondria

following exercise; thereby conferring that PCr resynthesis after exercise is mediated via

oxidative phosphorylation (Piiper and Spiller 1970; Meyer 1988; McCully, lotti et al. 1994;

Thompson, Kemp et al. 1995). Accordingly, PCr recovery rates have been conventionally

recognized as a non-invasive index of mitochondrial oxidative capacity and extensively used in

both healthy and diseased muscles as estimates of muscle oxidative capacity (Levy, Kushnir et

al. 1993; Paganini, Foley et al. 1997; McCully, Mancini et al. 1999; Argov and Arnold 2000;

Kent-Braun and Ng 2000; Pathare, Vandenborne et al. 2007).









The overall purpose of this study was to non-invasively assess muscle bioenergetics of

hind limb muscles after spinal cord contusion in rats. Specifically, we performed longitudinal

assessment of the oxidative capacity of rat gastrocnemius muscle for three weeks after spinal

contusion using an electrical stimulation protocol and 31P-MRS measures.

11.3 Specific Aims and Hypothesis

11.3.1 Specific Aim

a) To determine the impact of acute spinal contusion (one week) on oxidative capacity of

rat hind limb muscle after spinal cord contusion. b) To longitudinally monitor alterations in the

skeletal muscle oxidative capacity of the hind limb muscles.

Phosphorus magnetic resonance spectroscopy (31P-MRS) was performed on the animal

hind limb muscle using an electrical stimulation protocol to quantify in-vivo muscle

bioenergetics in real time. Measurements were obtained once weekly for three weeks starting at

one week post injury.

11.3.2 Hypotheses

a) Immediately after spinal cord contusion (one week), there is a decrease in the oxidative

capacity of the paralyzed hind limb muscle.

b) Muscle oxidative capacity approach recovery by three weeks of spinal cord contusion.

11.4 Methods

11.4.1 Experimental Design

Two groups of randomly distributed adult rats were studied. Animals either underwent a

spinal cord contusion (n=8) or served as controls for wet lab procedures relevant to the study (see

below). The spinal injured group underwent 31P-MRS measurements prior to injury and at

weekly intervals for three weeks after injury.









11.4.2 Animals

Sixteen adult Sprague Dawley female rats (12 week, 228-260g; Charles River, NJ) were

housed in a temperature controlled room at 210C with a 12:12 hours light: dark cycle and

provided with rodent chow and water ad libitum. Of these, 8 rats were moderately injured at the

T8-T10 thoracic spinal cord levels and 8 rats served as controls in providing healthy muscle

tissue to run biochemical assays for the quantification of phosphate compounds. The control

group also provided reproducibility results of our 31P data. All experimental procedures were

performed in accordance with the U.S. Government Principle for the Utilization and Care of

Vertebrate Animals by approval of the Institutional Animal Care & Use Committee at the

University of Florida.

Details about spinal cord contusion operation procedures have been described in detail in

chapter 5 (Section 5.2.2)

11.4.3 Data Collection

Combinations of an electrical stimulation and 31P-MRS were used to determine the in-vivo

skeletal muscle oxidative capacity of the rat gastrocnemius muscle before and after spinal cord

contusion injuries.

11.4.3.1 Experimental electrical stimulation protocol

An electrical muscle stimulation protocol was adopted to determine the mitochondrial

oxidative capacity of the rat hind limb muscle in-vivo. Animals were anesthesized using gaseous

isoflurane in oxygen (3% box induction), and maintained at 0.5%-2.5% during the MR

procedures. The limb was shaved and cleaned with alcohol and a circular 31P (190.5 MHz) tuned

surface coil was placed over the belly of the gastrocnemius muscle. A 1H surface coil was placed

underneath the hind limb to perform swimming. Two needle electrodes were placed

subcutaneously one over the region of the third lumbar vertebrae and the other land marked









over the greater trochanter to stimulate the hind limb plantarflexor muscles via stimulation of

the sciatic nerve. Electrical stimulation was carried out for four to six minutes to deplete PCr by

-30 to -40%. A Grass Stimulator (Quincy MA) with a Grass Model SIU8T stimulation isolation

unit (Grass Instruments, West Warwick, RI) was used to deliver a monophasic, rectangular pulse

with a 1ms pulse duration, 1Hz frequency and 10V. Following the electrical stimulation, the

muscle was allowed to recover for twenty minutes. During the entire duration of stimulation and

recovery, spectra were collected. No attempts were made to synchronize the radiofrequency

pulse with the muscle stimulation. The FIDs were multiplied by an exponential corresponding to

a 25Hz line broadening. Vital signs of the animal were monitored throughout the experimental

procedure.

11.4.3.2 31P magnetic resonance spectroscopy

The above electrical stimulation protocol along with combination of 31P-MRS was used to

assess the oxidative capacity of the hind limb rat muscle in a Bruker 1 1Tesla/470 MHz

spectrometer. A 1.5 x 1.7 cm oval surface coil tuned to 31P (190.5 MHz) was placed over the

belly of the gastrocnemius muscle. A 3-cm standard 1H surface coil was placed underneath the

hind limb to perform swimming and the animal's hind limb was extended such that the calf

muscles were centered over the surface coil. Spectra were acquired with a 50 |s square pulse, a

TR of 2s, spectral width of 10,000 Hz, 10 averages and 8000 complex data points. Spectra were

averaged into 20s bins and acquired at rest (5 min), electrical stimulation (4-6 min), and recovery

(20 min); thus amounting to a total of 93 fids. The partially relaxed spectra were then calibrated

by comparison with fully relaxed spectra (acquired at TR of 15seconds) to determine correction

factors (CF).









11.4.4 31P magnetic resonance spectroscopy: data analysis

The electrical stimulation protocol depletes PCr from skeletal muscle until stimulation is

on and MRS obtains this kinetic information in real time. Dynamic changes in PCr levels were

measured using complex principal component analysis (Elliott, Walter et al. 1999). Recovery

data were fitted to a single exponential curve, and the pseudo first-order rate constant for PCr

recovery (kPCr) was determined (Meyer 1988). End exercise [Pi] and [PCr] were measured by

area integration using 2D Bruker WinNMR data processing software. The maximal rate of PCr

resynthesis, a measure of mitochondrial oxidative capacity (Vmax-lin) was calculated based on kPcr

and baseline PCr values (Vmax-lin = kPCr. [PCr] rest) (Walter, Vandenborne et al. 1997). Initial rates

of PCr recovery (Vmeas mM/min, a direct measure of mitochondrial ATP synthesis) was

determined from the first three to four data points in recovery; depending upon the best linear

curve fit. The initial rate of PCr resynthesis was also extrapolated from the product of kpcr and

amount of PCr depletion (APCr). Thus, Vex (mM/min) = kpcr. APCr (Walter, Vandenborne et al.

1997).

Rates of PCr depletion at onset of stimulation (Vdep mM/min, a measure of ATP demand)

were determined from the first three to nine data points (first through three minutes) of PCr

declines during stimulation.

The maximum oxidative ATP synthesis rate (Qmax), which is a function of intrinsic

mitochondrial content and enzyme activity, oxygen and substrate supply to the mitochondrion,

and cytosolic redox state (Kemp, Sanderson et al. 1996), was calculated from the known

hyperbolic relationship between kpcr and cytosolic free [ADP] and from kpcr and

[ADP][Pi]/[ATP].









Qmax-ADP Vmeas (1 + Km/[ADP]) mM/min, where Km is the Michaelis constant and is

assumed as 50 [tM for rat leg muscle (Thompson, Kemp et al. 1995).

Qmax-[ADP][Pi]/[ATP]= Vmeas (1 + Km/[ADP][Pi]/[ATP]) mM/min, where Km is assumed as

0.11 mM.

11.4.5 Biochemical Assays

Details of biochemical analyses are described in chapter 5 (Section 5.2.5.1).

11.4.6 Statistical Analysis

Repeated measures ANOVA were used to statistically compare outcome measures from

kinetic data at rest, during electrical stimulation and during recovery. We compared the three-

minute Vdep, end exercise metabolite concentrations, Vmeas, Vex, kpcr and maximum

mitochondrial capacities (Vmax measures) and oxidative ATP synthesis rates (Qmax measures) of

the rat hind limb muscle prior to and along three weeks of spinal cord contusion. All hypotheses

were tested at an alpha level of 0.05. Post-hoc Bonferroni corrections were used for multiple

outcome comparisons. Analyses were performed using SPSS for Windows, Version 13.0.1.

11.5 Results

Dynamic relative changes in phosphate peaks in response to our stimulation protocol are

represented in Figure 11-1. Overall, results of our data show drastic differences between the pre

and post-injury outcome measures at one week after SCI (Table 1). Figure 11-2 shows the

average depletion and recovery graphs before and after one week of injury. For purposes of data

presentation, average results of our kinetic data are described separately in three steps as data at

onset of stimulation, at end exercise and during recovery.

Kinetic changes at onset of EMS: The absolute rates of PCr depletion (Vdep) at three

minutes of electrical stimulation were significant faster (p=0.02, by -30%) after one week of

spinal contusion than before injury (Figure 11-3, 11-4). Vdep at earlier times (one and two









minutes) did not reach statistical significance (Figure 11-3). While before injury, the time

required to deplete PCr was six minutes, after injury the PCr depleted to similar or more amounts

in around 4 minutes (Figure 11-4).

Kinetic changes at the end of EMS: The percentage PCr drop in the injured group was

lower by 39% in the injured group as versus the percentage drop before injury (0.41+2 % of

resting values versus 303% of resting values, p=0.022). Delta [PCr] values were however not

different between groups (Table 11-1). Due to large differences in baseline [PCr] after spinal

contusion injury at one week, the end stimulation [PCr] was lower by 30% (p=0.012) and end

stimulation [ADP] greater by 67% (p=0.021) after one week of injury (Table 11-1). Additionally,

this significantly affected end exercise [ADP][Pi]/[ATP] ratios (p = 0.011). The end pH after

stimulation was lower in the one week injured group by 0.05 units as compared to before injury

(6.98 versus 7.02 pH units). Importantly, end pH did not decrease by more than 0.2 units as

compared to before stimulation throughout the experiment.. End stimulation [Pi] + [PCr] taken

together were not different from baseline values of [Pi] + [PCr] taken together.

Kinetic changes during recovery: Following one week of spinal contusion, there was a

significant reduction (-24%) in kpcr measures of the paralyzed gastrocnemius muscle (p=0.001)

(Figure 11-5). However, no significant alterations were seen in Vmeas and Vex measures.

Consequently, maximum mitochondrial capacities (Vmax, Vmeas and Vex) and maximum

mitochondrial ATP synthesis rates (Qmax) were either significantly different or unaltered after the

injury (Figure 11-6, Figure 11-9, and Table 11-1). Moderate correlation (r=0.5) was seen

between Vmax and Vdep (Figure 11-7) and no correlation observed between end [ADP] and Vmeas

(r=0.016) (Figure 11-8).









All the measures that changed at one week, returned to near baseline values by three weeks

after injury. Biochemical assay results showed that there were no significant differences in the

[ATP] and [TCr] in the gastrocnemius muscle before and after spinal contusion injury.

Biochemical analyses revealed no differences in the total creatine and ATP content of

gastrocnemius muscle before and after injury. The TCr content (meanSEM) values from

gastrocnemius muscle assays were 44.22.6 mM and 44.23.9 mM for the control and injured

group respectively. The ATP content was 4.620.44 itmol/g wet weight (- 6.72mM) and

4.630.77 tmol/g wet weight (-6.85mM) from the control and injured gastrocnemius muscles

respectively.

11.6 Discussion

In the present work, we utilized combinations of an electrical stimulation protocol and 31p_

MRS to determine the in-vivo skeletal muscle bioenergetics of the rat hind limb muscle during

exercise after moderate thoracic spinal cord contusion injury. The key results of our study show

that there is a considerable elevation (-30%) in the PCr depletion rate (Vdep) and a decrease

(-24%) in the PCr resynthesis rate constants (kpcr) of the paralyzed muscle at one week after

injury that return to near normal values by three weeks. Our data suggests that there is a

significant imbalance in the energy producing and/or consuming states of the paralyzed muscle

along with a decrease in the overall mitochondrial oxidative capacity for oxidative

phosphorylation after one week of contusion injury.

An important and novel finding of our present study is that at one week after spinal cord

contusion, PCr depletes faster and to relatively lower levels than the pre-injury levels.

Specifically, with the protocol used in the study, the Vdep at one minute after electrical

stimulation is similar before and at after one week after the injury. However, by two minutes of

electrical stimulation, the PCr levels of the contused group begin to drop more rapidly and lower









as compared to that seen before the contusion. With the electrical stimulation parameters used in

the present study, duration of minutes was set to deplete the PCr content in our control group

(pre-injury) by -32%. However, after spinal contusion, we had to stop the stimulation at

minutes, because by this time the PCr had depleted by -42% and any further PCr depletion was

necessary to avoid. Moreover, while the control group reached a clear steady state by almost

three minutes into stimulation, the injured group never reached steady states. Parallel to our

findings, similar rapid and farther Vdep rates for a given exercise protocol have been documented

in a variety of disease states including chronic fatigue syndrome (Wong, Lopaschuk et al. 1992)

myotonic dystrophy (Taylor, Kemp et al. 1993), peripheral vascular diseases (Kemp, Hands et al.

1995), denervation (Hayashi, Ikata et al. 1997) and chronic heart failure (Toussaint, Kwong et al.

1996). We suppose that the likely mechanisms for the metabolic response of the paralyzed

muscle might be secondary to increases in ATP demand to perform similar work, a decrease in

local blood perfusion that impacts ATP synthesis via oxidative phosphorylation or a combination

of both. Our viewpoint is supported by the following explanations.

Homeostatic mechanisms within the myocyte couple overall ATP utilization with ATP

synthesis; thereby maintaining nearly steady concentrations of ATP during low intensity

exercises (Erecinska and Wilson 1982; Kushmerick 1995). Specifically, during muscle

contraction, increasing ATP demands in the myocyte are met by PCr breakdown via the creatine

kinase equilibrium reaction. This decrease in PCr content indicates the energy buffer role of PCr,

and consequently of the phosphorylation ratio ([ADP][Pi]/[ATP]) in response to ATP demands.

Accordingly, the magnitude of change in PCr concentration reflects the demand for oxygen and

substrates, and PCr depletion rates are purported to measure the ATP needed to meet cellular

demands (Kemp, Hands et al. 1995; Toussaint, Kwong et al. 1996). The finding that more









amount of PCr is utilized for hydrolysis to ATP implies that the ATP demands for similar

intensities of muscle contraction are higher than pre-injury levels. Indeed, sufficient evidence in

literature reveals that the paralyzed skeletal muscle is predisposed to increased fatigability,

deconditioning, declines in isometric force production over repetitive bouts of contraction and

decreases in muscle endurance (Gerrits, De Haan et al. 1999; Shields 2002). Consequently, the

paralyzed muscle will function less economically than normal and require more energy to

perform a given contraction. Moreover, increases in ATP consumption of paralyzed muscle can

also result from ATP consuming events in the tissue such as muscle atrophy (Erkintalo,

Bendahan et al. 1998) and the uncoupling of oxidative phosphorylation (Taylor, Kemp et al.

1993; Scheuermann-Freestone, Madsen et al. 2003) both of which are muscle adaptations after

spinal cord contusion that have been presented separately from past data in our lab (Liu, Bose et

al. 2008) and in the current study respectively. Taken together, our findings of an apparent

increase in ATP closely matches with the functional impairments posed by paralyzed muscles

and most likely provides a mechanistic explanation to those findings. However, confirmation

from actual measurements of the energy cost of contractions by the amount of ATP produced for

a given power output (Russ, Elliott et al. 2002) in the rat model of moderate spinal contusion are

warranted.

Another explanation for the faster Vdep after SCI is an apparent decrease in the local blood

perfusion that interferes with ATP synthesis via oxidative phosphorylation during muscle

contraction. During steady state exercises, there are increases in oxidative phosphorylation rates;

which are supplemented by an increase in the local blood perfusion. Marro et al have shown in

the rat hind limb muscle that during a range of low-level contractile activity, the declines in PCr

concentrations are the mirror image of the perfusion increases (Marro, Olive et al. 2007). A









correlation exists between energy metabolism and oxygen supply that is fueled by local blood

circulation in rat skeletal muscles during exercise (Idstrom, Subramanian et al. 1984; references

from Olive, Dudley et al. 2003). Steady state exercises are met by an initial rapid increase in

blood flow till oxygen delivery matches the exercise demands and then plateaus (Kushmerick

1981; Van Beekvelt, Shoemaker et al. 2001). Studies have shown that these initial rapid

increases in blood flow, in turn, depend upon muscle contractile efficiency and vasomotor tone

of the capillary pool (Tschakovsky, Shoemaker et al. 1996; Van Beekvelt, Shoemaker et al.

2001). Noticeably, these data imply that an inadequate blood flow during exercise will result in

inadequate delivery of oxygen to the contracting muscle cells and limit the ability of

mitochondria to produce ATP via oxidative phosphorylation denervationn paper). Indeed, after

SCI in humans, Olive et al have shown that there is an approximate five-fold increase in the time

to peak blood flow and a significant delay in the blood flow response at the onset of muscle

stimulation in persons with chronic SCI (Olive, Dudley et al. 2003). This increased time of blood

delivery, in turn, significantly compromises the ability of the paralyzed muscle to meet the

energy demands of muscle contraction secondary to electrical stimulation. Various mechanisms

for this deficiency in blood flow have been addressed including alterations in the vasomotor

tone, decreased contractile ability of the paralyzed muscle to deliver blood, decreased oxidative

capacity secondary to shift in muscle fiber type composition towards fast glycolytic fibers and a

decreased hyperemic response to muscle contractions (Olive, Dudley et al. 2003). Similar to

humans, the paralyzed mammalian rat muscle exhibits significant levels of muscle atrophy

(Hutchinson, Linderman et al. 2001; Liu, Bose et al. 2008), increase in the number of type II

glycolytical fibers of the paralyzed hind limb muscles (Stevens, Liu et al. 2006), alterations in in-

vitro measures of oxidative capacity (Gregory, Vandenborne et al. 2003), altered contractile









properties (Stevens, Liu et al. 2006) and vasomotor insufficiency (Guizar-Sahagun, Velasco-

Hernandez et al. 2004; Laird, Finch et al. 2008). Lastly, other disease states (including

denervation, heart failure and peripheral nerve diseases) that have shown sizably compromised

Vdep rates using similar in-vivo measures as ours, have demonstrated that the local perfusion to

the involved muscle is compromised; thereby suggesting that the oxygen delivery to the muscle

is a likely contributor of the abnormal metabolic response seen (Kemp, Hands et al. 1995;

Toussaint, Kwong et al. 1996; Hayashi, Ikata et al. 1997). In view of these findings, we deduce

that inherent characteristics of the paralyzed muscle potentially interfere with the local blood

perfusion, which in turn hampers the oxygen supply to the exercising muscle. Consequently,

ATP production from oxidative phosphorylation during exercise is compromised and is reflected

as faster Vdep rates.

Another principal finding of our study is that the rate constants of PCr recovery (kpcr) are

significantly decreased after one week of spinal contusion. Studies have well established that

during recovery from exercise, glycolysis ceases and PCr in the muscle cell is replenished at the

expense of ATP produced via mitochondrial oxidative phosphorylation (Taylor, Bore et al. 1983;

Meyer 1988; Quistorff, Johansen et al. 1993; Kemp, Roberts et al. 2001; Mattei, Bendahan et al.

2004). Accordingly, kpcr has been extensively used in both healthy and diseased muscles as

estimates of muscle oxidative capacity (Meyer 1988; Paganini, Foley et al. 1997; McCully,

Mancini et al. 1999; Argov and Arnold 2000; Kent-Braun and Ng 2000; Pathare, Vandenborne et

al. 2007). The declines in kpcr of the paralyzed muscle in the present study are parallel to various

disease states including the rat model of spinal transaction (Durozard, Gabrielle et al. 2000),

complete SCI in humans (Levy, Kushnir et al. 1993; Hartkopp, Harridge et al. 2003), peripheral

vascular diseases (Kemp, Hands et al. 1995) and models of muscle disuse including









immobilization in mice (Pathare, Vandenborne et al. 2007) and hind limb suspension in rats

(Yoshida, Ikata et al. 2001). In comparison with the percentage decline in the PCr recovery rates

of the paralyzed hind limb muscle after spinal contusion (-33 % decline in T1/2 recovery rates at

one week), an almost 134% decline in T1/2 is reported after 60 days of spinal cord transaction.

We believe that these discrepancies are largely due to the difference in severities of the two types

of cord injuries. Moreover, unlike our study, the pH in the transaction study was not controlled.

As a result, intracellular acidosis at the end of stimulation might have slowed down the rates of

PCr recovery after spinal transaction. When measuring oxidative capacity using MRS, it is

essential that the muscle does not get acidotic. This is because pH declines below 6.75 units

(Taylor, Bore et al. 1983; McCully, lotti et al. 1994; Boesch 2007) significantly impacts kpcr

rates and cannot adequately reflect skeletal muscle oxidative capacity. In our present study, the

end stimulation pH was 6.9 and did not decrease by more than 0.2 units as compared to the

baseline pH. We could achieve this by trading against shorter periods of electrical stimulation in

our injured group. Importantly, our control data show similar kpcr as those reported in literature

from healthy rat gastrocnemius muscles (Meyer 1988; Paganini, Foley et al. 1997). Collectively,

the decline in kpcr r and hence the Vmax measures in our present study suggest an overall decrease

in mitochondrial oxidative capacity of the paralyzed rat gastrocnemius muscle.

The maximum oxidative ATP synthesis rate (Qmax), is a function of intrinsic mitochondrial

content and enzyme activity, oxygen and substrate supply to the mitochondrion, and cytosolic

redox state (Kemp, Sanderson et al. 1996). Cytoplasmic [ADP] plays a crucial role in the

respiratory control in skeletal muscle; such that the Qmax largely depends on [ADP] in a

Michaelis-Menten fashion (Chance, Leigh et al. 1985; Kemp and Radda 1994; Paganini, Foley et

al. 1997). According to this model, Qmax derived from the known hyperbolic relationship









between kpcr and cytosolic free [ADP] adequately represents maximum oxidative ATP synthesis

rate (Paganini, Foley et al. 1997). Following one week of spinal cord contusion, various indices

of the PCr resynthesis rates including Vmax, Qmax-[ADP] and Qmax-[ADP][Pi]/[ATP] are reduced; thereby

suggesting that mitochondrial ATP production is considerably impaired in a paralyzed muscle. In

spite of an elevation in the end stimulation mitochondrial driving force ([ADP][Pi]/[ATP]), we

encountered a significant decline in the Qmax measures in our one week injured group. This most

likely suggests that the driving force is unable to trigger an adequate mitochondrial response in

enhancing oxidative phosphorylation. We believe that this is most likely due to a possible

uncoupling between oxygen fluxes in the mitochondria and subsequent ATP synthesis rates. In

fact, the finding from our present study that lower maximum mitochondrial capacity rates (Vmax)

accompany faster rates of PCr depletion rates (good negative correlation between Vmax and Vdep,

r=0.5, Figure 11-7) supports our view.

Our finding that the mitochondrial oxidative capacity of the partially paralyzed skeletal

muscle is compromised, is in concurrence with in-vivo and in-vitro models of complete SCI in

both humans and animal models. In an animal model of spinal transaction, Durozard et al have

used non-invasive magnetic resonance spectroscopy to demonstrate declines in oxidative

capacity of the gastrocnemius muscle in rats. Based upon drastic decreases in both kpcr and Qmax

measures, the authors suggest transition in the source of energy supply from oxidative to

anaerobic pathways for muscle metabolism following complete paralysis of the rat hind limb

muscle (Durozard, Gabrielle et al. 2000). Additionally, in vitro studies show that mixed muscle

such as the gastrocnemius and the vastus lateralis has significantly lower mitochondrial enzyme

activity following spinalization in both cats and rats (Jiang, Roy et al. 1990; Gregory,

Vandenborne et al. 2003). In addition, an overall decrease in the mitochondrial DNA content,









capillary density and blood flow of the paralyzed muscle, along with declines in skeletal muscle

oxygen uptake following exercise are reported following chronic SCI (Scelsi, Marchetti et al.

1982; Barstow, Scremin et al. 1995; Wang, Hiatt et al. 1999; Bhambhani, Tuchak et al. 2000).

Various mechanisms including skeletal muscle atrophy, change in muscle fiber type

composition, muscle inactivation and muscle deconditioning (as reflected in decreased functional

activity and limitation of exercise tolerance to daily activities) have been recognized as

precipitating factors in compromising the oxidative capacity of the paralyzed skeletal muscle. In

fact, since mitochondrial function can be impacted under low perfusion conditions such as those

experienced in peripheral vascular disease (Isbell, Berr et al. 2006; Greiner, Esterhammer et al.

2007; Kramer 2007) and heart failure (Toussaint, Kwong et al. 1996), deficits in local blood

perfusion after SCI (Olive, Dudley et al. 2003; Guizar-Sahagun, Velasco-Hernandez et al. 2004)

as a potential contributor to mitochondrial oxidative capacity cannot be ruled out.

Similar to rate constants of PCr recovery, initial rate of PCr recovery (Vmeas) has been

demonstrated to reflect mitochondrial oxidative capacity (Meyer 1988; Kemp, Thompson et al.

1994; Lodi, Kemp et al. 1997) and more specifically is an estimate of the end-exercise rate of

oxidative ATP synthesis (Kemp G, Hands L et al 1995). To our surprise however, we do did not

encounter simultaneous declines in Vmeas from our data at one-week post injury. We believe that

several factors could account for this discrepancy a) A decreased sensitivity of Vmeas as versus

kpcr: kpcr is calculated after fitting several points in recovery (60 fids in our data set) to a single

exponential function; whereas Vmeas is estimated from the first two-three data points in recovery.

Accordingly, spectral noise from Vmeas calculations leads to larger variability in Vmeas

calculations (CV=24%) than the kpcr calculations (CV=21%); thereby rendering Vmeas as a

relatively less sensitive measure. b) Biexponential function of kpcr recovery rates: Vmeas values









are based on a linear model of recovery while kpcr is extrapolated from a monoexponential

function. Given that both measures reflect similar physiological mechanisms in the skeletal

muscle, it is tempting to question if the recovery curves follows a biexponential function.

However, from careful visual inspection of our PCr recovery graphs (that show excellent

monoexponential fits; R2= 0.97 0.99) we do not believe that to be the case. c) End stimulation

elevations in [ADP]: At the end of electrical stimulation, the [ADP] is significantly elevated in

injured animals as versus before injury. Studies have shown that elevated [ADP] levels serve as a

strong respiratory drive (Kushmerick 1981; Paganini, Foley et al. 1997) and exert an influence in

enhancing the rates of PCr recovery via increased ATP synthesis. In fact, for similar rates of

Vmeas, we can infer from the Michelis-Mentel relationship between Vmeas and [ADP] that the

maximum rate of oxidative ATP synthesis (Qmax) should be higher in our injured group than

before SCI. However, our data show tendencies towards a decline in the Qmax measures when

predicted from both the Vmeas and Vex measures. Additionally, our data do not show any

correlation between end [ADP] and Vmeas (Figure 11-8). Collectively, we believe that the Vmeas

rates are most likely the result of an increased variability in our measurements.

The Vdep and Vmax measures in our study are predominantly altered at one week after injury

and return to baseline by two weeks of the injury. This reversal of metabolite adaptations of the

paralyzed rat gastrocnemius muscle to control values by two weeks after moderate spinal

contusion injury is remarkable suggestive of the extreme plasticity of the skeletal muscle in

response to neural input but not surprising. Others and we have previously reported an acute

response of the skeletal muscle to loss of descending neural input and recovery from this loss in

moderate spinal cord injury models (Hutchinson, Linderman et al. 2001; Liu, Bose et al. 2006;

Stevens, Liu et al. 2006; Liu, Bose et al. 2008). These studies demonstrate marked atrophy of the









paralyzed hind limb muscles as early as one week after spinal contusion; that starts to recover by

three weeks. Similarly, Liu et al have shown acute response in MR relaxation properties as early

as one weeks that recovers by three weeks after injury (Liu, Bose et al. 2006). We are not aware

of studies that have studied mechanisms related to the reversal of skeletal muscle adaptations

after spinal contusion injuries. Nevertheless, given that moderate SCI involves partial loss of

descending neural drive, spontaneous motor and physiological recovery after injury is expected.

We recognize potential limitations of the current study. We did not obtain force mechanics

data in these experiments to determine the intensity of stimulation voltage used for our muscle

stimulation protocol. However, the voltage was adjusted to give maximum contraction of the

gastrocnemius muscle as confirmed by palpation of the muscle and the stimulation parameters

kept constant throughout the experiment. Moreover, the protocol we used in the study is well

documented in literature and is established to stimulate the gastrocnemius, soleus and plantaris

muscle groups (Meyer, Brown et al. 1985; Meyer 1988; Paganini, Foley et al. 1997). In fact,

results from our pilot study show that the coil penetration was enough to obtain signal,

predominantly from the gastrocnemius muscle. Another reservation of the study is that fiber

atrophy might have caused the signal to bleed from surrounding slow muscle tissue (such as the

soleus); in which case the kpcr tend to slow down. However, the soleus muscle is a very small

part of the rat gastrocnemius-plantaris-soleus muscle complex and we expect minimal

contribution, if any, of the soleus muscle to our spectral signal. Moreover, previous reports from

our lab have demonstrated that muscle atrophy continues for at least two weeks after moderate

spinal contusion (Stevens, Liu et al. 2006; Liu, Bose et al. 2008). Since kpcr in our present study

recover to normal at two weeks after injury in spite of the atrophy, the lower recovery rates due

to atrophy alone are most likely ruled out. Lastly, in extrapolating the in-vivo absolute









concentrations of phosphate metabolites, the in-vitro [ATP] and [TCr] measures obtained at three

weeks after injury were assumed to be constant at all time points after injury. However, based

upon studies in complete spinal cord transaction in rats, Durozard et al do not show any

alterations in the [ATP] in the paralyzed gastrocnemius muscle at day 7, 30 and 60 after the

injury (Durozard, Gabrielle et al. 2000). Whether muscle [TCr] changes along the course of

spinal cord contusion injury needs to be verified in future studies.

In conclusion, results of our study show striking declines in the maximum mitochondrial

capacity and alteration in the ATP supply and/or demand characteristics of the paralyzed

gastrocnemius after one week of spinal cord contusion. By using in-vivo magnetic resonance

spectroscopic measures in the study, we have been able to circumvent limitations posed by

invasive and global measures of assessing the oxidative capacity of skeletal muscle. Specifically,

the non-invasiveness of 31P-MRS enabled us to longitudinally assess the cellular events that

define muscle oxidative capacity in real time and with high spectral resolution. Overall, our data

suggests an oxidative metabolic defect in the paralyzed hind limb muscle that might potentially

contribute to the host of motor dysfunctions seen after SCI (Waters, Adkins et al. 1994; Ulkar,

Yavuzer et al. 2003). Our findings support the potential usefulness of therapeutic interventions

aimed at improving aerobic muscle metabolism after this injury.










Table 11-1. Kinetic 31P-MRS data from the rat gastrocnemius muscle. Average data
(meanstandard error) during exercise and recovery from exercise is presented before
and for three weeks after moderate spinal contusion injury (n=8).
Pre SCI lwk SCI 2wk SCI 3wk SCI


I) Data at onset of EMS
3min [PCr] Vdep (mM.min-1)
Baseline [PCr] + [Pi] (mM)

II) End EMS data
End ex pH
End ex [PCr] (mM)
End ex [Pi] (mM)
End ex [ADP] (jiM)
End ex [ADP][Pi]/[ATP]
Delta [PCr] (mM)
Percentage PCr drop


-2.440.18
29.40+0.86



7.020.01
21.791.25
9.880.81
47.11+4.46
67.77+7.18
6.480.88
302.7%


III) Recovery data
a) PCr recovery constants
kpcr (min-1) 0.650.05
1/k (s) 96+8
Initial recovery rate (min-1) 0.240.02
b) Mitochondrial Oxidative Capacity (V)
Vmax-in (mM.min-1) 17.521.52
Vmeas (mM.min-1) 6.490.65
Vex (mM.min-1) 4.38+0.75
c) Oxidative ATP synthesis rates (Qmax)


Based on kpcr
Qmax-ADP (mM.min-1)
Qmax-ADP*Pi/ATP (mM.min-1)
Based on Vmeas
Qmax-ADP (mM.min-1)
Qmax-ADP*Pi/ATP (mM.min-1)
Based on Vex
Qmax-ADP (mM.min-1) (Vmeas)
Oma.-ADP*Pi/ATP (mM.min-1)


27.341.98
47.884.67

10.271.03
17.56+1.72

5.701.30
9.89+2.32


-3.160.18*
25.92+1.23*



6.970.02*
15.240.96*
9.590.76
78.678.02*
110.1212.51*
8.170.69
412%*


0.500.06*
13417*
0.260.01

11.641.42*
6.130.40
3.960.50



15.942.24*
24.933.79*

8.390.85
12.71+1.34*

5.390.75
8.41+1.30


-2.420.22
26.91+0.89



6.990.02
19.551.24
9.030.87
53.895.31
74.3812.97
5.740.99
354%


0.670.05
948
0.280.06

16.251.28
5.901.71
3.600.51



24.422.15
46.216.11

8.462.31
16.09+2.99

4.570.92
8.59+0.98


-2.680.22
28.01+0.67



7.000.01
19.030.50
9.520.68
54.432.55
77.066.25
7.090.71
344%


0.630.07
10818
0.270.01

15.971.59
6.960.31
4.330.66



23.142.34
39.41+4.69

10.220.45
17.28+0.84

6.280.91
10.48+1.46


*Statistically significant differences (p<0.03) between pre injury and lwk post SCI. Abbreviations: Vdep = PCr
depletion rate at onset of exercise; [Pi] = inorganic phosphate; [PCr] = phosphocreatine; [ADP]= cystolic adenosine
triphosphate; Delta [PCr] = resting [PCr] end exercise [PCr]; kPcr = rate constant; 1/k = time constant; Vmaxn-i=
mitochondrial oxidative capacity based on recovery rate constant; Vmeas = mitochondrial oxidative capacity based on










initial recovery rates; Vex = mitochondrial oxidative capacity extrapolated from rate constant and delta PCr; Qmax-ADp
= maximum oxidative ATP synthesis rates based on kpcr and [ADP]; Qmax-ADP*P/ATP = oxidative ATP synthesis rates
based on kPCr and phosphorylation potential.










PCr



ATP
6 Pi A Recovery at 20 min






4 \ A yJj End of EMS


3


1 /


A il~ ________


ASpectra at Rest


at is 10 s -5 -10 -1i -2a -as -3a
1
Figure 11-1. Representative kinetic 31P spectra of rat gastrocnemius muscle at 11T. Selective
spectra are shown from real-time in-vivo data obtained at rest (1,2), during electrical
stimulation (EMS) (3,4) and during recovery (5,6). Note the elevation in Pi at end of
EMS with concurrent decreases in PCr.











1.1 Rest Recovery



L 0.f -"






S0.6 wk



0.4
0 4 7 10 14 17 20 24
Minutes

Figure 11-2. Average PCr depletion and recovery graphs in response to the stimulation protocol.
Kinetic data during rest, electrical muscle stimulation (EMS) and recovery are
compared before and after one week of spinal cord contusion in eight rats.


C -
E
Z -2
E




c -4
1.


L --


I1


I1


* Pre SCI i1wk SCI E 2wk SCa N 3wk SCI


Figure 11-3. PCr depletion rates (1, 2, and 3min) before and after spinal cord contusion injuries
(SCI). *Significant differences in three-minute (3min) depletion rates are seen
between pre injury and Iweek SCI.


I













-9e-ProSC ---lwkSCI


1

0.9





3.-
u 0.6-

O.

0.4


EMS (mln)


Figure 11-4. Expanded view of PCr kinetic data obtained at the onset of electrical muscle
stimulation (EMS). PCr depletes earlier and farther after one week of spinal cord
contusion (SCI) than before injury.


I L


0.9

Q 08


S0.5

C. -.


-4- Pre SCI -A- infk SCI


I I I I I I I I I I I I I I
0 1 2 3 4 5 6 7 8 9 10 1 12 3 14 15 16 17
Recovery irmn EMS(m Inutes)


Figure 11-5. PCr recovery graphs obtained immediately after termination of electrical muscle
stimulation (EMS). kpcr are obtained after fitting the recovery graphs to a single
monoexponential function. Note that end stimulation PCr content is different before
and after injury.


1.21 -


o i : 4 i i
0 1 2 3 4 5 6












5 %d 2B% 35% 29% 3t3 47%


EPreStl *lwk SC


R3 Rd


R5 RG


R7 R$


Figure 11-6. Maximum mitochondrial oxidative capacity (Vmax mM.min-1) data from eight rats
(R1-R8). Percentages indicate declines in Vmax values after one week of spinal cord
injury.


Control
lwk SC
2wk SCI
3wk SCI


r=0.55
*


9
"9


-4.0 -3.5 -3.0 -2.5 -2.0
Vdep (rn M.mn n-1)


-1.5 -1.0 -0.5


Figure 11-7. Scatter-plot depicting relationship between maximum mitochondrial capacity
(Vmax) and rate of PCr depletion (Vdep). Data of all animals is pooled from each
time point (before SCI and 1, 2, and 3 weeks post injury).


30-


25



E




E

5.


0-


R1 R2


41%


7%a


;r,













R.2 0.0157 5
S iIm
*' *
~ ---- *i",


U 1
*


0.020 0.040 0.0650
End [ADP] (mp


0.B50 0.100 0.120


Figure 11-8. Relationship between end stimulation [ADP] and initial rates of PCr recovery
(Vex). Data of all animals is pooled from each time point (before SCI and 1, 2, and 3
weeks post injury).

G ,


I Pre SCr lwk SE

u wk, crI a 3wk SCI


LI


r


Qmax-ADP


Qmax-ADP*Pi/ATP


Figure 11-9. Maximal rates of oxidative capacity (Qmax) based on the [ADP] and
[ADP][Pi]/[ATP] models. *Significant declines are seen in the injured group at two
weeks after injury.


I F


04-.
0.000


E
E
i 40
E
x 30
E
"' 2
X
E
Cy in


0 4-









CHAPTER 12
CONCLUSION

Studying muscle adaptations in persons with incomplete SCI has been challenging because

of the "incomplete" nature of the injury and heterogeneity of this patient group. This dissertation

work demonstrates that despite the presence of relatively greater activity levels in persons with

incomplete spinal cord injuries (SCI), lower extremity muscles below the site of lesion exhibit

considerable motor impairments. Data from human studies in this work illustrate that the

incompletely paralyzed muscle displays marked muscle atrophy, significant fatty tissue

infiltration and a predisposition to muscle injury. These alterations are partially reversible by

physiologically based therapeutic interventions such as locomotor training. Similarly, data from

the animal studies demonstrate disturbances in the phosphorylation potential states and marked

alterations in the in-vivo mitochondrial metabolic capacity of the skeletal muscle after spinal

cord contusion in rats. An understanding of the underlying physiology of muscular adaptations

can assist clinicians and therapists in strategizing specific therapeutic interventions for this

patient cohort.

The present work will provide a foundation from which the relationship between skeletal

muscle adaptations and function in this population can be further explored. Moreover, the use of

sophisticated MR techniques will enable characterizing the paralyzed muscle non-invasively and

with high resolution; while also allowing longitudinal follow-ups all of which are crucial in

assessing injury mechanisms, disease progression and efficacy of therapeutic interventions.









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BIOGRAPHICAL SKETCH

Prithvi Shah was born in August 1978, in a small town of Gujarat state in India. At the age

of five years, Prithvi's parents sent her to a boarding school in western India. With a burning

desire to award the best education to their daughters and inculcate in them good English

speaking and writing skills, Mr and Mrs Shah let out their three girls from home for an

expedition of a lifetime.

Prithvi successfully completed her high school with distinction (equivalent to Honors in

the US) from St.Joseph's Convent School in Panchgani, Maharashtra. Having made up her mind

to have a career in the health sciences, she then attained her undergraduate degree in physical

therapy from Pune, India. While in the rehabilitation setting, her training encompassed

interactions with patients from diverse backgrounds. However, the fervor of understanding

mechanisms relevant to disease processes and strategizing therapies for patients led her to the

United States in pursuit of a higher education.

In her training as a doctoral student at the University of Florida, Prithvi has been a

recipient of numerous awards including scholarships for international students, nomination for

the best teaching assistant and research awards. Under the proficient tutelage of Dr. Krista

Vandenborne, Prithvi has had the unique opportunity to master her skills in both human and

animal research of spinal cord injury.

Her immediate short term goals vest in further exploring medical technology and learning

a variety of research tools to assess basic physiological processes concerning functioning of the

nervous system. Among the long term goals are pursuing an academic career. With a passion for

teaching and research, an academic career seems the right choice to make.

Prithvi Shah has a publication record of eight research articles (with two as the first author)

and looks forward to publications from her last two studies in the current dissertation.





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1 MAGNETIC RESONANCE CHARACTE RIZATION OF SKELETAL MUSCLE ADAPTATIONS AFTER INCOMP LETE SPINAL CORD INJURY By PRITHVI KRISHNAKANT SHAH A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2008

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2 2008 Prithvi Krishnakant Shah

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3 To My Uncle, Dinesh O Shah

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4 ACKNOWLEDGMENTS I can no other answ er make, but, thanks, and thanks. ~William Shakespeare I take this opportunity to thank the numer ous people involved in my journey towards obtaining a doctorate degree in this foreign land. This work, to a gr eat extent, is an outcome of the excellent professional guidanc e of several educators at the University of Florida and the unfaltering support, love and encouragement of my parents, family and friends. The contribution of my dissertation advisor, Dr Krista Vandenbor ne in chiseling my professional career throughout my doctorate studies is unparallele d. Equipped with her expertise and experience in the field, Dr.Vandenborne ha s guided and encouraged me to pursue the complex subjects of magnetic resonance (MR), mu scle physiology and animal studies with great persistence and simplicity. As a mentor, Dr. Vand enborne has introduced me to the concepts of being relentless yet patient and prolific while pr oducing high quality research. She has also been most influential in inculc ating into me the art of independent and scientific thinking. I next thank my teacher Dr. Andrea Behrman for first, givi ng me the opportunity to be a part of the Rehabilitation Science Program at the University and then, sharing with me her remarkable enthusiasm in the field of spinal cord injury research. Without the assistance of Dr. Glenn Walter, this dissertation work can be likened to a mechanic who possesses no knowledge of his tools. Discussions about MR a nd muscle physiology with Dr. Walte r have largely enabled me to better understand these concepts and implement complex theoreti cal knowledge into practical use. I also wish to thank Dr. David Fuller for his excellent ideas in en riching the quality of research in my work related to animal studies. His generosity and support in providing me with his lab resources are most appreciated. My co mmittee member, Dr. Lorie Richards, has been

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5 incredibly instrumental in direc ting me to think outside the box that helped this work take a more interdisciplinary approach. I wish to extend my special thanks to the pa st and present members in my laboratory To Chris, Jennifer, Arun, Mike, Shiv, Neeti and Sean for their assistance with the human studies; and to Min and Wendy for their excellent technical assistance with the animal experiments. The cheerful presence of Sunita, Donovon, Nathan, Fan and Ravneet made the endless hours in the laboratory far more enjoyable and science a lot mo re fun. I also wish to thank the Levine and Reier laboratories in the College of Medicine at the University of Florida for offering their resources to conduct part of th e animal studies. I exclusivel y thank Milap, Ravi, Geisha, Todd and Kevin for their help. I am most grateful to the Univ ersity of Florida for granting me the Grinter Scholarship for International students; to Dr. Teitelbaum for providing me a res earch assistantship during my first year as a doctorate student at the University of Florida; to the Department of Physical Therapy for assigning me a teachi ng assistantship to complete a major portion of my doctoral course work; and to the National Institutes of H ealth for the financial support awarded to Dr, Vandenborne for the present dissertation studies. My heartfelt thanks also go to my research subjects for their participation in the human studies. I extend my appreciation to the Department of Physical Therapys faculty and staff teams for creating an exceptionally dynamic and healthy learning environment and for maki ng my graduate student experi ence at the University most enjoyable. With genuine humbleness, I deeply thank all my friends who made the Unites States for me a home away from home. Sailing with me through times of frustration and nostalgia, and always attempting to encourage and inspire, thei r presence has proved matchless. I especially

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6 wish to convey my gratitude to Seemant, Rakesh Sheryl, Andrea, Shreya, Deepali, Avi, Jigesh, Preeti, Sunita, Surjit, and Bijoy. Without the unconditional love, strength and patience of my parents and sisters, this seemingly impossible task would not be made possible. Their enthusiasm and continual support often curtailed the perception of the thousands of miles distance from home. I wish to thank my parents, sisters and relative s for having the confiden ce in me and relentle ssly encouraging me to remain persistent with my research efforts. Lastly, I wish to thank my role model and unc le, Dr. Dinesh Shah, from the bottom of my heart. I dedicate this dissertation thesis to him. In the truest sense, he has been my mentor, guide, friend, and a parent cruising along with me thr ough the ups and downs of my doctorate studies. From him, I have learned much of life's philos ophy and in him I have witnessed the essence of a true scientist.

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7 TABLE OF CONTENTS page ACKNOWLEDGMENTS...............................................................................................................4 LIST OF TABLES................................................................................................................. ........12 LIST OF FIGURES.......................................................................................................................13 LIST OF ABBREVIATIONS........................................................................................................ 16 ABSTRACT...................................................................................................................................18 CHAP TER 1 SPINAL CORD INJURY AND SKELETAL MUSCLE....................................................... 20 1.1 Significance......................................................................................................................20 1.2 Spinal Cord Injury in Humans.......................................................................................... 21 1.3 Spinal Cord Injury in Animal Models.............................................................................. 22 1.3.1 Spinal Cord Injury in the Rat Model...................................................................... 23 1.3.1.1 Spinal cord contusion model in rats............................................................. 24 1.3.1.2 Spinal contusion by impaction..................................................................... 25 1.3.2 Assessment of Locomotor Behavior in Rats.......................................................... 26 1.4 Skeletal Muscle Adaptations after SCI............................................................................. 28 1.4.1 Morphological Adaptations: Muscle Atrophy........................................................ 28 1.4.2 Morphological Adaptations: Mu scle Fiber Type Conversion ................................ 31 1.4.3 Adaptations to Cont ractile Properties .....................................................................34 1.4.4 Metabolic Adaptations: Altered Oxidative Capacity ............................................. 36 1.4.5 Metabolic Adaptations: Alte red Glucose Hom eostasis.......................................... 39 1.5 Summary...........................................................................................................................44 2 SPINAL CORD INJURY AND LOCOMOTOR TRAINING............................................... 46 2.1. Locomotor Function after SCI.........................................................................................46 2.1.1. Conventional Rehabilitati on Therapies after SCI .................................................. 47 2.1.2 Limitations of Compensatory Rehabilitation Strategies ......................................... 49 2.2 Spinal Cord Plasticity.......................................................................................................50 2.3 Locomotor Training: A Paradigm Shift............................................................................ 54 2.3.1 A Historical Perspective and Locomoto r Training in the Spinal Cord Injury Animal Model..................................................................................................................55 2.3.2 Locomotor training in Individua ls with Spinal Cord Injury ...................................58 2.3.3 Central Pattern Generators and Locomotion.......................................................... 61 2.4 Locomotor Training Effects on Paralyzed Skeletal Muscle .............................................63 2.5 Summary...........................................................................................................................65 3 MAGNETIC RESONANCE A ND SKELETAL MUSCLE .................................................. 66

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8 3.1 Introduction............................................................................................................... ........66 3.2 Basics of Magnetic Resonance......................................................................................... 66 3.2.1 Magnetic Property of Nuclear Spins...................................................................... 67 3.2.2 Larmor Frequency..................................................................................................68 3.2.3 Longitudinal and Transverse Magnetizations........................................................69 3.2.4 Relaxation Times....................................................................................................71 3.2.5 Fourier Transform..................................................................................................73 3.3 Magnetic Resonance Imaging...........................................................................................73 3.3.1 T1 and T2 Weighted Images.................................................................................. 74 3.3.2 Image Construction................................................................................................ 75 3.4 MRI Applications in Skeletal Muscle ............................................................................... 78 3.5 Magnetic Resonance Spectroscopy..................................................................................82 3.5.1 Contrasting MRI and MRS.....................................................................................82 3.5.2 Nuclei Studied with MRS.......................................................................................83 3.5.3 Spectral Components of MRS................................................................................84 3.5.4 Chemical Shift in MRS.......................................................................................... 84 3.5.5 Correction of Saturation Effects.............................................................................86 3.5.6 Proton Spectroscopy (1H-MRS).............................................................................86 3.5.7 Phosphorus Spectroscopy (31P-MRS).....................................................................87 3.6 Application of Magnetic Resonan ce Spectroscopy in Skeletal Muscle ........................... 89 3.6.1 Role of 1H-MRS : Quantify Intramuscular Fat....................................................... 90 3.6.2 Role of 1H-MRS: Assess Muscle T2 Characteristics............................................. 90 3.6.3 Role of 31P-MRS: Quantify Resting Muscle Metabolites.....................................91 3.6.4 Role of 31P-MRS: Identify Fiber-type and Muscle Fatigue.................................... 92 3.6.5 Role of 31P-MRS: Measure Muscle Oxidative Capacity........................................ 93 3.6.6 Relationship between PCr Recovery Ra tes and Muscle Oxidative C apacity.........95 3.7 Summary...........................................................................................................................98 4 OUTLINE OF EXPERIMENTS.......................................................................................... 107 4.1 Experiment One..............................................................................................................107 4.1.1 Specific aim..........................................................................................................107 4.1.2 Hypotheses...........................................................................................................107 4.2. Experiment Two............................................................................................................107 4.2.1 Specific Aim.........................................................................................................107 4.2.2 Hypotheses...........................................................................................................108 4.3 Experiment Three........................................................................................................... 108 4.3.1 Specific Aim.........................................................................................................108 4.3.2 Hypothesis............................................................................................................108 4.4. Experiment Four............................................................................................................108 4.4.1 Specific Aim.........................................................................................................108 4.4.2 Hypotheses...........................................................................................................109 4.5. Experiment Five.............................................................................................................109 4.5.1 Specific Aim.........................................................................................................109 4.5.2 Hypotheses...........................................................................................................109 4.6. Experiment Six..............................................................................................................110 4.6.1 Specific Aim:........................................................................................................110

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9 4.6.2 Hypotheses...........................................................................................................110 5 METHODOLOGY............................................................................................................... 111 5.1 Human Studies.............................................................................................................. ..111 5.1.1 Subjects.................................................................................................................111 5.1.2 Clinical Assessments............................................................................................ 112 5.1.3 Locomotor Training..............................................................................................113 5.1.4 Proton Magnetic Resonance Imaging (1H-MRI).................................................. 114 5.1.4.1 Muscle cross sectional area: data collection and analysis .......................... 115 5.1.4.2 T2 relaxation times: data collection and analysis....................................... 116 5.1.5 Proton Magnetic Resonance Spectroscopy (1H-MRS)......................................... 116 5.1.5.1 Muscle lipid: data analysis......................................................................... 117 5.1.5.2 Muscle T2 relaxation times: data analysis................................................. 118 5.2 Animal Studies............................................................................................................. ...118 5.2.1 Animals.................................................................................................................118 5.2.2 Gender Differences in Animal Models of SCI..................................................... 119 5.2.3 Spinal Cord Contusion Injury............................................................................... 119 5.2.4 Experimental Electrical Stimulation Protocol...................................................... 120 5.2.5 Phosphorus Magnetic Resonance Spectroscopy (31P-MRS): Data Collection..... 121 5.2.5.1 31P-MRS spectral analysis at rest............................................................... 121 5.2.5.2 31P-MRS spectral analysis of electrical stimulation protocol..................... 122 5.2.6 Biochemical Assays..............................................................................................123 5.2.6.1 ATP measurements.................................................................................... 124 5.2.6.2 Total creatine measurements...................................................................... 124 6 EXPERIMENT ONE LOWER EXTR EMITY MUSCLE C ROSS-SECTIONAL AREA AFTER INCOMPLETE SPINAL CORD INJURY................................................. 129 6.1 Summary.........................................................................................................................129 6.2 Introduction............................................................................................................... ......130 6.3 Methods..........................................................................................................................132 6.3.1 Subjects.................................................................................................................132 6.3.2 Maximum Muscle Cross-sectional Area.............................................................. 133 6.3.3 Data Analysis........................................................................................................ 133 6.4 Results.............................................................................................................................134 6.5 Discussion.......................................................................................................................136 7 EXPERIMENT TWO NON-INVASIVE A SSESSMENT OF LOWER EXTREMITY MUSCLE COMPOSITION AFTER INCO MPLETE SPINAL CORD INJURY ............... 147 7.1 Summary.........................................................................................................................147 7.2 Background.....................................................................................................................147 7.3 Methods..........................................................................................................................149 7.4 Results.............................................................................................................................150 7.5 Discussion.......................................................................................................................151

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10 8 EXPERIMENT THREE MAGNETIC RESONANCE ASSESSMENT OF MUSCLE DAMAGE DURING L OCOMOTOR TRAI NING IN PERSONS WITH INCOMPLETE SPINAL CORD INJURY........................................................................... 162 8.1 Summary.........................................................................................................................162 8.2 Introduction............................................................................................................... ......162 8.3 Methods..........................................................................................................................163 8.4 Results.............................................................................................................................164 8.5 Discussion.......................................................................................................................164 8.6 Conclusion......................................................................................................................165 9 EXPERIMENT FOUR IMPACT OF LOCOMOTOR TRAINING ON MUSCLE SIZE AND I NTRAMUSCULAR FAT AFTER SPINAL CORD INJURY........................ 167 9.1 Summary.........................................................................................................................167 9.2 Introduction............................................................................................................... ......167 9.3 Methods..........................................................................................................................169 9.4 Results.............................................................................................................................169 9.5 Discussion.......................................................................................................................170 10 EXPERIMENT FIVE MONITORI NG AL TERATIONS IN INORGANIC PHOSPHATE OF HINDLIMB MUSCLE AF TER SPINAL CORD CONTUSION IN RATS....................................................................................................................................176 10.1 Summary.......................................................................................................................176 10.2 Introduction.............................................................................................................. .....176 10.3 Specific Aims and Hypothesis...................................................................................... 178 10.3.1 Specific Aim.......................................................................................................178 10.3.2 Hypotheses.........................................................................................................178 10.4 Methods........................................................................................................................179 10.4.1 Experimental Design.......................................................................................... 179 10.4.2 Animals...............................................................................................................179 10.4.3 Magnetic Resonance Spectroscopy: Data Collection and Analysis ...................179 10.4.3.1 31P-MRS spectral analysis at rest............................................................. 180 10.4.4 Biochemical Assays............................................................................................ 181 10.4.5 Data Analysis...................................................................................................... 181 10.5 Results...........................................................................................................................181 10.6 Discussion.....................................................................................................................182 11 EXPERIMENT SIX IN-VIVO ASSESSMENT OF SKELETAL MUSCLE BIOENERGETICS AFTER SPINAL CORD CONTUSION IN RATS ..............................191 11.1 Summary.......................................................................................................................191 11.2 Introduction.............................................................................................................. .....191 11.3 Specific Aims and Hypothesis...................................................................................... 194 11.3.1 Specific Aim.......................................................................................................194 11.3.2 Hypotheses.........................................................................................................194

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11 11.4 Methods........................................................................................................................194 11.4.1 Experimental Design.......................................................................................... 194 11.4.2 Animals...............................................................................................................195 11.4.3 Data Collection................................................................................................... 195 11.4.3.1 Experimental electrical stimulation protocol........................................... 195 11.4.3.2 31P magnetic resonance spectroscopy...................................................... 196 11.4.4 31P magnetic resonance spectr oscopy: data analysis.......................................... 197 11.4.5 Biochemical Assays............................................................................................ 198 11.4.6 Statistical Analysis............................................................................................. 198 11.5 Results...........................................................................................................................198 11.6 Discussion.....................................................................................................................200 12 CONCLUSION..................................................................................................................... 218 LIST OF REFERENCES.............................................................................................................219 BIOGRAPHICAL SKETCH.......................................................................................................245

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12 LIST OF TABLES Table page 3-1 Longitudinal (T1) relaxation times (in m illis econds) of water and lipid components...... 99 3-2 Transverse (T2) relaxation times (in m illiseconds) of water and lipid components.......... 99 6-1 Characteristics of subjects after incomplete-SCI. ............................................................ 142 6-2 Percentage differences between th e lower extremity maximum muscle CSA............... 142 6-3 Relative proportions of muscles in the pooled i-SCI and control groups. .......................143 7-1 Characteristics of indivi duals with incom plete-SCI........................................................ 157 7-2 Percent differences between the T2 relaxation times....................................................... 157 8MR measures of T2 relaxation times of lower extremity muscles before and after LT.....................................................................................................................................166 10-1 Absolute phosphate metabolite concentrati ons after spinal cord contusion in rats. ........ 187 11-1 Kinetic 31P-MRS data from the rat gastrocnemius muscle.............................................. 211

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13 LIST OF FIGURES Figure page 3-1 Schematic representation of a nucle us and its behavior as a bar m agnet........................ 100 3-2 Application of a 90o RF pulse.......................................................................................... 100 3-3 T1 relaxation time......................................................................................................... ...101 3-4 T2 relaxation time......................................................................................................... ...101 3-5 Fourier transform of the FID signal................................................................................. 102 3-6 Schematic representation of two tissu es with different T1 relaxation tim es................... 102 3-7 Schematic representation of two tissu es with different T2 relaxation tim es................... 103 3-8 Bandwidth of frequencies excites a sp ecific width of slice in the sam ple....................... 103 3-9 Frequency and phase encoding gradient effects on spins.. ..............................................104 3-10 Representative 1H-MR spectrum of a healthy human soleus muscle at 1.5Tesla............ 104 3-11 Representative 1H-MR spectrum of a human skeletal muscle at 1.5Tesla...................... 105 3-12 Representative typical 31P-MR spectrum of a rat calf muscle at 11Tesla........................105 3-13 Schematic representation of the creatin e-phosphocreatine shuttl e and buffer role of the creatine-kinase reaction m uscle................................................................................. 106 5-1 Representative 1H-MRI coronal image of the calf mu scles from a healthy control........ 126 5-2 Representative 1H-MRI trans-axial image of the calf muscles from a healthy control... 126 5-3 A) Representative 1H-MRI T2 weighed images B) Indi vidual pixel signal intensities C) Representative T2 map...............................................................................................127 5-4 Representative trans-axial image presents a voxel prescribed ove r the soleus m uscle... 127 5-5 Decomposition of a lipid peak obtained from a healthy soleus muscle........................... 128 5-9 Experimental-setup for electrical stimulation protocol during 31P-MRS data acquisition.................................................................................................................... ....128 6-1 Representative trans-axial proton magnetic resonance im ages obtained at 1.5Tesla...... 144 6-2 Muscle CSA in the pooled inco m plete-SCI and control groups...................................... 145

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14 6-3 Proportion ratios of muscle gr oups w ithin the leg and thigh...........................................146 6-4 Ratio of individual plantar flexor mu scles to the m ax CSA of the posterior compartment of the leg.................................................................................................... 146 7-1 Representative T2 weighted trans-axial proton m agnetic resonance images of the lower leg...........................................................................................................................158 72 Representative proton magnetic resonance spectra obtained from the soleus muscle..... 159 7-3 Box-plot depicting variability in EMCL/w ater, I MCL/water and total soleus muscle lipid/water ratios............................................................................................................. .160 7-4 Individual data comparisons of IMCL to water ratio (IMCL/water) ............................... 161 7-5 Individual data comparisons of EMCL to water ratio (EMCL/water) ............................. 161 9-1 Cross sectional area of lower leg muscles after nine weeks of locom otor training......... 174 9-2 Cross sectional area of thigh muscles after nine weeks of locom otor training................ 174 9-3 Estimates of soleus muscle lipid before (Pre LT ) and after (Post LT) nine weeks of locomotor training............................................................................................................ 175 10-1 Percent change in resting phosphate me tabolites of the rat h ind limb muscle one week after spinal cord contusion...................................................................................... 188 10-2 Change in phosphorylation ratios [ADP ][Pi]/[ATP] be fore and after spinal cord contusion...................................................................................................................... ....188 10-3 Representative 31P-spectra before and after one week SCI obtained at 11T................... 189 10-4 Change in [PCr] before and after spinal co rd con tusion*Statistically significant difference. 189 10-5 [PCr] in individual rat hind limb muscle af ter one week of spinal cord contusion. ........ 190 11-1 Representative kinetic 31P spectra of rat gastrocnemius muscle at 11T..........................213 11-2 Average PCr depletion and recovery graphs in response to the stim ulation protocol..... 214 11-3 PCr depletion rates (1, 2, and 3m in) before and after spinal cord contusion injuries (SCI).................................................................................................................................214 11-4 Expanded view of PCr kinetic data obta ined at th e onset of electrical muscle stimulation (EMS)............................................................................................................ 215 11-5 PCr recovery graphs obtained immediatel y af ter termination of electrical muscle stimulation.................................................................................................................... ....215

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15 11-6 Maximum mitochondrial oxidative capacity (Vmax mM.min-1) data from eight rats (R1-R8)............................................................................................................................216 11-7 Scatter-plot depicting relationship between m aximum mitochondrial capacity (Vmax) and rate of PCr depletion (Vdep)........................................................................ 216 11-8 Relationship between end stimulation [A DP] and initial rates of PCr recovery (Vex).... 217 11-9 Maximal rates of oxidative capacity (Qmax) based on the [ADP] and [ADP][Pi]/[ATP] models................................................................................................. 217

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16 LIST OF ABBREVIATIONS ASIA American spinal cord injury association [ADP] Absolute concentration of free cystolic adenosine diphosphate [ATP] Absolute concentrati on of adenosine triphosphate [ADP][Pi]/[ATP] Phosphorylation potential CSA Maximum cross sectional area EMCL Extramyocellular lipid EMS Electrical muscle stimulation End ex End exercise (EMS) FID Free induction decay GAS Gastrocnemius muscle 1H-MRS Proton magnetic resonance spectroscopy IMCL Intramyocellular lipid kPCr Rate constant of PCr recovery LT Locomotor training mM milimoles/liter of intracellular water MR Magnetic resonance MRI Magnetic resonance imaging MRS Magnetic resonance spectroscopy [Pi] Absolute concentration of inorganic phosphate [PCr] Absolute concentr ation of phosphocreatine 31P-MRS Phosphorus magnetic resonance spectroscopy Qmax Maximum oxidative ATP synthesis rate Qmax-ADP Maximum oxidative ATP synthesis rates based on kPCr and [ADP]

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17 Qmax-[ADP][Pi]/[ATP] Maximum oxidative ATP synthesis rates based on kPCr and phosphorylation potential. SCI Spinal cord injury T2 T2 relaxation times TR Repetition time Vex Extrapolated initial ra tes of PCr recovery (kPCr* PCr) Vdep Rates of PCr depletion at onset of EMS Vmax-lin Maximal rate of PCr resynthesis Vmeas Initial rates of PCr recovery (f irst three points in recovery) PCr [PCr]rest [PCr]end ex ( mol/g wet wt) micromol/gram wet weight

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18 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 MAGNETIC RESONANCE CHARACTE RIZATION OF SKELETAL MUSCLE ADAPTATIONS AFTER INCO MPLETE SPINAL CORD INURY By Prithvi Krishnakant Shah May 2008 Chair: Krista Vandenborne Major: Rehabilitation Science Spinal cord injury is one of the most disabling health relate d problems that often results in paralysis and paresis of body musculature below the lesion site. Persons with incomplete-SCI typically exhibit impaired moto r performance and varying degr ees of functional limitations. Despite the obvious motor dysfunctions, physiologi cal muscle adaptations following incompleteSCI are relatively unstudied. An understandi ng of the muscular adaptations following an incomplete-SCI will help in the development of therapies aimed at reducing the secondary effects of paralysis and paresis. The overall objective of this disserta tion was to investigate skeletal muscle adaptations following incomplete -SCI using combinations of non-invasive MRI and MRS techniques. Findings from our human studies reveal that chronic incomplete-SCI is associated with significant muscle atrophy in the affected lower extremity that is uniform between limbs and somewhat influenced by mobility status. In addi tion, persons with incomplete-SCI demonstrate an increase in the total lipid, IMCL and extramyocellular EMCL content and enhancements in the T2 relaxation properties of the lower leg muscles. Moreover, repetitive locomotor training with body weight support and a treadmill are associated with significant increases in the

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19 plantarflexor muscle size. Data from our animal experiments reveal that the paralyzed rat hindlimb muscle show faster ra tes of PCr depletion, thereby suggesting that, after SCI, there is either an increase in ATP requirement for similar demands in muscle contraction or the overall supply of ATP is compromised follo wing the injury. In addition, a pronounced decrease in PCr recovery rates implies a le ss effective oxidative phosphorlyation and a reduction in the mitochondrial oxidativ e capacity of skeletal muscle. Collectively, findings from this dissertation work reveal that the para lyzed skeletal muscle shows drastic alterations in its morphological and metabolic properties af ter a SCI and that these adaptations can be successfully characterized by the us e of non-invasive MR techniques. The present work will provide a foundation from which the relationship between skeletal muscle adaptations and function in this population can be further explored. Moreover, the use of sophisticated MR techniques will enable characte rizing the paralyzed muscle non-invasively and with high resolution; while also allowing longitudinal follow-ups all of which are crucial in the assessment of injury mechanisms, disease progre ssion and efficacy of therapeutic interventions.

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20 CHAPTER 1 SPINAL CORD INJURY AND SKELETAL MUSCLE 1.1 Significance Spinal co rd injury (SCI) results in paralysis and paresis of muscles below the injury site making movement difficult. Impairments in sk eletal muscle contribute to a host of musculoskeletal deficits that lead to secondary health related c onditions that cost an estimated $220,000 and $750,000 per person annually (NSCISC 2006). The clinically relevant musculoskeletal and movement disorders in pe rsons with SCI include muscle weakness and paralysis of affected extremities (Gordon and Ma o 1994; Kim, Eng et al. 2004), early muscle fatigue and increased energy demands for simple functional activities (Hopman, Dueck et al. 1998; Ulkar, Yavuzer et al. 2003) diminished capacity or inability to ambulate (Burns, Golding et al. 1997; Stein, Chong et al. 2002), and overall decreas ed endurance and dependence on assistive aids for locomotion (Waters, Adkins et al. 1994; Ulkar, Ya vuzer et al. 2003). Furthermore, inactivation of paralyzed skelet al muscle interferes with implementation of therapeutic interventions (Subbarao 1991). Inactivity and se dentary lifestyle in this patient cohort leads to unloading of paralyzed muscles. As a re sult, a myriad of muscle adaptations including morphological, contractile, metabolic and neural alterations ensue following SCI (Castro, Apple et al. 1999; Shields 2002). Note worthily, most of these adaptations have been studied following a complete SCI. Understanding these muscular adaptations following an incomplete SCI will help in the development of therapies aimed at reducing the secondary e ffects of paralysis and paresis. The purpose of this work is to i nvestigate skeletal muscle adaptations following incomplete SCI using non-invasive magnetic re sonance imaging (MRI) and spectroscopy (MRS) measures. Combinations of human and animal mode ls of incomplete SCI are utilized to achieve this goal.

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21 1.2 Spinal Cord Injury in Humans The curren t gold standard classification system for SCI, American Spinal Injury Association Classification System ASIA is based on the presence, absence and degree of normality of motor and sensory impairments (ASIA 2001). Since these impairments are highly variable, classifying SCI has posed a major challenge for clinicians. According to the ASIA classification system, human SCI is classified as either complete or incomplete. Complete SCI involves total absence of sensor y and motor functions in the lowe st sacral segments, whereas an incomplete SCI is characterized by partiall y preserved motor and/or sensory function below the level of lesion, including spar ing of the lowest sacral segmen ts (ASIA 2001). Irrespective of the severity of injury, the majority of human spinal cord injuries leave the spinal cord tissue relatively intact (Kakulas 1987). As a result, often after infarction, contusion and/or mechanical deformation, much of the spinal cord tissue is spar ed. A severed cord is se en only in rare injury types such as sword injuries, bullet wounds or penetration of bone fragments into the cord. Interestingly, while some individu als with significant sparing of sp inal cord tissue may present as clinically complete, others with very litt le spinal cord tissue sparing are considered incomplete. These varying clinical signs make classifying SCI difficult and leaves clinicians guessing as to the integrity of the spinal cord tissue. With technological advancements in the manage ment of acute SCI, and with an increased life expectancy of persons with an incomplete in jury, there is an emerging healthcare trend in the treatment of persons with chronic SCI (NSCISC 2006). The majority of new spinal cord injuries (~53%) occurring annually are now classified as incomplete. Given their unique injuries and varying degrees of tissue sparing, persons with incomplete SCI constitute an extremely heterogeneous group. Individuals after this type of injury exhibit a continuum of ambulatory abilities ranging from being completely wheelcha ir dependent to nearly normal walking without

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22 the use of assistive devices (Waters, Adkins et al. 1994; Melis, Torre s-Moreno et al. 1999). Accordingly, due to their variable paralysis a nd paresis, they present with impaired motor performance and varying degrees of functiona l limitations (Subbarao 1991; Tang, Tuel et al. 1994; Burns, Golding et al. 1997). Despite the obvious motor dysfunctions in persons with incomplete SCI, muscle adaptations following the injury have not been well described. 1.3 Spinal Cord Injury in Animal Models The benefits of utilizing experimental animals is reflected in the more than hundred years of ongoing research in animal models of SC I. Current understanding of anatomical and molecular changes in response to human SCI has largely been derived from parallel lesion models of experimental SCI in animals th at mimic human SCI, both histologically and behaviorally. Animal models of SCI enable inves tigation of SCI at a leve l of detail that would not be possible in human studies. A variety of experimental animal models of adult SCI involving rats, mice, pigs, monkeys and cats have been utilized for this purpose (Wrathall 1992; Anderson, Howland et al. 1995; Qayumi, Janus z et al. 1997; Stokes and Jakeman 2002; Rosenzweig and McDonald 2004; Ni, Li et al. 2005). The majority of these studies have utilized various injury paradigms ranging from sharp trans ections to blunt contusion of the spinal cord that simulate the variations in the severity of human SCIs. Animal models of SCI have provided insights into, and continue to c ontribute greatly towards our cu rrent understanding of the human spinal cord injury mechanisms, anatomical and pathophysiological se quels of the injury, neuromuscular adaptations followi ng the injury, regeneration and repair of spinal cord, and effectiveness of novel th erapeutic interventions following inju ry. Furthermore, investigations have also attempted to answer the questi on of effective duration and time frames of implementing these interventions (Rosenzweig a nd McDonald 2004). Goals and objectives of a study dictate which animal and lesion mode l is best suited for a given study.

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23 1.3.1 Spinal Cord Injury in the Rat Model Of the various anim al models mentioned a bove, research on studying the rat model of SCI has gained tremendous momentum in the recen t years (Wrathall 1992; Kwon, Oxland et al. 2002; Young 2002). In the 1980s, the feline spinal cord model dominated the field of SCI research in investigating the wide gamut of neurophys iological changes including biochemical, physiological, vascular and metabolic changes fo llowing the injury (Young 2002). In fact, the cat spinal model yielded several therapies that went to clinical trial in humans. One of the most classic examples is the first clin ical trial of methylprednisolone in 1985 (Bracken, Shepard et al. 1985). However, in recent years th e trend has been towards the use of rats for SCI studies. Two major interconnected determinants of this transition have been recognized. Firstly, greater percentages of SCI in humans are now being classified as incomple te injuries. Consequently, the weight drop device similar to that used for the feline spinal cord contus ion model was developed by Noble and Wrathall in 1987 for use in rats. This device induces spinal cord contusion in rats that morphologically and behaviorally mimics huma n incomplete spinal cord injuries (Wrathall, Pettegrew et al. 1985; Noble and Wrathall 1989) (see below for more details). Secondly, rat models are advantageous because they are easily accessible and large groups of rats are easy to manage in a laboratory setting. Rat models allow for practical post-operative animal care, conduction of longitudinal studies in a feasible time frame due to relatively shorter lifespan of animals, and considerably lower initial costs (Khan, Havey et al. 1999); while simultaneously serving the purpose of mimicking an injury that corresponds to human SCI. Assessment of postinjury symptoms in the rat model is reliably and validly tested using the Basso Bresnahan, Beattie scale (BBB) (Basso, Beattie et al. 1995). Lastly, with the explosion of genetic rese arch in neuroscience, SCI mice models are becoming more prevalent (Stokes and Jakeman 2002). However, this model has not as well been

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24 validated and suffers from relatively larger variability in injury production and locomotor functional assessments post-injury (Rosen zweig and McDonald 2004; IOM 2005). 1.3.1.1 Spinal cord contusion model in rats The common experim ental rat models of SCI include transection, compression and contusion (Kwon, Oxland et al. 2002; Rosenzweig and McDonald 2004). Each of these models exhibit unique characteristics and are used as pe r the experimental objectives. A transection rat model produces complete injury to the spinal cord and is more commonly used to study the neurophysiological processes/mechanisms above or below cord lesion site s. Transection model studies overcome the confounding effects of spared neural pathways that are commonly present in studies using incomplete injury models. A compression model, on the other hand, causes cord compression and better simulates ischemic injuries to the spinal cord. This model is commonly used to study the acute vascular pathophysio logy following SCI (Kwon, Oxland et al. 2002; Young 2002). Finally, the SCI contusion model produ ces mechanical impaction of the cord and most closely imitates an incomplete SCI in humans both pathophysiologically and behaviorally and can create graded focal injuries similar to humans (Metz, Curt et al. 2000). In the present dissertation work, SCI contusion injury in rats is utilized as the model of choice. Various approaches have been devised to indu ce an experimental cont usion injury in the rat model including weight drop impaction of an e xposed spinal cord (Wrathall, Pettegrew et al. 1985), contusion by compressing the cord between th e arms of an aneurysmal clip (Rivlin and Tator 1978), arterial occlusion ((IOM 2005) or chemical toxicity (M agnuson, Trinder et al. 1999). Of these, induction of cont usion injury to the cord by imp action has proven to be most relevant to incomplete, traumatic SCI in humans. The mechanical impact by a weight drop on the exposed cord reflects the dynamics of clinical injury that occurs in humans (Khan, Havey et al. 1999; Metz, Curt et al. 2000; Kwon, Oxland et al. 2002; Young 2002). The majority of human

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25 incomplete spinal injuries are a direct result of trauma to the cord. Traumatic SCI in humans causes an initial mechanical insu lt to the cord that involves a pr imary lesion, termed the umbra region, followed by a penumbra or secondary injury that involves either further damage to already damaged tissue or new damage to otherwise healthy tissue (Hall and Springer 2004). The underlying molecular and pathophysiologica l mechanisms and the functional symptoms following an incomplete SCI in humans are well reproducible in the contusion rat model. In fact, electrophysiological outcomes su ch as motor evoked potentials (MEP) and somatosensory evoked potentials (SSEP), spinal cord lesion morphology, relationship between the morphology and functional measures such as functional loco motor capacity have been demonstrated to be analogous in humans with SCI and spinally contused rats (Noble and Wrathall 1985; Noble and Wrathall 1989; Metz, Curt et al. 2000). As a result, the contusion animal model of SCI has been established as a valid model of SCI fo r comparison between rats and humans. 1.3.1.2 Spinal contusion by impaction The weight drop device used to induce spinal contusion injury was first introduced by Allen in 1911 where weight was dropped onto canine cords exposed by lam inectomy (Young 2002). Unfortunately, Allens death in World War II created a void in the use of this model. It was only in the late 1960s, that investigators revived the cont usion model in the primate and feline spinal cord to describe th e histopathological effects, evoked potentials and assess effects of corticosteroids and hypothermia in response to SCI. However, owing to questionable reproducibility of the weight drop technique, a standard grad ed and reproducible spinal contusion injury procedure by impaction was warr anted. By making defin ite alterations in the height at which a 10g weight was dropped on the exposed dura, a reproduci ble injury to the rat spinal cord was established by Wrathall et al in 1985 (Wrathall, Pettegrew et al. 1985). Depending upon the desired severity of injury to the cord, the 10g we ight is dropped from

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26 heights of 2.5cm, 5cm or 17.5 cm on the exposed dura. Accordingly, injury to the cord is considered mild, moderate or severe respectivel y, corresponding to simila r severity of spinal injuries in humans. Before experimental therapeutic interventions can be implemented, standard protocols are required to help minimize variability in injury between animals. Indeed, the magnitude of mechanical injury produced by these methods have been validated and proven to closely correlate with histological, behavioral, el ectrophysiological eval uations and functional measurements following SCI (Gale, Kerasidis et al. 1985; Noble and Wrat hall 1985; Metz, Curt et al. 2000). Three standard devi ces are now used to produce this injury in rodents: Multicenter Animal Spinal Cord Injury Study (MASCIS) imp actor formerly called the New York impactor; the Ohio State University impactor and the Infinite Horizons device (Wrathall 1992; Basso, Beattie et al. 1996; Khan, Ha vey et al. 1999; IOM 2005). 1.3.2 Assessment of Locomotor Behavior in Rats One of the first behavioral test scores to a sses s recovery in spinal injured animals was established by Tarlov and Klinger in 1954. Briefly, the testing used a six-point scale to assess motor function recovery after sp inal injury in dogs (grade 0 = no spontaneous movement; grade 5 = normal motor function) (Tarlov and Klinge r 1954). The Tarlov score, as it is commonly named today, has emerged as a popular tool to as sess motor recovery in a variety of spinal injured animals including rats. In addition, vari ous other motor scoring approaches have been specifically formulated for the spinal injured ra t. Bresnahan et al in 1987 compiled a behavioral testing strategy that included assessments of ge neral locomotor skill (in an open field), fine locomotor skills (through grid walking task) an d postural adjustment to displacement (in an inclined plane) after SCI in rats (Bresnahan, B eattie et al. 1987). However, a valid and reliable

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27 tool the Basso, Beattie and Bresnahan score, also commonly known as the BBB score was launched only in 1995 (Basso, Beattie et al. 1995). The BBB score is an operationally defined or dinal scale that assesses hind limb locomotor recovery of thoracic spinal cord injured rats. It is the first assessment tool that has been validated with testers from several laboratories across the United States (Basso, Beat tie et al. 1996). Each of the components of the 21 point scale is based on specific features of locomotor recovery after spinal cord contusion in rats including joint movements, trunk posture, weight support, stepping and weight support ability, coordination between forelimbs and hind limbs, and tail position. The score order from 0 to 20 assumes progressi ve recovery with every score representing a unique and sequential stage of locomotor recovery : a) scores 1 through 8 center around recovery of joint movements unique to the early phase of recovery, b) scores 9-13 focus upon initial recovery of stepping ability and coordination that represents an intermediate stage of recovery and c) scores 14-21 focus on the progression in f oot placement, toe clearance trunk stability and tail position during stepping that re presents the late and final phase of recovery. Testing consists of placing the animal in an open field beginning as early as one day post injury and observing it for 4 minutes. Behavioral scoring is done by two observers in real time with repeated testing every week for 6-9 weeks. Used extensively throughout the neurotrauma literature, the scoring system is sensitive enough to differentiate between severities of the spinal injury including mild to severe spinal contusion and tran section (Basso, B eattie et al. 1996). Though the versatility of the BBB score makes it a valuable locomotor assessment tool, investigators have also questioned its accuracy in assessing specific categories such as coordination. Accordingly, measures such as the Catwalk analysis have been proposed as potential addendums to the standard BBB scale (Koopmans, Deumens et al. 2005). The Catwalk

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28 analysis allows collec tion of data on dynamics of locomotion such as degree of coordination and weight bearing, duration of gait cycles etc. In addition, various other measures have been identified to assess locomotor recovery after SCI in the rat; the use of which depends on the research question posed by investigators. The inclined plane test for example measures the animals ability to maintain posi tion in an inclined plane and is commonly used to assess balance and posture (Rivlin and Tator 1977; Bresnahan, B eattie et al. 1987). The cr ossway tests involves crossing runways such as a beam or grid (Kunkel-Bagden and Bregman 1990). The substantial motor control required to pass this test makes it suitable for assessing limb coordination. Video recordings from these tests yield data such as time taken and number of errors made during crossing. Treadmill walking is yet another measure of assessing locomotor recovery and is often used for assessment of footprint and kinemati cal analysis of indi vidual joint movements (Kunkel-Bagden and Bregman 1990). 1.4 Skeletal Muscle Adaptations after SCI Skeletal m uscle adaptations following SCI ar e a direct consequence of neuronal damage within the spinal cord and an indirect result of prolonged peri ods of muscle inactivation. In addition, paralyzed skeletal muscle is subject to altered loading conditions and muscle length (Alaimo, Smith et al. 1984; Gordon and Mao 19 94; Dietz, Colombo et al. 1995; Shields 2002). Ultimately, a myriad of muscle adaptations incl uding morphological, contractile and metabolic muscle alterations occur following SCI. The fo llowing discussion is focused on adaptations of paralyzed muscles that are partially or complete ly devoid of a descending spinal drive and yet have an intact peripheral nerve supply. 1.4.1 Morphological Adaptations: Muscle Atrophy Like any other m odel of muscle disuse, such as cast immobilization (Vandenborne, Elliott et al. 1998; Stevens, Walter et al. 2004), unilateral lower limb su spension (Berg, Dudley et al.

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29 1991; Hather, Adams et al. 1992), bed rest (Berg, Larsson et al. 1997; Alkner and Tesch 2004), space flight (Akima, Kawakami et al. 2000; Tesch, Berg et al. 2005) and inactivity secondary to injury or disease processes (Arokoski, Arokoski et al. 2002; Johansen, Shubert et al. 2003), SCI is accompanied by marked atrophy of the paralyzed muscles. The abovementioned human models of disuse atrophy result in 15% 32% decrease in muscle size of the immobilized lower extremity muscles, depending upon the muscles tested and time of testing. A number of studies have reported effects of disuse ranging from immobilization periods of 1 week to almost 17 weeks. Generally, greater declines in muscle size are reported for postural and antigravity muscles such as the soleus and vasti versus th e knee and ankle flexors of the lower extremity (Hather, Adams et al. 1992; Akima, Kawakami et al. 2000; Tesch, Berg et al. 2005). In comparison to other disuse models, atrophy of th e paralyzed muscles following SCI is markedly higher. An overall 40-80% decline in the aver age CSA of human lower extremity muscles has been reported 24 weeks after complete SCI. The degree of atrophy typi cally depends upon the functional role, shortened muscle length related to posture assumed by paralyzed limbs and anatomical location of muscles (Castro, Apple et al. 1999). Using magnetic resonance imaging (MRI) techniques, Castro et al reported that the antigravity plantarflexor muscles show greater decline in CSA as compared to the dorsiflexors ( 40% versus 20%). On the other hand, the thigh antigravity knee extensors and knee flexors, unlik e in other models of muscle disuse (Tesch, Berg et al. 2005), show similar degrees of at rophy (~42%) (Castro, Appl e et al. 1999). While this initial study by Castro et al reported the muscle CSA inclusiv e of fat within the muscle, the same research group subsequently reported an almo st 38% decline in total thigh fat-free muscle CSA of the injured group as compared to contro ls (Elder, Apple et al. 2004). Modelesky et al also estimated a 38-44% decline in the fat-free muscle mass in individuals with complete SCI

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30 two years after injury using MRI and Dual X-Ra y absorptiometry (Modlesky, Bickel et al. 2004). Other investigators have studied alterations in muscle fiber size based on needle biopsies. Declines of 54-74% in muscle fiber size are observed by 24 w eeks after SCI with maximum declines seen in muscle fiber types that have a greater fiber CSA initially (i.e. fast twitch fibers) (Scelsi, Marchetti et al. 1982; Castro, Apple et al. 1999). While maximum rates of decline in whole muscle size (60%) and muscle fiber size (74%) are seen during the first 6 weeks after injury, progressive atrophy is observed for as long as one year after SCI (Scelsi, Marchetti et al. 1982). Furthermore, atrophy appears to be musc le specific: the gast rocnemius muscle, for example, continues to atrophy for as long as 6 months, whereas the tibialis anterior and semimembranosus reach a plateau by as early as 6 weeks after injury (Cas tro, Apple et al. 1999). While maximum emphasis has been placed on st udying atrophic adaptations in the lower extremity paralyzed muscles, a couple of studies have also reported at rophy of upper extremity muscles (Thomas, Zaidner et al. 1997) and a bdominal muscles (Estenne, Pinet et al. 2000) following SCI. Atrophy following animal models of SCI is in concurrence with humans with SCI where almost 10% to 56% of whole muscle and fiber atrophy is observed depending upon the muscles studied and the model of injury based on injury severity (Roy and Acosta 1986; West, Roy et al. 1986; Dupont-Versteegden, Houle et al. 1998; Gregor y, Vandenborne et al. 2003; Otis, Roy et al. 2004). Similar to human studies, larger degrees of atrophy are observed in the slow antigravity soleus muscle as compared to the fast flexor mu scles such as the tibialis anterior or extensor digitorum longus. Moreover, similar to humans, an tigravity muscles continue to atrophy for at least 6 months after SCI. Ho wever, unlike the paralyzed kne e flexors in humans, minimal declines are seen in the non-antigravity knee flexor muscles (semitendinosus) following SCI in

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31 animal models (Roy and Acosta 1986; Roy, Ta lmadge et al. 1998; Otis, Roy et al. 2004). While all the above-mentioned studies have focused on the complete SCI human and animal models, no studies in humans and just few sp inal contusion animal model investigations have reported the impact on skeletal muscle after incomplete spinal cord injuries. Hutchinson et al studied lower extremity muscle atrophy following contusion injury in rats over the course of ten weeks (Hutchinson, Linderman et al. 2001). At one week, they found a 20-25% decline in the wet weight of lower leg muscles (soleus, plantaris, gastrocnemius and tibialis anterior) and no change in the EDL muscle wet weight. While spontaneous recovery occurred in the soleus, plantaris and tibialis anterior musc les by three weeks, the gastrocnemius muscles continued to show declines for as long as ten weeks (Hutchinson, Linderman et al. 2001). Similarly, Liu et al have reporte d an 11%-26% decline in the triceps surae and tibialis anterior cross sectional areas two weeks after spinal contusion injury. Spontaneous recovery in the paralyzed muscle was seen by four weeks (Liu, Bose et al. 2008). In the present work, muscle cross-sectional area of paralyzed lower extremity muscle is studied in humans with incomplete SCI. In addi tion, the impact of ambulat ory status of persons with incomplete SCI on muscle size is determine d. A detailed explanation of this study follows in Chapter 6. 1.4.2 Morphological Adaptations: Muscle Fiber Type Conversion Muscle fiber type conversion is another hallm a rk of chronically paralyzed skeletal muscle. Muscle fibers can be categori zed using several cla ssification systems. The most common and recent refinement for characterization of muscle fibers is based upon the myosin heavy chain (MHC) expression of muscle fibers. MHC is a cr itical structural and en zymatic muscle protein that possesses distinct molecular forms (isoform s). MHC isoforms control the pH of the myosin ATPase reaction and accordingly are responsible for the degree of histochemical staining of

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32 muscle fibers during immunocytochemistry (M cComas 1996). Depending upon the staining, four main isoforms of the MHC corresponding to I, IIa, IIx, IIb fiber t ypes have been identified in the mammalian skeletal muscle. Thus, fibers that stain strongly at pH 9.4 are refe rred to as type II in contrast to the poorly reacting t ype I fibers (McComas 1996). Type II fibers are further classified as IIa, IIb or IIx on the basis of the strength of staining. (Note: IIb fiber type in humans is now termed as type IIx fibers rendering humans wit hout type IIb fibers). Im portantly, MHC isoforms also determine the rate of cross-bridge reactions with actin filaments and therefore the speed of muscle shortening. Accordingly, type I fibers generally are the slow twitch fibers that generate twitch forces slowly and are less fatigable. By an d large, type I fibers possess relatively higher concentrations of oxidative phosphorylation enzymes, mitochondr ial content, capillary density and redox proteins; thereby making them more su itable for sustained periods of ATP production. Type IIb fibers on the other hand quickly generate peak tension and are easily fatigable. They are generally equipped with relatively larger concentrations of glycol ytic enzymes, phosphocreatine, glycogen, calcium sequestrating proteins and sarc oplasmic reticulum; thereby making them more suitable for burst activities that demand rapid pr oduction of ATP. Fast twitch fibers contain relatively lower mitochondrial cont ent, blood capillaries and hence redox proteins. Finally, Type IIa and IIx muscle fibers possess intermediate char acteristics. Many studies also report existence of a mixed/hybrid phenotype such as type 1/IIa and IIx/IIb; es pecially in paralyzed muscles (Roy, Talmadge et al. 1999). While the above-men tioned classification is confined to muscle fibers, categorization of whole muscle is not uncommon. Whole muscles ar e generally termed as slow or fast twitch based upon the proportion of fi ber types, though both fibe r types co-exist in the same muscle. Thus, a slow muscle is ge nerally recognized as ha ving relatively greater oxidative capacity, predominantly c onsists of type 1 fibers, is func tionally more fatigue resistant,

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33 has slow twitch muscle properties (see below) and participates in slow phasic muscle activities. Based on the resting content of bioenergetically important metabolites, slow muscles also have relatively higher Pi/PCr ratios as compared to wh ite muscles; the differences being largely due to a higher Pi content (Meyer, Brow n et al. 1985; Kushmerick, Moer land et al. 1992). Examples of such a muscle include the soleus and vastus media lis. A fast muscle in co ntrast, demonstrates an overall faster contractile prope rty, is more fatigable, with relatively lower mitochondrial oxidative capacity and larger gl ycolytic capacity, predominantly consists of type II fibers and plays a major role in fast tonic activities (such as sprinting). Metabolically, fast muscles have relatively lower Pi/PCr ratios as compared to slow muscles; the differences being largely due to a higher PCr content (Kushmerick, Moerland et al 1992). Examples include the gastrocnemius and vastus lateralis muscles. While skeletal muscles of animals are more homogenous in the fiber type composition and characteristics, the human skeletal muscle is extremely heterogeneous. Usually, muscle inactivity due to disuse, imm obilization or even decreased neural activity causes muscle fiber type conversion from the slow to fast phenotype. Reverse expression of muscle fiber type is observed with increased muscle activity. There is a decrease in the expression of type I muscle fibers and an increas e in the proportion of hybrid type I+IIa and pure type IIb and/or IIx muscle fibers after chronic SCI in both animal models (Mayer, Burke et al. 1984; Lieber, Friden et al. 1986; West, Roy et al 1986; Roy, Talmadge et al. 1999; Otis, Roy et al. 2004) and in humans (Scelsi, Marchetti et al. 1982; Lotta, Scelsi et al 1991; Burnham, Martin et al. 1997; Castro, Apple et al. 1999). Similar to complete SCI, recent studies using the rat model of incomplete SCI have also demonstrated a similar shift in fiber type expression with declines in the levels of type IIa, elevations in type IIb MHC along with the presence of a

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34 transitional type IIx MHC (that is normally not seen in the control soleus muscle) after incomplete injuries (Hutchinson, Linderman et al 2001; Stevens, Liu et al. 2006). In incomplete models of SCI, this conversion occurs as ea rly as 1-3weeks and is reversible (Hutchinson, Linderman et al. 2001). This fi ber type conversion after SCI leads to significant alterations in the characteristics of paralyzed muscle including a change in contractile properties, metabolic capacities and an impaired endurance capacity (Hopman, Nommensen et al. 1994; Burnham, Martin et al. 1997). 1.4.3 Adaptations to Contractile Properties After SCI, muscles with a greater proportion of type I fibers a nd slower contractile properties (s low twitch muscles) ge nerally exhibit characteristics analogous to those of muscles with a greater proportion of type II fibers and relatively faster c ontractile properties (fast twitch muscle). Most human and animal studies concur that the contraction and relaxation speeds of slow twitch muscle, but not fast twitch muscles, are significantly faster following chronic SCI (one year after injury) when compared to befo re injury (Lieber, Frid en et al. 1986; Roy and Acosta 1986; Gerrits, De Haan et al. 1999; Roy, Talmadge et al. 1999; Shields 2002). In the spinalized animal model, contractile properties of the paralyzed soleus muscle (an otherwise slow muscle) are consis tent with those of a primarily fast muscle. Elevations in the slow muscle contraction speeds af ter SCI are reflected as decreases in time to peak tension (by almost 20ms), increase in specific tension (by almost 100%), a nd increases in fusion frequency (by almost 100%). In addition, paralyzed muscle de monstrates faster twitch half-relaxation times (Lieber, Johansson et al. 1986; Roy and Acosta 1986). While adaptations following a complete SCI are quite drastic, contractile properties afte r an incomplete SCI are relatively less marked. A decrease in halfrelaxation time by 20% in the soleus muscle that recovers to control values by ten weeks is reported in rodents wi th a moderate spinal cord cont usion (Stevens, Liu et al. 2006).

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35 Though conversion of muscle fiber type is genera lly purported as reasons for alterations in contractile properties, various molecular mech anisms are suggested for this change. These mechanisms include decreases in Ca2+ uptake required for the actin-myosin coupling mechanism and change in endoplasmic reticulum functioning secondary to altered motor neuron activity and muscle activation. Because the soleus muscle is well established to be more sensitive to decreased neuromuscular activation (Roy and Ac osta 1986; Roy, Talmadge et al. 1998), it is studied more extensively to explore the contract ile properties of the skeletal muscle following SCI. Similar to animal studies, chronically paraly zed individuals with complete SCI exhibit faster contractile properties of skeletal musc les. Depending upon the muscle studied, rates of force rise (contraction rates) ha ve been reported to be signifi cantly greater (+52%) and halfrelaxation times are markedly shorter (-19% to 2fold) as compared to a healthy muscle. Similarly, contractile properties from chronically paralyzed upper extremity thenar muscles also show fast muscle characterist ics (Thomas 1997). Note worthily, at relatively earlier time points after injury (within 6-24 weeks post SCI), contractile properties of the human paralyzed muscles are rather variable. Time to peak tension of the paralyzed soleus and quadriceps muscles, for example, has been demonstrated to remain the same or even slower than control values (Shields, Law et al. 1997; Castro, Apple et al. 2000). This disparity in the contractile prope rties of acute and chronic paralyzed muscles is attributed to the relationshi p between contractile speed and MHC isoform expression (Burnham, Martin et al. 1997; Dupont-Versteegde n, Houle et al. 1998). At 6-24 weeks, fiber type conversion and hen ce MHC type expression has not occurred thereby retaining the contractile characteri stics of the paralyzed muscle (S hields, Law et al. 1997; Castro, Apple et al. 1999). As far as the relaxation time is concerned, the half-relaxation times are slower

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36 in the skeletal muscle of acutely injured subjects (-47%). Briefly, the rate of Ca+2 uptake or the rate of the cross-bridge detach ment is surmised to have been compromised thereby slowing the overall muscle relaxation rates (Shields 2002). Altered contractile properties of a para lyzed muscle have important functional implications. Paralyzed human muscle fatigues much more rapidly than a healthy muscle. Increased fatigability of skeletal muscle as char acterized by force loss over repeated contractions with otherwise non-fatigable electrical stimulation parame ters is a common phenomenon of chronically paralyzed slow muscles (Shields 200 2). Declines in isomet ric force production over repetitive bouts of contraction ar e as large as 60% in the paralyzed skeletal muscle as compared to 40% declines in controls. Furthermore, muscle endurance, as measured by the muscles ability to maintain force levels when stimulated repeat edly, is drastically reduced following SCI. While a healthy quadriceps muscle can maintain force levels of more than 30% of maximum isometric contraction for as long as 10 minutes, a paralyzed quadriceps muscle can maintain similar force levels for only less than 4 minutes (Gerrits, De Haan et al. 1999) Such weakness and fatigability of locomotor muscles potentially limits the use of rehabilitation programs that emphasize using functional electrical stimulation to achieve standing and walking. 1.4.4 Metabolic Adaptations: Alter ed Oxidative Capacity Alteration in skeletal muscle mitochondrial oxidative capacity and subsequent impairments in endurance are reflected by a variety of mu scle adaptations following SCI. Mitochondrial oxidative capacity is a functi on of mitochondrial volume, comp etence and oxygen delivery to the mitochondria (McCully, Mancini et al. 1999; Kemp, Roberts et al. 2001 ). It is commonly used as an estimate of oxidative energy supply to the muscle and refl ects overall muscle endurance. Mitochondrial oxidative cap acity is altered follo wing SCI (Jiang, Roy et al. 1990; Jiang, Roy et al. 1990; Otis, Roy et al. 2004). Histochemical measurements of various oxidative enzyme

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37 activities from muscle homogenates or biopsy sa mples serve as markers of muscle metabolic capacities. Additionally, non-invasive measurements of metabolic capacities by magnetic resonance spectroscopy have been well established and are found to significantly correlate with mitochondrial enzymes activity measured with biopsy specimens (McCully, Fielding et al. 1993). Generally, marked alteration in muscle oxida tive capacity is reported following SCI in humans. Martin et al and Roch ester et al have shown that the chronically paralyzed human tibialis anterior muscle shows marked decrease in succinate dehydrogenase (SDH) activity as compared to able-bodied individuals (Martin, St ein et al. 1992; Rocheste r, Barron et al. 1995). Kjaer et al have demonstrated a marked decrease in activity of a variet y of oxidative enzymes (almost 1.5 to two fold) from the vastus lateralis muscle of men who sustained a complete spinal cord injury for almost 12 years (Kjaer, Mohr et al. 2001). Hartkopp et al report a significantly lower oxidative capacity in the upper extremity wris t extensor muscles of pe rsons with more than five years of SCI (Hartkopp, Harridge et al. 2003). An overall decrease in mitochondrial oxidative capacity, also measured by decrease s in mitochondrial DNA content, (reflective of mitochondrial protein content), capillary densit y and blood flow are reported following chronic SCI in humans (Scelsi, Marchetti et al. 1982; Wang, Hiatt et al. 1999). Furthermore, as early as 11 weeks after complete SCI in humans, Gregor y et al have shown decease in SDH enzyme activity by almost 41% (Gregory, Vandenborne et al. 2003). However, in a follow up study at 24 weeks after injury in the same subjects the S DH activity returned to control values (Castro, Apple et al. 1999). Interestingly, oxidative enzyme activity in anim al models of SCI is rather muscle specific. After six months of both spinal cord transection a nd spinal isolation, the cat soleus muscle either

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38 enhances or maintains SDH activity in comparison to control rats implying maintenance of muscle oxidative potential after ch ronic SCI in the slow skeletal muscle (Jiang, Roy et al. 1990; Graham, Roy et al. 1992). Elevated SDH activity is also demonstrated in the spinalized rat soleus muscle 6 months after injury (Otis, Roy et al. 2004). Though mechanisms to elucidate the elevated oxidative enzyme activ ity are unknown, investigators ha ve attributed the greater oxidative capacity in slow muscle to the pres ence of different fiber type composition of the paralyzed slow muscle. Apparen tly, the paralyzed slow soleus muscle possesses an unusual proportion of hybrid fibers (Roy, Talmadge et al 1999) that in turn are purported to have inherently larger oxidative capacities than the typi cal type 1 muscle fibers (Rochester, Barron et al. 1995; Otis, Roy et al. 2004). In fact, mixed muscle such as the gastrocnemius and the vastus lateralis with predominantly fast muscle fibers (type IIx and IIb) show significant declines in SDH activity following spinalization in both ca ts and rats (Jiang, R oy et al. 1990; Gregory, Vandenborne et al. 2003). In an animal model of spinal transection, Duroza rd et al have used non-invasive magnetic resonance spectroscopy to de monstrate declines in oxidative capacity of the gastrocnemius muscle in rats. Based upon their data, the authors also suggest transition in source of energy supply from oxidative to anae robic pathways for muscle metabolism following paralysis (Durozard, Gabrielle et al. 2000). Though ch ange in enzyme activity is associated with phenotypic alterations following SCI (Martin, Stein et al. 1992; Rocheste r, Barron et al. 1995), change in muscle enzyme activitie s is also thought to occur indepe ndent of shifts in fiber types composition (Castro, Apple et al. 1999; Gregor y, Vandenborne et al. 2003). Accordingly, the existence of any association between enzyme activ ity and fiber type alteration after SCI remains a subject for debate.

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39 Irrespective of the cause, decrease in skelet al muscle oxidative capacity following muscle inactivity is purported to ultimately associat e with sub-maximal endurance and exercise performance. Decrease in overall muscle enduran ce as measured by the fatigue index (defined as the decrease in force production with repeated muscle contraction) accompanies muscle inactivity following spinaliz ation (Roy 1998, Shields 2002, Gerri ts 1998). Researchers have partly attributed muscle fatigability following SCI to altered muscle oxidative capacity. This premise is somewhat supported by the observation that reduced physical tr aining in able-bodied individuals causes significant dec lines in oxidative enzyme activity, which in turn drops long term skeletal muscle endurance (Houston 1979, Hickson 1982 from Kjaer 2001). Secondly, conversion to faster muscle fiber phenotype at the cost of slower muscle fibers predisposes the paralyzed muscle to acquire fast er contractile speeds thereby maki ng it easily fatigable (Shields, Law et al. 1997; Gerrits, De Haan et al. 1999; Hutchinson, Li nderman et al. 2001). Lastly, studies have also linked reduced skeletal muscle oxidative capacity in the development of a major contributor of cardiovascular complications, namely altered insuli n resistance (see below for more on insulin resistance) (Brehm, Krssak et al. 2006; Schrauwen-Hinderling, Kooi et al. 2007). 1.4.5 Metabolic Adaptations: Altered Glucose Homeostasis Cardiovascular com plications were the second leading cause of death in individuals with SCI until 1990 (Bravo, Guizar-Sahagun et al. 20 04; Jacobs and Nash 2004). With increasing survival rates of SCI and the deve lopment of multiple cardiovascula r risk factors, it is now the leading cause of death after chr onic injury (Bravo, Guizar-Sahagun et al. 2004). Risk factors for these complications include development of insulin resistance, dyslipidemia (elevated lowdensity lipoproteins, depleted high-density lipopro teins and increase in total cholesterol), overall obesity and diabetes, a sedentary lifestyle and limited exercise (Bauman and Spungen 2001;

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40 Bravo, Guizar-Sahagun et al. 2004; Jacobs and Nash 2004). Of these, glucos e intolerance due to development of insulin resistance is suggested as the main cause of cardiovascular disease after SCI (Bravo, Guizar-Sahagun et al. 20 04). Insulin resistance is the re sistance of target cells such as muscle or liver tissue to insulin. The target ce lls (muscle and fat that ta ke up glucose) are said to be resistant to glucose uptake and/or are less sensitive to the peripheral uptake of blood insulin. Elevated blood glucose and insulin levels s ubsequently precipitate as one of the earliest hallmarks in the development of type 2 diabet es mellitus (Jacob, Machann et al. 1999). In fact, persons with complete and incomplete SCI are predisposed to hyperinsulinemia and an imbalance in glucose homeostasis with subseque nt development of insulin resistance (Bauman, Spungen et al. 1999; Bauman and Spungen 2001). Additional risk factors include relative increases in adiposity, a sedent ary lifestyle and muscle atroph y. The association of skeletal muscle and insulin resistance is explained below. Skeletal muscle tissue is responsible for the majority of the insulin mediated glucose disposal in the body (Goodpaster and Kelley 1998; SchrauwenHinderling, Hesselink et al. 2006). The study of intramuscular fat content has ga ined considerable attention because of the association of high levels of skel etal muscle lipid with insulin re sistance. Existence of lipid in skeletal muscle was first identified by De nton and Randle in 1967 (Denton and Randle 1967). However, it is only since the last decade, that wi de arrays of studies ha ve reported associations between intramuscular fat and insu lin resistance (Perseghin, Scifo et al. 1999; Furler, Poynten et al. 2001). Pan et al first established this relationship; th ey found that muscle triglyceride content correlates with insulin resistance irrespective of total body adiposity (Pan, Lillioja et al. 1997). Lipids are typically stored in the skeletal muscle in the form of intramyocellular lipid (IMCL) or extramyocellular lipid (EMCL). EMCL is the plate or tube shaped fatty infiltrate located outside

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41 the myocyte and between muscle fibers (Szczepan iak, Babcock et al. 1999; Boesch, Machann et al. 2006). EMCL can also be located intramus cularly as adipose tissue along with fascia separating fascicles of the same muscle, inte r-muscularly along with fa sciae between adjacent muscles and in subcutaneous fat layers. EMCL is purported to be a long-term storage depository and suggested as being metabolically relatively in ert. It is utilized for energy production during very low intensity exercises and has no known correlation with insulin resistance (Boesch, Slotboom et al. 1997). In contrast, electron microscopic studies have established IMCL to be the fat located within the cytoplasm of muscle cell close to the mitochondria (Boesch, Slotboom et al. 1997; Schrauwen-Hinderling, Hesselink et al. 2006). Because of its proximity to the mitochondria and its relatively larg er content in oxidative fibers, IMCL is regarded as an energy source for mitochondrial fat oxidation during rest and long-term endurance activities (Krssak, Petersen et al. 2000; Boesch, Machann et al 2006; Schrauwen-Hinderling, Hesselink et al. 2006). IMCL can be mobilized and utilized with in hours of physical act ivity, recovers after several hours following exercise and finally r eaches normal levels within days (Boesch, Machann et al. 2006). However, excessive accumula tion of IMCL can have a negative impact on insulin signaling to induce insulin resistance (see below). A variety of studies have confirmed the correlation of IMCL with insulin resistance in healthy persons as well wi th obesity and Type 2 diabetes mellitus (Jacob, Machann et al. 1999; Ke lley, Goodpaster et al. 1999; Krssak, Falk Petersen et al. 1999; Perseghin, Scifo et al. 1999; Sinha, Dufour et al. 2002; Goodpaster and Wolf 2004; Schrauwen-Hinderling, Hesselink et al. 2006). Investigat ors have reported moderate to strong associations of IMCL with several markers of coronary artery disease (interleukin -6, homocysteine) and pre-diabetes states (insulin, insulin resistance) (Krssak, Falk Petersen et al. 1999; Weiss, Dufour et al. 2003; White, Ferguson et al. 2006). Accordingly, IMCL content is

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42 suggested to serve as a potent ial non-invasive marker of insulin resistance. However, one limitation of using IMCL to describe insulin resi stance is that IMCL is largely influenced by a host of factors including age, gender, diet, obe sity, physical inactivity, exercise, genetics, ethnicity as well as the muscle s studied (Forouhi, Jenkinson et al. 1999; Sinha, Dufour et al. 2002; Goodpaster and Brown 2005; St ettler, Ith et al. 2005; Schrauwen-Hinderling, Hesselink et al. 2006; Boesch 2007). IMCL leve ls are reported to be elevated in older people compared to young (Cree, Newcomer et al. 2004), females than males (White, Ferguson et al. 2006), people with high fat diets compared to lo w fat diets (Stettler, Ith et al 2005) and higher levels in obese as compared to lean persons (Sinha, Dufour et al. 2002). Additionally, lipid content varies from muscle to muscle with the slow soleus muscle for example, showing almost three times the IMCL content as compared to the fast tibialis ante rior in the same subject (Rico-Sanz, Thomas et al. 1999; Hwang, Pan et al. 2001; Vermathen, Kreis et al. 2004). Accordingly, large variations in IMCL continue to exist even in non-diseased hea lthy individuals, making it difficult to relate it with insulin resistance. These f actors therefore make it important to have stringent inclusion criteria even for healthy controls in a study de sign. Nevertheless, stud ies have attempted to document inter and intra-subject variations in the IMCL content of more than one muscle, making it feasible to still conduct these measures a nd reliably relate them with insulin resistance (Boesch, Decombaz et al. 1999; Torriani, Thomas et al. 2005). Positive correlations between IMCL and insulin resistance have been reported in sedentary individu als irrespective of sex, body weight and physical fitness (Sinha, Dufour et al. 2002; White, Ferguson et al. 2006). Presently, the advent of new sp ectroscopic techniques continues to encourage investigators to explore the relationship between IMCL and in sulin resistance. Accordingly, simple ratio

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43 measurements that yield intramyocellular fat content has the potential to serve as an in vivo biomarker of insulin resistance, which might prove beneficial in a variety of patient populations. In persons with either complete or incomplete SCI, a three to four fold increase in thigh intramuscular fat is reported as compared to able bodied adults (E lder, Apple et al. 2004; Gorgey and Dudley 2006). These studies also reported that their patient group had high plasma glucose and insulin levels that were posi tively correlated to intramuscular content. Moreover, a decrease in skeletal muscle mass in this patient populati on was inversely correlated with plasma glucose levels suggesting that skeletal muscle atrophy and intramuscular fa t both contributed towards the decrease in insulin sensitivit y. Taken together, it appears th at persons with SCI may be predisposed to the development of this health rela ted risk factor and may also be at a relatively greater risk for the development of type 2 diabetes. In the present work, an attempt is made to qua ntify IMCL in the skeletal muscle of persons with incomplete SCI (Chapter 7). Mechanisms linking decreased insulin sensitivit y (or increases in insulin resistance) with elevated IMCL remain ambiguous, but a variety of factors, either singly or in combination have been surmised for this association. An increased level of IMCL is a possible resu lt of greater uptake of fatty acid in muscle (Hegarty, Furler et al. 2003; Hulver and Dohm 2004). Studies have found strong negative correlations between adiponectin protein and IMCL measures in both obese and non-obese adolescent and adult populations (Stefan, Vozarova et al. 2002). Adiponectin, a protein released from adipose tissue is suggested to maintain normal triglyceride levels in blood. Elevated IMCL content from the skeletal muscle is establishe d as a strong predictor of adiponectin levels suggesting a potential role of the protein in the accumulation of tr iglycerides in skeletal muscle

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44 tissue (Stefan, Vozarova et al. 2002; Weiss, Dufour et al. 2003). Elev ated IMCL content, in turn, increases intracellular fatty acids and their derivatives. Intermediates of fatty acid metabolism at the intramyocellular level (triacylglycerols, diacylglycerol, ceramide and fatty acyl CoAs) inactivate insulin action by inhibi tion of specific steps in the insulin-signaling pathway that is normally responsible for glucose uptake in the myocyte (Itani, Ruderman et al. 2002; Hegarty, Furler et al. 2003; Hulver and Dohm 2004). Studies report that th e elevated diacylglycerol and fatty acyl CoA levels secondary to increase in musc le triglyceride, increase s the protein kinase C activity. This enzyme can directly inactivate insu lin receptors for uptake of insulin in muscle. Similarly, elevated ceramide levels mediate i nhibition of signaling pa thways by release of specific enzymes (Hegarty, Furler et al. 2003; Hulver and Dohm 2004). Another proposed mechanism suggests that increase in IMCL cont ent leads to a decrease in insulin receptor synthesis in the skeletal muscle. Interference with insulin receptor function ultimately leads to an increase in insulin resistance, which in turn stimulates hepatocytes to increase serum triglycerides and decrease serum HDL (White, Fe rguson et al. 2006). Theref ore it appears that accumulation of IMCL serves as a mediator rather than being a direct cause of decreased insulin action. Collectively, decreased insulin action (upt ake in muscle) is proposed as the major contributor of insulin resistan ce in the myocyte. Though elevated plasma fatty acid levels ultimately increase IMCL content, which can lead to the development of insulin resistance, the reverse route that insulin resistance can lead to an elevation in IM CL content cannot be ruled out. 1.5 Summary W ith technological advancements in the effici ent management of acute SCI, the proportion of and life expectancy of persons with an incomp lete injury has considerably increased. Varying degrees of tissue sparing and muscle loading presents the incomple te SCI population with

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45 variable degrees of paralysis and paresis and he nce distinct degrees of functional limitations. A host of skeletal muscle adaptations including muscle atrophy, muscle fiber type conversion, declines in contractile function, predisposition to muscle injury, fatty tissu e infiltration, altered oxidative capacity and glucose homeostasis are well established after complete SCI in animals and humans. Despite the obvious motor dysf unctions, physiological muscle adaptations following incomplete spinal cord injury are relatively unstudied. With the advent of new therapeutic strategies and advances in SCI resear ch aimed at recovery of lost functions, it is imperative that the machinery for movement and locomotion remain intact. The overall objective of the present work was to elucidate muscle adap tations after incomplete spinal cord injury in humans and in the rat model of incomplete SCI.

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46 CHAPTER 2 SPINAL CORD INJURY AND LOCOMOTOR TRAINING 2.1. Locomotor Function after SCI Initial insult to the spinal cord profoundly im pacts almost all biological systems of the body. Specifically, neuromusculoskeletal deficits following the injury tremendously impair the motor performance and locomotor capabilities of individuals with SCI. Following incomplete SCI; there always exists some degree of spont aneous recovery. As a result, persons with incomplete SCI exhibit variable paralysis and pare sis of affected muscles, typically resulting in impaired motor performance and varying degr ees of functional limitations (Subbarao 1991; Tang, Tuel et al. 1994; Burns, Golding et al. 1997). Similar to persons with complete-SCI, persons with incomplete-SCI exhibit a variety of clinically relevant motor and functional deficits, including local muscle fatigue, weakness of affected muscles (Sloan, Bremner et al. 1994; Johnston, Finson et al. 2003) and diminished capacity to ambulate (Waters, Adkins et al. 1994; Ulkar, Yavuzer et al. 2003). Other studies have show n a significant reduction in ambulatory capacity, with functional deficits in gait including a reduced gait speed, step frequency; stride length and longer durations spent in the dou ble support phase of gait cycle (Melis, Torres-Moreno et al. 1999; van der Salm Nene et al. 2005). Loss of ambulation is the most obvious functional limitation as sociated with a SCI and regaining walking capability one of the main aims of this patient cohort (Kilgore, Sc herer et al. 2001). In a report by Iezzoni et al, Walking holds profound symbolic importance. Nowadays, upright movement permeates American aphorisms, connoting independence, au tonomy, self-reliance and strength. Inability to walking tall creates a physical need to rest ore mobility and an emotional need to restore ones core sense of value and place in the wo rld (Iezzoni 1996). With the remarkable focus that is now spent for new treatments for SCI, a lot of energy is spent in exploring the

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47 neuromuscular physiological impairments asso ciated with locomoti on (Dobkin 2000; Wolpaw and Tennissen 2001; Dobkin and Havton 2004; Fouad and Pearson 2004). This approach has in turn held optimism in determining mechanisms associated with other functional impairments seen in this patient population such as urinati on, sexual, and bowel functi on that at present are less understood. Over the past tw o decades, researchers in the field of SCI have come to recognize the remarkable potential of the central nervous system to regenerate following its injury. Accordingly, investigators are discove ring new therapies to improve the locomotor capability of persons with SCI. The rest of this chapter includes a brief review of: a) Conventional rehabilitation for persons with SCI b) limitations of cu rrent clinical practice in SCI c) spinal cord plasticity and principles of ne uroplasticity d) a potential paradigm shift in enhancing locomotor function after SCI e) locomotor training in animal and humans 2.1.1. Conventional Rehabilitation Therapies after SCI Present reha bilitation goals in a clinical setup for individual s with SCI are re-entry into community, energy conservation and functional inde pendence. To achieve these goals, much of the rehabilitation is focused on task-oriented tr aining along with compensation of lost abilities (Thompson 2000; Dobkin and Havton 2004). Patients are trained to perfor m a functional task that is specific to the goal to be achieved, but at the same time, achievement of the task accompanies using spared residual function and/or modifications in a persons environment. Thus, patients use whatever residual sensorimotor functions persist after the injury along with use of assistive aids to maximize their self-c are, mobility and commun ity roles. These task specific strategies are also called compensatory techniques in current pr actice (Mathiowetz and Haugen 1994). Examples of compensatory strategi es that are commonly s een and/or taught in clinical practice for individuals with SCI include use of assistive aids (example: orthoses/crutches/wheelchair), ne w movement strategies (example: hip hiking) (Bell 1955) and

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48 electrical stimulation induced movements (Pec kham, Keith et al. 2001), (Dobkin and Havton 2004). Use of a wheelchair after a SCI is one of the most common compensatory strategies. A lower extremity motor score (LEMS) of 20/50 or le ss indicates that persons with SCI are likely to be limited ambulators (ASIA 2001). Clinically a wheelchair prescription is a common choice for such persons. However, sometimes, wheelchair use stems from the need to enhance the mobility and independence in the community [for reference see (Minkel 2000). Functional electrical stimulation (FES) is bein g used extensively in improving functional motor performance of persons with SCI. Generall y, FES devices are designed to comprise of a control unit and stimulating electrodes. The cont rol unit translates signals from sensors or a voluntary movement onto the stimulating electrodes that are taped over th e skin or surgically implanted near the nerve for specific muscle contraction. Mild electrical stimulus elicits muscle contraction that translates into muscle movement. By adequate alterations in stimulus parameters of the FES device, persons with SCI gain assistance in movement initiation and control. FES has been used widely to facilita te a variety of functional ac tivities following SCI including controlling upper extremity movements and gras ps, improving cardiovascular conditioning and breathing, training to walk for short distances, standing up for transfers and c ontrolling bowel and bladder function (Creasey, Grill et al. 2001; Johnston, Fi nson et al. 2003; IOM 2005). Physiological effects of FES involve increase in muscle mass, aerobic metabolism and maintenance of bone density (Postans, Hasler et al. 2004); which ultimately play an important role in prevention of musculoskeletal complicatio ns after SCI. However, the role of FES in improving the ambulatory capability after SCI is limited. Expense of the equipment, assistance needed for set up, time taken from other daily ac tivities, and the meager daily effects of FES

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49 exercise deter patients from its use (Dobkin a nd Havton 2004). The gait pa tterns with use of FES are unrefined and allow for short-distance ambula tion. Moreover, two major deterrents to the use of FES for functional performance include a) presence of an optim al degree of initial motor and sensory control in the lower extremities and b) a relatively lower level of incomplete SCI (lower thoracic); thereby making it suitable for use for only a limited population of SCI. Therefore, FES devices for assisting in ambulation are limited in scope. Most importantly, though FES signals are proposed to alter central activ ity dependent plasticity, sensory feedback induced by FES is not likely to cause any persistent central sensori-motor activati on or motor skills learning [for review see (Dobkin and Havton 2004). So ultimat ely, FES still represents a compensatory strategy that leads to non -use of the persons neurological systems and added disability in the long-term still persists. [Note: Though numerous other compensatory strategies concerning other body systems are commonly implemented in rehabilitation (5), in this discussion, the primary focus is made on techniques aimed at enhancing locomotor performance]. 2.1.2 Limitations of Compensatory Rehabilitation Strategies Com pensatory techniques are focused little, if any on the neural processes necessary or neurophysiological adaptations occu rring secondary to performance of a functional task. In fact, there is no evidence that programs of rehabil itation have any effect on restoring impaired nervous system function or enha ncing natural recovery following disease or injury (McDowell 1994). It appears that by using compensatory strategies and provi sions in the environment, one completely ignores what a persons body is capab le or might be capable of doing. Any potential for the person to explore the cap abilities of his or her body system s to regenerate or restore is completely neglected. Today community environments are becoming easily accessible for people using wheeled devices as their primary means of mobility. Though this approach definitely

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50 permits successful mobility of individuals in th e community, long-term effects of such therapy can precipitate further disability. In fact, impact of long-term us e of a wheelchair causes further disuse and negative plasticity of the neuromus cular system, which in turn creates further dependence on the use of a wheelchair for mobility. As it turns out, such interdependence leads to a host of psychosocial and physiological cons equences that further enhance disability. For a therapist, wheelchair provision for hi s/her client might be perceived as an opportunity of mobility, but a young mans opinion about his wheelchair has been You might as well stick me in a damn closet. That wheel chair just makes me th ink of how hopeless I am (Minkel 2000). Thus, psychologica lly, many people interpret depende nce on assistive aids as an indication of greater disability rather than an option for increased mobility (Iezzoni 1996). Physiologically, inactivity/disuse of the affected body part leads to more systemic complications (muscle atrophy, more predispositio ns to fractures, contractures, etc) and further dependence on walking aids for mobility. Overcompensating with spared motor function limits the capacity of the central nervous system to adapt (Barbea u, Fung et al. 2002). As such, prolonged disuse conforms to the frequently used adage-use it or lose it. Few studies also associate disuse with suppression of genes that attempt axonal rege neration (for reference see (Dobkin and Havton 2004). 2.2 Spinal Cord Plasticity The m ain reason for use of compensatory t echniques to recover function after SCI has evolved from the doctrine that the spinal cord is nothing more than a hard-wired system that simply serves as a conduit for ascending and descending axons. More than 3500 years ago, SCI was referred to as A disease that cannot be treated (from (Vikhanski 2001). This dogma dominated for several centuries. In fact, Ram on Cajal in 1906 was awarde d the Nobel Prize for discovering the regeneration capabi lities of peripheral nerves wh ile simultaneously confirming

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51 that as is well known, the central tracts are in capable of repair (Vikhanski 2001). A true paradigm shift in the field of spinal cord ne uroscience occurred in th e 1950s. Windle, Liu and Chambers first suggested sprouting (referred to as growth of short shoots or sprouts from healthy nerves to compensate for damaged fibers ) of healthy nerves within the central nervous system as a mechanism of recovery in experime ntally spinalized animals (Windle 1954; Liu and Chambers 1958). However, scientific proof for ne rve repair and regeneration within the spinal cord emerged only in the 1980s (Richardson, McGu inness et al. 1980). In the past two decades, animal and human research has revealed the potential capability of the CNS to repair and regenerate following its injury (Dobkin 2000; Thompson 2000; Rosenzweig and McDonald 2004). Currently, convincing evidence from spinal cord repair lit erature suggests that the most important contributors inhibiti ng growth of neurons is the presence of an non-permissive environment that consists of a mechanical barrier such as a scar tissue, insufficient neurotrophic factors for axonal regeneration and the presence of inhibitory molecules within the scar tissue (Fouad and Pearson 2004; Rosenberg, Zai et al. 2005). In the field of SCI research, vigorous attempts have been made in understanding the ba sic mechanisms of neuronal cell growth, death and repair; influence of central and periphe ral neural signals on motor neuronal output; identifying spinal pathways (such as the central pattern generators) for locomotor function and strategizing new therapeutic modalities based on understanding of these mechanism. Neurons within the spinal cord have a complex interac tion with cortical neurons sub-cortical neurons (neurons within brainstem), motor neurons and a fferent pathways. Indeed, the spinal cord has a large potential for plasticity at multiple levels of its architecture including afferent neurons that enter the cord, inter-neu ronal population interposed between these inputs and the motor neurons, ascending axons within the cord, descending axons within the cord, synap tic connections on the

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52 motor neurons and motor neurons themselves. Furt hermore, alterations in neurotransmitter and neuro-modulator activity along with cortical ch anges accompany SCI (Bregman, Coumans et al. 2002; Dobkin and Havton 2004). Rightfully, the term neuroplasticity has al so been extended to the spinal cord. Neuroplasticity is a permanent change in the st ructure and function of the nervous system in response to experience, ones state of health and disease and experimental manipulation or injury. It is important to note that persistent changes both in the peripheral and descending input to the cord via myriad of factors such as prac tice, trauma, and disease will cause plasticity at multiple levels of the nervous system both spinal and supraspinal. Therefore, acquisition of any new behavior whether it is skilled learned be havior through prolonged pract ice (or disuse) or an abnormal response associated with CNS disease or compensatory movements is inevitably associated with neuroplasticity. Interestingly, th e spinal cord is so flexible that spinal neuroplasticity is a function of experiences throughout ones subsequent life (Wolpaw and Tennissen 2001). In this section, ma jor focus is made on a specific, activity-dependent plasticity (walking) of the spinal cord. Activity dependent plas ticity is the lasting change that occurs in the spinal cord because of activity (sensorimotor or sensory inputs) from the periphery or the brain. Such a change finally affects output of the spinal cord. However, to retain neuroplasticity, few basic rules are essential for its expression. These rules, fo cused on physiological recovery following neural injury eventually form the funda mental principles of current neurorehabilitation techniques. a) Repetition/practice: Repeated performance of a task is invariably associated with learning. Thus, practice makes perfect. Physio logically, learning is bu t a reorganization of neuronal mechanisms (Kandel 2001). Repetition stimulates multiple neurons and consequently

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53 alters neural activity that pe rsists for long durations even after the activity ceases. The physiological mechanisms associated with learni ng range from neuronal pre-synaptic and/or post-synaptic structural changes, modulation of neurotransmitters, expression of new proteins, activation of silent synapses, growth of new ne ural spines (Kandel; Kandel 2001) and changes in representation of the cortical homunculus (Byl and Melnick 1997; Byl 2003). Therefore, for such permanent physiological changes to manifest, the stimulus that causes it must be repetitive. Moreover, whether the repetitive experience is in the form of excess or diminished stimuli (example activity versus disuse), plasticity is in evitable. Several studies have demonstrated the critical role of repetition for neural plasticity (Kandel; Wolf and Segal 1996; Beaumont and Gardiner 2003). For example: sing le shock of tactile electrical stimulus to the aplysia-siphon gives rise to a memory lasting only minutes, while four or five spaced shocks gives rise to a memory lasting several days (Kandel 2001). So leus H-reflex modulation in monkeys was demonstrated after 3000-6000 trials/day (Wolpa w 1987). Therefore, to promote learning of a new motor task or for retraining, tremendous emphasi s is placed on repetiti on as one of the major treatment principles in neurorehabilitation research (Edgerton, Roy et al. 1992; Behrman, Lawless-Dixon et al. 2005). b) Task specificity: The nervous system will respond to whatever stimulus it is exposed to. Specific sensory stimulus will stimulate plasticity of appropria te neuronal circuits. In fact, only the activated neural pathways will respond to the stimulus. Moreover, any motor output is generally associated with and/or is a result of precise sensor y experience. For example: the normal pattern of walking requires afferent sensory stimulus from the feet (Rossignol, Chau et al. 1996; Dietz and Harkema 2004; Duysens, Bastiaanse et al. 2004). To enha nce firing of specific neuronal circuits and hence strengt hen their synaptic efficiency, the same activity needs to be

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54 entrained. For example: spinalized cats that were trained to walk had faster walking speeds than animals that were trained to stand only (Hodgs on, Roy et al. 1994). In fact, locomotor training that closely simulates normal walking pattern has been suggested to improve functional locomotor outcomes while conventional rehabilitati on approaches have not shown similar effects of training (Barbeau, Norman et al. 1998; Dobkin BH 2003, Sep). c) Pattern of practice: Whether stimulus of practice is continuous or intermittent clearly dictates plasticity. Generally, interm ittent practice is a more appropriate stimulus than continuous under wide circumstances of plasticity. For example: intermittent and not continuous hypoxic exposure elicits long term enhancement of inspiratory motor output known as long term facilitation (LTF) (Baker and Mitchell 2000). Also, continuous sessions of ta ctile stimulus to the aplysia-siphon leads to a short-term memory for habituation of the gill-w ithdrawal reflex, while intermittent sessions produces a long-term memo ry (Kandel 2001). One of the probable reasons for this pattern is the molecular neural mechanism associated with plasticity. Mechanisms of phrenic motor neuron LTF for instance, involve in termittent release of se rotonin, which in turn initiates a cascade of events for protein synthe sis. The elevated protein increases synaptic strength and hence phrenic motor output (Bake r-Herman and Mitchell 2002). In fact, continuous release of serotonin (due to continuous hypoxia) may have an inhibitory effect on the presynaptic receptors for protein synthesis, there by hindering plasticity. Th us, based on findings in lower animals, it appears that interval training might be better preferred over massed practice. 2.3 Locomotor Training: A Paradigm Shift With the above background in mind, one can de duce that rehabilitation techniques aimed at enhancing locomotor function following SCI should minimize the use of compensatory strategies. As such, a physiologi cally based intervention program se ems to be a more scientific approach towards recovery. As is well rec ognized now, regaining neuromuscular functions

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55 following a neurological injury largely depends upon recognizing the pathophysiology of injury. The current scientists perspective of recovery based therapy dwells from the notion Much needs to be done in the management of neurol ogical disability and it will only be through basic science clarifying the mechanisms of disability and the application of scientifically sound outcome measures that interventions w ill make a real difference (Thompson 2000). Consequently, approaches for rehabilitation of the SCI cohort needs to be emphasized on taskspecific training that can trigge r appropriate neural mechanisms When developing therapeutic interventions to enhance functional recovery after SCI, an understa nding of the underlying physiology of neuromuscular responses that occur af ter this type of injury will promote different interventions that rely less heavily on compen satory rehabilitation. Locomotor training using treadmill and overhead body weight support (LT) is one such rehabilitation strategy that is based on the neuroplasticity principles of walking r ecovery and has gained tremendous momentum in improving walking ability of persons with SCI (see below). The LT approach has proved more beneficial than conventional therapy in chronic incomplete spinal cord injuries. The following section includes a brief historical perspective an d description of LT in an imals and in individuals with SCI. 2.3.1 A Historical Perspective and Locomotor Training in the Spinal Cord Injury Animal Model One of the most rem arkable finding in spinal cord injury rehabilitation research is that spinalized cats were able to step with thei r hind limbs on a moving treadmill. This phenomenon evolved from the pioneering works of Shurrager and Dykman who emphasized the importance of regular repetitive locomotor movements in sp inalized mammals (Shurrager and Dykman 1951). Later in the 1970s, Grillner et al and Edgerton et al were able to demonstrate the ability of the isolated spinal cord to produ ce cyclic activity between agonist and antagonist muscle groups

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56 with levels of coordination that mimicked normal locomotion (Grillner and Zangger 1975; Edgerton, Roy et al. 1992). Since then, numerous investigators have demonstrated the stepping phenomenon in spinalized mammals on a movi ng treadmill (Forssberg, Grillner et al. 1980; Lovely, Gregor et al. 1986; Barbeau and Rossignol 1987; Edgert on, Roy et al. 1992). A typical training protocol in the animal model involves walking of a thoracic spinal cord lesioned animal on a treadmill. Initially stepping is possible only with manual assi stance by trainers that assist with stepping of the hind limbs and applica tion of a non-specific sensory input from the perineum, abdomen or the tail. Generally, after three months of daily weight supported treadmill training, the amount of support required to step declines and hind limbs move into a more rhythmic alternate pattern that closely rese mbles normal walking (Edgerton, Roy et al. 1992). Comparative data show that trained spinalized cats show better ability to st ep than those that are allowed to recover spontaneously (de Leon, Hodgson et al. 1998). Stepping is also associated with recovery of EMG patterns in the paralyzed muscles close to the pre-spinalized patterns (Lovely, Gregor et al. 1990; Hodgson, Roy et al. 1994). Note wo rthily, training outcomes in incompletely injured cats differ from spinalized cats. Incompletely injured cats eventually recover voluntary quadrupedal locomotion overgr ound and on the treadmill within three days to three weeks depending upon the seve rity of injury (Ro ssignol, Chau et al. 1996; Rossignol, Drew et al. 1999). As mentioned in Section 1.3.1, the SCI rat mode l has gained enormous recognition for use in SCI research. Accordingly, t hough much of the basic concepts in the neuronal control of walking were established in the cat model, these findings have also been tr anslated to the spinal rat model. Over the past few years, investigators have assessed the effects of treadmill training on the recovery of rats following spinal tr ansection (Moshonkina, Avelev et al. 2002;

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57 Moshonkina, Gilerovich et al. 2004) as well as contusion (Thota, Carl son et al. 2001; Multon, Franzen et al. 2003; Fouad and Pearson 2004; Stevens, Liu et al. 2006). In ra ts spinalized at the thoracic level as adults, nine weeks of LT (5days/week, 10mi nutes, starting on day one after operation) has been demonstrated to hasten hind limb joint movements in the spinalized rats, generate separate voluntary joint movements in the hind limbs and produce coordinated stepwise limb movements on a treadmill. This locomoto r function has been shown to coincide with morphologically intact motor neurons in the sp inal cord (Moshonkina, Avelev et al. 2002; Moshonkina, Gilerovich et al. 2004). In spinally contused rats, fe w studies in recent years have shown that LT improves overground hind limb locomotor function. Thota et al reported improvements in lower limb joint kinematics of spinally contused rats that led to recovery of coordinated locomotor function after 7 weeks, al beit with deformities in gait (Thota, Carlson et al. 2001). In 2003, Multon et al induced spinal contusion by compression and found significant functional gains in the trained animals throughout a training period of 9 weeks (30 min/day, 5/week). The training starting at 3 da ys post injury enabled the trained rats to voluntarily support their body weight at the end of training; wh ile the untrained group showed spontaneous recovery just enough to move their hind limb joints (Multon, Franzen et al. 2003) Stevens et al in 2006 have observed the effects of a relatively shorter dur ation of LT (1 week, 20min/trial, 2trials/day) starti ng one week after spinal contusion in rats. An overall 32% in overall locomotor function (BBB score) was observed in the trained rats that correlated well with peak muscle force measurements (Stevens Liu et al. 2006). Though LT has enhanced the functional locomotor capabilities after incomplete injuries, it is important to note that several factors including the severity of injury, dose of therapy, initiation of training, etc. can largely dictate the effect of LT in these incomplete inju ry models. Fouad et al for example, have shown

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58 that LT (2*15min for 5 weeks) did not add to the locomotor functional improvements in the trained rats versus the untr ained group. Even untrained c ontused rats show functional improvements as early as 7-14 days post training. However, compar ed to other studies of spinal contusion, this group utilized a different degree and kind of inco mplete injury to the cord (relatively less severe dorsal incomplete transaction of the cord as versus cord contusion or compression). The authors suggested that sponta neous recovery in the control group probably curbed differences between the two groups and th eir findings could be at tributed to either insufficient amount of training and/or insensitiv e testing outcome measur es (Fouad, Metz et al. 2000). Collectively, these findings suggest that LT enha nces locomotor capabili ties in spinal cord injured animal models. However, an important note to make is that though LT has shown to evoke stepping patterns in the mammalian an imal model of complete SCI on the moving treadmill, studies have failed to demonstrate their translation into independent overground walking. Nevertheless, animal studies have laid the foundations for a potential new therapeutic strategy for locomotion recovery in humans with SCI and have yielded valuable information about spinal cord plasticity and its response to training. 2.3.2 Locomotor training in Individuals with Spinal Cord Injury Based on the principles of locom otor training established in the animal model of SCI, the training has been transferred to humans with SCI. Barbeau et al performed one of the initial studies in humans to study the effects of locomo tor therapy in individu als with SCI (Barbeau, Wainberg et al. 1987; Visintin and Barbeau 1989). Subsequently, va rious investigators worldwide demonstrated impact of LT in th e walking capability of this patient population (Wernig and Muller 1992; Dietz, Colombo et al. 1995; Dobkin, Harkema et al. 1995; Harkema, Hurley et al. 1997; Behrman and Harkema 2000). Briefly, a typical LT protocol in humans

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59 comprises of placing subjects on a treadmill while 0-50% of their body weight is supported by an overhead climbing harness. Therapists manually assist in stepping over the moving treadmill while attempting to maintain appropriate join t kinematics. Note that LT uses body weight support to support body weight dur ing training; but the training involves utilizing numerous principles of neuroplasticity to enhance locomotion. The principles of LT, in which the stepping pattern is repeatedly trained while body weight support is provided, are based on basic science research demonstrating the role of the spinal cord in controlling locomotion in animal models of SCI. In humans, attempts are made to encompas s several basic principles that facilitate the kinetic and kinematical parameters associated with phases of st epping. Briefly, these include: a) adequate limb loading on the stance limb; b) maintain appropriate body kinematics including appropriate joint angles and an upright and extended trunk and head; c) generate stepping speeds approximating normal walking speeds (0.75-1.25 m/s); d) coordinate timing of hip extension and unloading of limb in stance with simultaneous loadi ng of the contra lateral limb; e) avoid weight bearing on the arms and facilitate reciprocal arm swing; f) aid symmetrical inter-limb coordination; and g) minimize sensory stimulatio n that would conflict with sensory information associated with locomotion. These principles not onl y facilitate balance cont rol, but also provide the ensemble of appropriate input for motor out put (Harkema, Hurley et al. 1997; Behrman and Harkema 2000; Behrman, LawlessDixon et al. 2005). Such an appr oach also ensures training consistency across researchers. Thus, LT is not to be considered as a modality, but is rather a training strategy that is based on neurophysiological principles de rived from animal studies and whose implementation necessitates adequate professional training, knowledge and skill. Though investigators have demonstrated EMG activity from the lowe r extremity muscles following LT in persons with complete SCI, unlik e the animal spinalized model however, LT has

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60 failed to demonstrate initiation of voluntary stepping pattern in humans with complete SCI (Dobkin, Harkema et al. 1995). Nevertheless, the tr aining has shown significant improvements in the locomotor capabilities in se lective populations of incomplete SCI. Accordingly; therapies directed towards recovery of walking function have focused extensively on the use of LT in persons with incomplete SCI (Wernig, Nanassy et al. 1998; Behrman, Lawless-Dixon et al. 2005; Behrman, Bowden et al. 2006). Considerable research efforts have been directed in revealing the major characteristics of the training, potential neuronal mechanisms associated with the training along with its poten tial role as a therapeutic strategy (Dobkin and Havton 2004; Hicks, Adams et al. 2005). Locomotor training ha s been suggested to have a positive impact on the walking ability, walking speed, kinematical parameters, distance walked, limb coordination, functional independence, ability to walk with fewer assistive aids, transition from use of a wheelchair to upright walking with assistive ai ds and subjective well being (Wernig and Muller 1992; Behrman and Harkema 2000; Hicks, Adam s et al. 2005; Hannold, Young et al. 2006)). Locomotor training has also shown to induce mo dulation of H-reflex and EMG patterns towards close to control values that accompany improve d walking capabilities (Dietz, Colombo et al. 1995; Trimble, Behrman et al. 2001). As a result of these behavioral and neurological benefits, the training has also been subject to a large mu lti-center randomized clinical trial the SCILT (Spinal cord injury LT). This trial involved participation of around 140 persons with incomplete SCI with ASIA grades B, C or D within 8 week s of injury. The experimental group received LT and the second group a similar intensity of stan ding and overground mobility training. However, no significant differences were observed in th e primary outcome measures of walking speed, distance walked in 6 minutes and the functi onal independence measure scores for lower extremity (FIM-L) between the two groups (Dobkin, Apple et al 2006). Nonetheless, LT has

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61 shown tremendous potential as a therapeutic st rategy in improving loco motor capabilities of persons with chronic incomplete SCI. In fact, LT has gained a new direction in the treatment of SCI in that: a) investig ators are now proposing a st rict patient selection criteria for conducting studies aimed at functional recovery of a hete rogeneous population such as the incomplete SCI (Dobkin 2007) and b) various researchers are favoring combining LT with other training strategies for maximum locomotor gains. LT alo ng with micro-stimulation of the spinal cord, pharmacological agents such as clon idine, and repair with nerve gr afts are suggested as potential future strategies to enhance locomotor function after SCI (Herman, He et al. 2002; Dobkin and Havton 2004; Field-Fote 2004). 2.3.3 Central Pattern Generators and Locomotion One of the principal neuronal recovery m echan isms promoting repair/regeneration of the injured spinal cord following LT is suggested to be the reestablishm ent of inter-neuronal connections known as the central pattern generators (CPG) within the spin al cord. The discovery of CPG in the quadruped spinal cord has stimul ated much interest in current research on promoting neural regeneration following SCI. In quadrupeds, CPGs are established to exist as a group of inter-neuronal networks within the spinal cord that produce a rhythmic motor pattern resembling normal locomotion (Edgerton, Leon et al. 2001; Fouad and Pearson 2004). In spinalized cats, an approximate 25% of total da ily-integrated EMG activity can be elicited from the soleus muscle during treadmill stepping; thereby implying the presence of a partial functioning spinal network even after a complete SCI. This fictive locomotion is produced independent of supraspinal and phasic a fferent input (Grillner and Zangger 1975). Though their presence is well known in rats and ca ts, presence of CPG in the human spinal cord is debatable. Based on the inability of pe rsons with complete SC I to generate stepping, some investigators have refuted it s presence in the human spinal co rd. In contrast, support for the

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62 existence of CPG in humans is well reported (Dimitrijevic, Gerasimenko et al. 1998; Lamb and Yang 2000; Dietz and Muller 2004; Edgerton, Tilla karatne et al. 2004). Dietz et al have demonstrated LT induced increase in leg muscle EMG activity when persons with complete SCI are made to step on a treadmill with body wei ght support and manual assistance (Dietz and Muller 2004). The authors suggest existence of neur onal networks within the spinal cord as the source of this enhanced neuronal activity. Furthermore, direct electrical stimulation of the lumbar spinal cord in humans with complete SCI ha s been shown to evoke locomotor-like rhythmic activity. This seemingly alternating pattern of the lower limbs has been attributed to the presence of a programmed inter-neuronal network within the sp inal cord that via input from the electrical stimulation receives its drive to produce th e motor output (Dimitrijevic, Gerasimenko et al. 1998). Lamb and Yang in 2000 further show that infants at birth are capable of stepping continuously when their feet are placed on a tr eadmill. Since infants do not have a functional descending spinal pathway, this locomotor behavior is attributed to a functional CPG within the spinal cord (Lamb and Yang 2000). Note worthily, presence of some kind of sensory stimulation (peripherally or supraspinally) presents as an essential prerequisite for mammalian locomotion. It is well known that descending pathways in the ventro lateral region of the spinal cord play a significant role in transmitting voluntary commands from the motor cortex to the spinal cord and are involved in initiation of locomotion (Noga, Kriellaars et al. 1991; Brustein and Rossignol 1998). These pathways most likely provide the stimulus necessary for CPG stimulation during normal walking. On the other hand, phasic afferent input ha s been demonstrated to play a key role for stepping in spinalized cats (Bouyer and Rossignol 2003), rats (Timosz yk, De Leon et al. 2002), as well as in humans with incomplete SCI (Dietz and Duysens 2000; Harkema 2001).

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63 Investigators purport that one of the main sources of proprioceptive feedback during stepping is probably provided by the stretch sensitive and load sensitive receptors in the lower extremity muscles. While cutaneous receptors in animals ar e also influential in producing a motor pattern, they are implicated to play a larger role in sk illed locomotor activities such as beam walking or paw placement on rungs of a horizonta l ladder (Bouyer and Rossignol 2003). Therefore, neuronal networks within the sp inal cord (CPGs) can be regarded as autonomous; but sensory input is nevertheless nece ssary for both normal and spinal locomotion. In addition to peripheral input, the ensemble of supraspinal input is also necessary in the initiation, maintenance, balance and vestibular control of locomotion so as to adapt to environmental constraints. Locomotor function th en is a consequence of neuronal interaction between a wide ensemble of information coming fr om supraspinal centers, afferent signals and CPGs (Edgerton, Tillakaratne et al. 2004). 2.4 Locomotor Training Effects on Paralyz ed Skeletal Muscle While locomotor training has proven to yiel d much neuronal plas ticity, the following paragraphs discuss the effect of LT on skeletal muscle morphol ogy and function in animal and human models of SCI. Locomotor training eff ects on the paralyzed skeletal muscle holds importance for two major reasons: 1) with increase in new therapeutic interventions for SCI, it is necessary that an intact machinery for limb move ment is maintained; 2) current studies purport that exercise in normal rats increases the level of neurotrophic factors and proteins associated with neuronal growth and plasti city. Thus, exercise induced in crease in neurotrophic factors produced in the muscle might, via their retrogr ade transfer, promote neurite outgrowth or synaptic plasticity within the injured spinal cord (Fouad and Pearson 2004; Hutchinson, GomezPinilla et al. 2004).

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64 Locomotor training has an overall ameliora ting effect on the spinal transection or contusion induced muscle alterati ons. Generally, maximum effect of the training is seen in the slow extensor muscles of the lower extremities with minimal or no effect on the fast extensor or flexor muscles (Roy and Acosta 1986). Five we eks of daily locomotor stepping with emphasis on load bearing 30min/day, 5days/week, beginni ng one month after transection has shown to markedly alleviate the atrophic response in the lower extremity muscles of spinalized cats (Roy and Acosta 1986; Roy, Talmadge et al. 1998). In addition, a relatively higher proportion of Type 1a fibers are expressed in the pa ralyzed soleus muscle after LT as compared to before initiation of the training. Such conversion is reflective of transition to a healthie r muscle. Furthermore, these studies show that the peak isometric forces produced by slow extensor muscles (soleus and vastus intermedius) increases to control values at the end of th e training period. In addition, there is a concurrent increase in the overall oxidative enzyme activity after LT. Recently; Stevens et al have elucidated the impact of one week of LT, starting at 1week after contusion (20min/trial, 2 trials/day at 11mpm) on the skeletal muscles of contused rats. Significan t increases in soleus muscle fiber cross-sectional area, peak tetanic force and decreases in muscle fatigue measurements have been demonstrated in tr ained rats versus untra ined injured rats. Measurements of force improvement correlated well with functional performance (BBB score); implying marked improvement in motor recovery by LT of as short as one week (Stevens, Liu et al. 2006). In humans with incomplete traumatic SCI (ASIA C), a couple of recent studies have reported the morphological and metabolic effects of LT on skeletal muscle. In nine persons with incomplete SCI, sixty-eight trai ning sessions of LT spanned over si x months increased the vastus lateralis fiber cross-sectional area, an overall incr ease in the expression of Type Ia muscle fibers

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65 and increases in muscle oxidative capacity (Stewart, Tarnopolsky et al 2004). These measures were accompanied by concurrent increases in the walking speed and locomotor capacities of injured subjects Subsequently, the same research group also observed increased vastus lateralis muscle glycogen stores (suggestive of increase in the gl ycogenolytic capacity of the muscle) and hexokinase enzyme activity (reflective of improvem ents in insulin sensitiv ity) in their subjects (Phillips, Stewart et al. 2004). 2.5 Summary Persons with both complete and incomplete SCI display significant locomotor deficits following the injury. Current advances in the field of neuroscience and reha bilitation research are providing a new dimension to ther apeutic approaches in SCI. A ccordingly; physiological based restorative interventions have the tremendous potential to replace current conventional therapies. Locomotor training, based on the principles of ne uroplasticity is one such intervention and has been used extensively to improve locomotor capabil ities in select populations of incomplete SCI. In addition to alleviating various functional de ficits, LT has the potenti al to induce several muscle adaptations including increases in mu scle fiber size, fiber type conversion and improvements in muscle oxidative capacity. In th e light of maintaining an intact peripheral machinery after SCI while also revealing potential effects of the trained muscular system on the neurological system, more studies are necess itated that focus on studying muscle adaptations following LT in persons with SCI.

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66 CHAPTER 3 MAGNETIC RESONANCE AND SKELETAL MUSCLE 3.1 Introduction Throughout the present work on both hum an and animal models of SCI, magnetic resonance (MR) is used as a key methodological tool to characterize lo wer extremity skeletal muscle. Skeletal muscle function depends both on its morphological and metabolic properties. Traditionally, muscle properties are studied us ing non-specific measurem ents (for example, anthropometric measures to determine muscle morphology), invasive tech niques (for example, muscle biopsy to estimate fiber types or muscle enzyme profile) and/or global measures (for example, maximum oxygen consumption levels to reflect muscle oxidative capacity). In the past decade however, MR has gained tremendous momentum in characterizing skeletal muscle in healthy as well as a variety of pa tient populations. One of the most critical features of MR that makes it an extremely valuable tool in studying mu scle is that it is non-invasive. In addition, the specificity, sensitivity and high resolution nature of MR makes it highly suitable for the assessment of a wide range of skeletal muscle ch aracteristics including st ructural, functional and metabolic properties. Repetitive measurements en able users to determine disease progression and allow for follow up of various therapeutic inte rventions. Lastly, obtaini ng information about a physiological process from a functioning muscle in real time makes MR a unique non-invasive measurement technique in modern science. The following sections review the basics of MR, followed by distinct features of magnetic resonance imaging (MRI), magnetic resonance spectroscopy (MRS) and their app lication in skeletal muscle. 3.2 Basics of Magnetic Resonance The term Magnetic Resonance (MR) refers to th e magnetic properties of the nucleus that is utilized in magnetic resonance imaging (MRI) and magnetic resonance spectroscopy (MRS).

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67 MRI is an imaging tool that is capable of imag ing soft tissues in the body; MRS, on the other hand, is used to study the metabolis m of human tissues and organs. Generally, a diagnostic image uses some physical property of the substance that is being imaged. For example, a photograph uses reflected light from the object that is pictured while ultrasonography uses reflected sound from th e body part under study. Similarly, current MR techniques are based on receiv ing and processing signals fr om atomic protons. The exact molecular environment where these protons are lo cated has a profound effect on the nature of the magnetic resonance signals created and thus gives rise to the remarkable power and versatility of MR. The following sections focus on the fundament als of MR. However, it should be recognized that various complexities including nuclear spin physics theories and mathematical calculations that more completely explain MR are beyond the scope of this dissertation. 3.2.1 Magnetic Property of Nuclear Spins An atom consists of a nucleus that cont ains positively charge d protons and neutral neutrons. Surrounding the atomic nuc leus is a cloud of negatively charged electrons that are located in orbits around this nucleus. In 1946, Nobel prize winners Felix Bloch and Edward Purcell theorized that any spinning charged part icle (for example, specific charged atomic nuclei) when placed in a strong magnetic field cr eates an electromagnetic field around it (Bloch 1953) The fact that these spinning particles behave as tiny bar magnets and can emit signal when subjected to a radiofrequency pulse has fo rmed the basic concept of magnetic resonance (Figure 3-1). Existence of a nuclear magnetic field depends on the number of unpaired protons in an atomic nucleus. According to quantum physics, pr otons in a nucleus are paired. For every proton spin with a magnetic field in one direction, a paired proton aligns in the opposite direction having an opposing magnetic fiel d. Consequently, magnetic mome nts of a proton pair cancel

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68 each other out and the net magnetic field is zero. In other words, when the number of protons in an atomic nucleus is even, the magnetic mome nts created by the paired protons cancel out each other and the net magnetic moment is zero. In co ntrast, when the number of protons is odd, there is always one proton that is unpaired and this gives rise to a net magnetic field or non-zero magnetic dipole moment (MDM) in the nucleus. Th e unpaired protons of an element can have their nuclear MDM oriented in e ither a parallel or anti-parallel direction. Th is alignment follows the Boltzmann distribution eE/kT (k stands for the Boltzmann constant) such that, at thermal equilibrium, part of the nuclei aligns anti-parallel (higher energy state), and a larg er part aligns parallel (lower energy state). Th e net magnetization of a sample is equal to the sum of these individual nuclear magnetic moments. Generally, a MDM exists in any nucleus that has an odd number of protons. Nuclei of certain elements like Hydrogen (1H), Phosphorus (31P), Flourine (19 F), Sodium (23Na), Carbon (13C), Nitrogen isotopes (14N and 15N), Deutrium (2D) and Oxygen isotope (17O) have a MDM property (Garlick and Mais ey 1992; Andrew 1994; Kevin K McCully 1994). Each of these nuclei can be used for MR purposes, but we use hydrogen atom for most MRI purposes and hydrogen, phosphorus and carbon for most MRS purposes. Hydrogen is the simplest and the most abundant element in the human body, since almost 60% of the human body is made up of water. Every water molecule has two hydrogen atoms and larger biological molecules such as lipids and proteins cont ain many hydrogen atoms. Sometimes enrichment (adding an extra proton to the nucle us) of nuclei is required to enab le them for use in MR. Thus, enriching the 12C atom to 13C does not alter the chemical prope rties of the carbon atom much, but enrichment gives it a nuclear magnetic moment (Mulkern and Chung 2000). 3.2.2 Larmor Frequency Each proton behaves like a bar magnet and has its own magnetic axis. In nature, the orientation of these axes is random. In the pres ence of a magnetic field (Bo) however, the axes

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69 are aligned parallel to the axis of the main magnetic field. Spi nning of protons around this axis when placed in a magnetic field is called precessi on. The frequency at which the protons precess in the magnetic field is directly proportional to the strength of the main magnetic field. This frequency is called the Larmor or precessiona l frequency. The Larmor equation expresses the relationship between the precessional fr equency and magnetic field strength. = x Bo Where is the Larmor frequency, Bo is the magnetic field strength and is the gyro magnetic constant. The gyro magnetic constant is a number without units that describes an intrinsic characteristic of a nucle us in a given environment. For water protons, the gyro magnetic constant is 42.6. At 0.5T the re sonant frequency of water prot ons is therefore 21.3 MHz and at 1.5T it is around 63.9MHz. The gyro magnetic constant of protons within lipids and other nonwater molecules is somewhat different. Thereby, th e resonant frequencies of protons in different chemical environments are close, but not identical to that of water protons. 3.2.3 Longitudinal and Transverse Magnetizations At the core of all MRI instruments is a homogenous magnetic field (Bo). The purpose of the magnetic field is to cause magnetization of pr otons within it. The pr ecessing of protons gives rise to small secondary magnetic fields, or ma gnetization. The average ma gnetization of protons at a given time is referred to as net magnetization. At equilibrium, protons precess with their net magnetization align longitudinally along the axis of the main ma gnetic field and therefore the magnetization is more precisely referred to as net longitudinal magnetization, Mz. This equilibrium/net longitudinal magnetizati on (Mz) can be considered as potential energy. Strength of Mz from tissues is dwar fed by the strength of main magnetic field Bo Since we can only transmit and receive signals that osci llate, and the longitudinal magnetization is not an oscillating function, a receiver cannot read it. This severely limits detection of signal from

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70 tissue protons at equilibrium. To detect MR si gnals for imaging, it is therefore necessary to disturb this equilibrium (Ray H Hashemi 1997). An excitation pulse, called the radio-frequency (RF) pulse is used to excite protons to emit radiations and caus e disequilibrium. MR electromagnetic radiations are ca lled RF waves and the pulse is called RF pulse because these are low frequency waves in the ra nge of frequencies typically us ed by radio stations. These low frequencies are not capable of a ny DNA or biological tissue damage that is otherwise associated with high frequency ionizing radiations like X-Ra ys; a unique feature of MR that makes it exceptionally useful for use in living tissues. Note worthily, to excite a proton precessing within a magnetic field, the frequency of the RF pulse ha s to be similar to the precession frequency at which the proton is precessing. A resonance condition is one in which energy may be transferred to and from a system very efficien tly under unique conditions (Gift, Pera et al. 1989). In absence of the unique conditions, energy transfer does not occur. Nuclear magnetic resonance in particular, involves measurement of signals coming from the atomic nuclei in response to radio waves that have the same na tural frequency (precessi onal frequency) as the nuclei themselves. That is, nuclei may absorb energy in the form of electromagnetic waves from the RF pulse and give rise to a signal only when the frequency of the radio pulse exactly matches the nuclear magnetic moment precessional frequenc y. The Larmor or precessional frequency is called the resonance frequency because it is equal to the frequency of the radio pulse that induces this resonance (reverberati ons/echoes) in the protons. Application of the radio pulse causes the net longitudinal magnetization Mz to rotate into the transverse plane, producing tran sverse magnetization Mxy (Figure 3-2). Since the transverse magnetization continues to precess in the x-y plane and is not obscured by the longitudinal magnetization of the main magnetic field, it is now cap able of inducing current in a coil placed in

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71 its vicinity. The coil is called the RF receiver co il and the induced current in the coil (i.e. the signal) is called an echo. Echo amplitude is greate st when it is first cr eated. Once the excitation pulse is switched off, the amplitude of this si gnal rapidly decays and b ecomes weaker with time. Thus, the final signal is an oscillating decaying signal and is called free induction decay (FID). This signal is recorded and stored in the computer to construct an image. 3.2.4 Relaxation Times Imm ediately after application of the RF pulse, Mxy precesses in the x-y plane around the z-axis. This happens only as long as the pulse is on. As soon as th e RF pulse is switched off, the protons tend to regain their state of equilibrium, and hence their lowest energy state, by realigning themselves in the di rection of the main magnetic field, Bo. The net axis of proton magnetization, Mz, eventually returns back to equ ilibrium. This return to equilibrium is referred to as relaxation. Relaxation is the process in which the spins are rela xing back towards their equilibrium state in the direction of the main magnetic field. Explanations of the two relaxation times associated with MR follow. Longitudinal relaxation ti me (T1 relaxation time): T1 or longitudinal relaxation time is the time it takes to regain the longitudinal magneti zation (Figure 3-3). As s hown in the figure, at time T1, 63% of Mz is regained. That is, T1 rela xation time can also be referred to as the time required for the longitudinal magnetization to reac h 63% of its original equilibrium level after complete flip by a 90 pulse. T1 relaxation rate (1/T1) is the rate at which the longitudinal magnetization Mz recovers along the z-axis after saturation by the RF pulse. Also, note that at time 2*T1, the longitudinal magnetization has recovere d to 91% of its original equilibrium value and after three relaxation times, Mz has recove red to 97% of its orig inal net magnetization (Mitchell 1999). T1 is also called the spin-latti ce relaxation time because it refers to the energy that the protons have to give away to their su rrounding (lattice) before gaining the equilibrium

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72 state. Rapid transfer of energy to the surrounding lattice results in a shorter T1 and slow energy transfer to the lattice produces a long T1. Transverse relaxation time (T2 relaxation time): As soon as the RF pulse is switched off, protons spin out of phase resulting in a ra pid decay of the transverse magnetization Mxy. T2 relaxation time is the time it takes for the M xy component to decay. Thus, in addition to T1 relaxation, a simultaneous but separa te process is happening after the RF pulse (Gift, Pera et al. 1989). Mxy decay is a result of dephasing of protons once the RF pulse is removed. Dephasing occurs because of a) spin-spin interaction: because of their proximity, protons within the same molecule and between two molecules interact with each other. The spins dephase because of the consequent spin-spin interactions; b) external magnetic field inhomogeneity: inhomogeneties in the main magnetic field causes the protons to spin at slightly di fferent frequencies. T2 relaxation rate is the rate of decay of the transverse magnetization Mxy. T2 relaxation time can also be referred to as the time require d for the transverse magnetization to decay to 37% of its signal (Figure 3-4). Like T1, T2 relaxati on depends on inherent prop erties of the tissue and is fixed for a specific tissue. T2 of a tissue de pends on how fast the prot on spins in the tissue dephase. Rapid spin dephasing leads to a short T2 and slow dephasing causes a long T2. Tables 3-1 and 3-2 give a brief summary of the T1 and T2 relaxation times of human skeletal muscle (bound water), muscle lipid, intramyocellular lip ids (IMCL) and extramyoc ellular lipid s (EMCL) at various magnetic field strengths. Note that at a specific magnetic field strength, the T2 relaxation times of a tissue are 5-10 times sma ller than the T1 relaxation times (Boesch 1999). Moreover, both T1 and T2 are affected by magnetic field strengths such that T1 gets longer and T2 decreases with increase in magnetic field strength).

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73 3.2.5 Fourier Transform Fourier transfor m is an extremely important an d useful tool in MR that was introduced by Jean Baptiste Fourier. The MR signal is acquired in the time domain i.e. a waveform that varies with time. Hence, every signal is composed of a series of frequencies. The RF receiver coil detects and records not one Larmor frequency, but sum of all the possible Larmor frequencies produced from the frequency encoding gradients. To disentangle frequencies in the signal and therefore determine precessional frequencies of nuclear spins in the x and y directions, a mathematical manipulation of the signal called the Fourier transform (F T) is performed. FT basically decomposes arbitrary signals of time into familiar sine and cosine waves. In MR, FT enables one to determine which Larmor frequenc ies are present in any signal (Figure 3-5). Fourier transform of the digitized MRI data is the final image and that of the raw MRS data the final spectrum. 3.3 Magnetic Resonance Imaging Magnetic Resonance Imaging (MRI) is a noninvasive, non-ionizing and powerful imaging tool that is capable of imaging soft tissues in the body and is now being proposed as the im aging modality of first choice for a wide array of di seases. MRI can produce contrasts between soft tissues with great resolution. Cont rast on MR images is a direct result of the different relaxation times of protons in body tissues. Though these times per se cannot be altered, two important MR parameters repetition time (TR) and echo tim e (TE) enable tissue contrast. Appropriate adjustments of TR and TE allow putting more weight on T1 or T2 relaxation times of a tissue thereby yielding the most common T1 weighted, T2 weighted and proton density weighted (PDW) images. The choice of imaging is based upon the tissue desired for study.

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74 3.3.1 T1 and T2 Weighted Images Figure 3-6 shows T1 relaxation tim es of two tissues, one with a long T1 relaxation time and the other with a short T1 relaxation time When the repetition time (time between two RF pulses; TR) is short, then the Mz component of the tissue with long T1 time possesses a smaller magnitude of recovery as compared to Mz comp onent of tissue with shor t T1. Consequently, the magnitude of Mxy will differ largely between two tissues show larger differences in signal intensities. It is however necessary that the TR s hould be at least close or similar to the T1 of one of the tissues. This allows for best contrast between the two tissues. Also, a very short TR will not produce any signal because of insufficient recove ry of the Mz component of either tissue. On the other hand, a very long TR will allow close to complete relaxation of both tissues such that their signal intensities will be similar (not shown here). A long TR therefore prevents differentiation between the two tissues. In this way, based on T1 of a tissue, tissue contrast is obtained using an appropriate TR. Because T1 of tissues dictates final image contrast, such images are called T1 weighted images. TE for such images is usually short. Figure 3-7 shows T2 relaxation times of two tissues, one with a long T2 relaxation time and one with a short T2. After the Mz component is flipped to the transverse plane, spin dephasing of both tissue occurs. The tissue with long T2 will take longer to dephase as compared to tissue with a short T2. When images are acqui red at an optimal TE, adequate differences in signal intensity exist between the two tissues. Therefore, differences in T2 relaxation will produce contrast between two tissues if the signal is collected at an appropriate TE. When TE is too short, the difference in SI is not much. For best T2 contrast a TE is chosen that balances sufficient decay of transverse magnetization fr om one tissue against ad equate presence of transverse magnetization from another tissue. Generally, images with TEs that approximate T2 relaxation times of tissues of interest have a si gnificant T2 weighting. Lastly, very long TEs will

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75 cause almost complete decay of transverse magnetization of both tissues a nd the ratios of their SI and hence contrasts between the two tissues are lo st. In this way, based on T2 of a tissue, tissue contrast is obtained using an appropriate TE. Beca use T2 of tissues dictates final image contrast, such images are called T2 weighted images. TR and TE for such images are usually long. Lastly, consider a sequence ut ilizing a long TR and short TE that yields a proton density image (Figure 3-7). When the TE is too short, enough time is not allowed for spin dephasing and the differences in signal inte nsity are purely based on differences in proton concentration of tissues. The T2 relaxation property of the tissue in this case is not explored. Furthermore, the long TR eliminates differences in contrast base d on T1 relaxation rates of tissues. This is a proton density weighted image (PDW). For PDW image, one eliminates T1 effect by using a long TR and eliminates a T2 effect by using a short TE (The two factors that determine the respective image weighting). In such an image water will show up bright and fat will be less bright. 3.3.2 Image Construction MR signal, like other radio waves, do es not possess any directional information. Therefore, signals received contain information from the entire part of the body imaged. The fundamental process used to determine the lo cation of the sources of MR signal and hence identify the specific body part imaged, is by a pplication of magnetic field gradients. In a homogeneous magnetic field, wate r protons resonate at the same frequency, regardless of location. If a second magnetic field is now superimposed upon the main magnetic field, a predictable variation is observed in the magnetic field along a predetermined axis. The resulting magnetic field is highest at one end of the gr adient and lowest at the other; between are intermediate values along the axis of the gradient Thus, protons at one end of the gradient spin slower and protons at the other end spin fa ster. Gradients therefore create temporary

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76 inhomogeneties in the main magnetic field to obtain spatial information. These signals from the protons can now be measured and used to constr uct images. The three basic steps in an imaging process are discussed below: Slice selection : This process involves use of gradient during application of an excitation or refocusing pulse. A slice is selected by simulta neous application of a selective RF-pulse and gradient along the z-axis (conventionally defined) such that alignment of protons in a specific width of tissue is disturbed. The pulse excites certain portion of tissue that has the same resonant frequency as the gradient. At th e end of the brief RF pulse, speci fic spins that are exposed to a specific gradient in a sample are excited. Magnetization vectors representing out-of-phase locations remain undisturbed and therefore do not contribute to the signal following the sliceselective excitation process. Applic ation of a gradient causes tippi ng of protons only in a specific slice (Figure 3-8). The RF pulse has a specific frequency (called the central frequency) and a range of frequencies around this central frequency (bandwidth of frequencies) that excites a specific slice (as described above). The read out phase takes approximately 3msec. Though the signal is obtained from the entire s lice, the slice image is not yet seen because of lack of in-plane spatial information about the slice. The signa ls are phase and frequency encoded for this purpose(Gift, Pera et al. 1989; Ray H Hashemi 1997; Mitchell 1999; Mulkern and Chung 2000). Phase encoding: Phase encoding conventionally define s application of magnetic field gradients and hence image construction in the ydirection. To get spatial information in the ydirection, a gradient is applied in this direction. The phase enc oding gradient (Bruhn, Frahm et al.) is turned on before applica tion of the frequency encoding gradie nt (Gx) (explained next). It is usually applied right after the RF pulse or just before the Gx gradient or anywhere in between. Consider three rows of spins as in Figure 3-9. The left panel shows the sp ins before application

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77 of Gy and the right panel shows th e spins after application of Gy (arrows depict spin phases). After application of Gy, spin s in the upper row experience a higher magnetic field and precess faster, spins in the lowest row experience the least magnetic fiel d and precess slower. Spins in the center row experience no change in magne tic field and therefore do not change their precessional frequencies. Consequently, after a short time, protons in the three rows are left with different phases of their spins. When the Gy gr adient is switched off, the precessional frequency of protons in each row becomes the same. Howeve r, the gradient has brought about a permanent shift in the spin phases. So the protons precess at same frequencies but are out of phase from each other. Because the gradient brings about a change in the pha se of proton spins this process of image construction is termed phase encoding. Note that a separate phase encoding step is needed for each row of pixels that needs to be discriminated in the slice. Therefore to discriminate between 256 rows in a slice, the process has to be repeated 256 times, each with a unique Gy gradient (phase shift). Frequency encoding: Frequency encoding conventionally de fines application of magnetic field gradients and hence image construction in th e x-direction. This process occurs after the application of Gy and it is during this cycle that the signal is r ecorded as a function of time and stored in a computer. Consider the same matrix as defined above. To get spatial information in the x-direction of this matrix, a gradient is applied in the x direction called the frequencyencoding gradient (Gx). The left panel shows the spins before a pplication of Gx and the right panel shows the spins after applic ation of Gx. Note that applica tion of Gy had already caused a phase shift. Application of Gx gradient varies the Larmor freq uency of the spins in the three columns so that the spins in the right column have a higher frequency than the spins in the left column. Therefore, the central column of spins appears not to precess at all, the one on the right

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78 has the highest frequency and the one on the left has the smallest frequency. In addition, because of the phase shift, each cell in the matrix ultimately experiences a unique phase shift and frequency. The read out phase takes approxima tely 10msec. The different frequencies are summed to form a signal that is detected by the re ceiver coil. The signal amp litude is a sum of all the frequencies and is recorded as a function of time. Note th at the signal is collected only once during the read-out phase. That is, after one RF pul se, the Gy gradient is applied. This is then followed by the Gx gradient and subsequent m easurement of the signa l. Each signal is a representation of the entire image. However, a single RF pulse and hence one signal is not enough to produce the image. To get spatial info rmation from body-tissue a series of RF pulses and phase encoding steps are necessary to reso lve the image (Gift, Pera et al. 1989; Ray H Hashemi 1997; Mitchell 1999; Mulkern and Chung 2000). 3.4 MRI Applications in Skeletal Muscle Skeletal m uscle function depends on the morphological and metabolic properties of the muscle. MRI has the distinct ability to provide high-resolution spatial information and possesses the ability to specifically measure muscle size, quantify muscle damage, and visualize muscle recruitment patterns and intramuscular fat. Th e following paragraphs discuss the present and potential role of MRI in ch aracterizing skeletal muscle. Muscle size significantly impacts muscle f unction (Berg, Dudley et al. 1991; PloutzSnyder, Tesch et al. 1995; St evens, Walter et al. 2004). Musc le size measurements include anatomical and physiological cros s-sectional area (CSA), musc le thickness and length, and muscle volume. Traditional methods of assessi ng muscle size involve using anthropometric measurements like skin fold thickness and lim b circumference, ultrasonography and dual X-Ray absorptiometry (DEXA). These measurements however, do not clearly differentiate between muscle and non-muscle tissue. Consequently, in clusion of non-muscle tissue like intramuscular

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79 fat and connective tissue in muscle size measurements can overestimate muscle size. Other shortcomings include use of ionizing radiations (DEXA), limited field of view (ultrasonography) and inability to distinguish between individual muscles and/or muscle groups (anthropometric measures). MRI circumvents many of these di sadvantages. MRI is nonionizing, can clearly contrast between fat and muscle tissue, differe ntiates between contract ile and non-contractile tissues within a muscle and can image the entire le ngth of more than one muscle at the same time (Boesch 1999; Mulkern and Chung 2000). These a dvantages have established MRI as a gold standard of assessing skeletal muscle size. Numerous studies have us ed MRI as a standard technique to assess skeletal muscle size followi ng disease processes (Mulkern and Chung 2000) progression of muscle atrophy following disuse (Vandenborne, Elliott et al. 1998; Kitahara, Hamaoka et al. 2003) and effects of interventi ons (Stevens, Pathare et al. 2006). Moreover, anatomical and physiological CSA measures by MRI have been shown to correlate significantly higher with muscle strength measures su ch as maximum voluntary contraction than anthropometric and DEXA indices (Bamman, Newcomer et al. 2000). Muscle T2 relaxation properties are sensitive to muscle contraction and therefore possess widespread applications in studyi ng muscle characteristics. One of the initial studies reporting exercise induced increase in signal intensity of skeletal muscle T2 in humans was conducted by Fleckenstein in 1988 (Fleckenstein, Canby et al. 1988) Fleckenstein et al showed that depending upon exercise intensity, contraction induced in crease in T2 relaxation times can occur as early as after two muscle contractions, reach a plateau by few contractions and return to baseline values after 10-40 minutes of exerci se. Interestingly, studies have successfully demonstrated positive correlations between elevated T2 times following exercise with integrated EMG patterns (Adams, Duvoisin et al. 1992). Fu rthermore, the exerci se induced contrast

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80 increases with exercise intensity so that the act ive area of the muscle is mapped to detect muscle use (Ploutz-Snyder, Tesc h et al. 1995). Consequently, if a muscle shows more contrast shift, it reflects more use of the muscle and vice versa. (Adams, Duvoisin et al. 1992; Fleckenstein, Watumull et al 1993; Ploutz-Snyder, Tesch et al. 1995). This phenomenon of muscle T2 has been used to infer which muscles are used during activity, the extent of their contribution, presence of any substituted activ ity by other muscles; in cluding contribution of multiple and deep anatomical muscle groups at th e same time. Thus, T2 changes are sensitive to the activity status of the muscle and therefore hold tremendous potential in identifying muscle activation patterns in both nor mal and diseased conditions. Currently, studies indicate that the mechanisms of change in muscle T2 following rest to work transitions are a consequence of increase in T2 relaxation times of muscle water (Saab, Thompson et al. 2000; Patten, Meyer et al. 2003). This is probably a consequence of osmotically driven shifts of water into intramyocellular sp aces secondary to accumulation of end products of muscle metabolism and/or intracellular aci dosis (Ploutz-Snyder Nyren et al. 1997; Vandenborne, Walter et al. 2000; Pa tten, Meyer et al. 2003). Note worthily, the increase in T2 values af ter normal activity is transient and typically resolves within minutes to a couple of hours (Fleckenstein, Canby et al. 1988; Hayashi, Hanakawa et al. 1998). However, enhancement of and subsequent persistence of T2 values for longer periods (as long as two to three months) may indicate muscle da mage. This is commonly observed following eccentric exercise protocols. Several studies have reported that eccentric exercise-induced muscle injury is associated with marked elevations in T2 values in both healthy and patient populations (Ploutz-Snyder, Tesch et al. 1995; Ploutz-Snyder, Nyren et al. 1997; Bickel, Slade et al. 2004). Elevated T2 valu es following strenuous exercises correlate with

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81 specific markers of muscle damage including serum creatine kinase ac tivity, plasma myosin heavy chain fragments (a specific marker of slow fiber muscle damage), muscle soreness and maximal isometric force (Foley, Jayaraman et al 1999; Sorichter, Mair et al. 2001). Repeated MRI measurements of skeletal muscle for as long as two to three months following the exercise continue to show elevated T2 relaxation times; thereby suggestive of a long lasting change in the muscle (damage). Interestingly, similar long last ing elevated T2 relaxa tion times reflective of muscle damage have also been reported follo wing reloading after hind limb immobilization, spaceflight and disease processes in both human s and animals (Bryan, Reisch et al. 1998; LeBlanc, Lin et al. 2000; Frimel, Walter et al. 2005). Muscle damage following reloading is attributed to mechanical disrupti on of muscle fibers and inflamma tion in muscle (Kasper, Talbot et al. 2002). Frimel et al ha ve shown strong correlations betw een the elevated T2 relaxation times and histological markers of muscle damage (Frimel, Walter et al 2005). Relatively large areas of T2 signal contrast have also been observed in the quadriceps muscle of persons with SCI as compared to control subjects after electrically stimulated mu scle contraction. The authors in this study suggest that the increased recruitment of muscle in the SCI group not only implies more use of the muscle to evoke force; but also that persistence of the elevated T2 values most likely implies muscle damage (Slade, Bickel et al. 2004). In a follow up study, the same research group found that the thigh muscle areas showed a similar enhancem ent of signal in tensities after a repeated bout of exercise that was followed 8 weeks after the in itial bout. Since the controls did not show similar areas of elevated pixel signal intensities, the aut hors surmised that the skeletal muscle of persons with SCI remained injured and that probably the paralyzed muscles did not develop a protective effect following the first bout of exercise (Bickel, Slade et al. 2004).

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82 Lastly, based on the high-resoluti on feature and ability of MRI to contrast between tissues; soft tissues within the muscle can be well demarcated using dis tinct MRI techniques. Accordingly, MRI has served as a non-invasi ve measure to identify, quantify and monitor progression of fatty tissue infiltra tion within skeletal muscle during diseased conditions (Huang, Majumdar et al. 1994; Elder, Apple et al. 2004). However, similar to other imaging techniques, T2 weighted MRI also suffers from partial vo lume filling, making it challenging to reliably assess the inherent T2 of skeletal muscle in the presence of increased amounts of intramuscular lipid. Since elevated T2 values can reflect a vari ety of muscle proce sses including damage (Bryan, Reisch et al. 1998; LeBl anc, Lin et al. 2000; Frimel, Walter et al. 2005), edema (PloutzSnyder, Nyren et al. 1997; Patten, Meyer et al. 2003), fibrosis and fat (Huang, Majumdar et al. 1994), investigators are now making attempts to discriminate between these physiological processes utilizing a variety of advanced MR techniques includ ing diffusion weighted imaging, magnetization transfer contrast and spectroscopy. Thus, the scope of MR in studying skeletal muscle is on a constant rise. 3.5 Magnetic Resonance Spectroscopy While MRI provides spatial inform ation, magnetic resona nce spectroscopy (MRS) has been used for more than two decades to st udy the metabolism of human tissues and organs (Heerschap, Houtman et al. 1999). MRS is used to identify metabolites and monitor the absolute and relative concentrations of metabolites; thereby providing a non-invasive measure of physiology and pathology in body tissues. 3.5.1 Contrasting MRI and MRS The main difference between MRI and MRS is that MRS does not result in images with spatial information, but rather results in a set of spectral peaks. The frequency encoding gradient

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83 (read out) during MRI serves as a giant chemical shift in spatially discriminating proton spins along the conventional x-axis (Boe sch 1999). In MRS, chemical shift is produced by the inherent nature of metabolites. Such inhere nt nature of the metabolites such as water and fat represent an artifact and spatial mis-registration in MRI. For example: artifacts at organ fat interface in the abdomen, vertebral body and intervertebral disc interface, bone muscle interface etc. Secondly, MRI uses the protons in the bodys water molecule s that have a concentr ation of 110M to obtain information about anatomy and pathology (Garlic k and Maisey 1992; Boesch 1999). The sample to be imaged is placed in a magnetic field gr adient and depending upon th e frequency of protons, spatial information of the structure under is ob tained. In MRS, proton nu clei and a variety of magnetic nuclei present at concentr ations of 2-10mM are used to obtain information about tissue biochemistry. Precise localizat ion to small volumes of body tissue are achieved using surface/volume coils in combination with single volume or multiple volume spectroscopic techniques (Alger 1994). Accordingly, MRS can quantify smaller concen trations of muscle tissue (example lipid) that might not be feasible with imaging t echniques (Schick, Machann et al. 2002). 3.5.2 Nuclei Studied with MRS Hydrogen, phosphorus and carbon are widely used in MRS because these nuclei produce strong MR signal and are of biomedical intere st. Hydrogen is used to study hydrogen compounds such as lipids. Phosphorus is used for phosphate compounds such as ATP and PCr that play a key role in the bioenergetics of resting and exercising muscle states. Carbon is especially used for studying the metabolic fate of glycogen and me tabolites of the tricarbo xylic acid, which play a major role in carbohydrate metabolism (Boesc h 1999). For detection by MRS, the nuclei need to be in adequate concentrations as well as fairly mobile. For example, phosphorus nuclei at 370C in a 1.5T magnet have 1,000,026 nuclei in th e low energy state and 1,000, 000 nuclei in the

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84 high-energy state. The MR signal therefore comes from 0.0001% of the total nuclei (Garlick and Maisey 1992). In most experiments, a threshol d concentration of 0.5mmo l of metabolite per kg of wet weight is required for detection with MRS (Heerschap, Houtman et al. 1999). Metabolites present in a concentration lower than milli mo lars (mM) are not detectable by MRS. Also, molecules that are in a bound state are not detectable. For example: ADP is a phosphate compound not detectable by 31P-MRS because most of the ADP exists in the bound form. 3.5.3 Spectral Components of MRS Identifying different nuclei in the form of different peak resonances is an outstanding feature of MRS. Each spectral peak is defined by a specific resonance frequency, height and width. The height of the spectral peak or the area unde r it yields relativ e measurements of metabolite concentrations. The area under a fully rela xed spectral peak is directly proportional to the concentration of nuclei that make up the peak. The spectral width at half its height is called the line width and gives relaxation time information because it is proportional to 1/ T2 (Garlick and Maisey 1992). Spectral widt h is influenced by magne tic field inhomogeneties. The position of the spectral peak with a specific resonant frequency on the plot is expressed as parts per million. Therefore, the peaks have a specific positiona l relation with respect to each other in the spectra (see below for detailed description of chem ical shift). Thus, the ch emical shift difference between fat and water peaks will always remain 3.5 ppm in all strength fields. Accordingly, metabolites can be identified from the position of spectral peaks. Figure 3-10 shows a typical proton spectrum with its principle components. 3.5.4 Chemical Shift in MRS The distinct peaks seen in spectro scopy can be a ttributed to the inherent chemical shift of nuclei that are tested. Even in a perfectly homogenous magnetic fiel d, not all protons resonate at the same frequency. Depending upon the chemical environment of the nucleus in different

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85 biochemical compounds, the same nuclei are subject to very small variations in frequency. For example: hydrogen protons in water and fat have different chemical environments and therefore their precessional/resonant frequenc ies are also different. In fact, protons at different sites within the same complex molecule will have different precessional frequencies. Precessional frequency of phosphorus nuclei (31P) at 1T is approximately 17.18 MHz and that of hydrogen nucleus at 1T is approx 42.6MHz. As seen in Figure 3-10, the two resonance sign als of fat and water p eaks are distinct. In different magnetic field strengths, these resonance frequencies will vary and therefore the numerical locator representing the two peaks will ch ange. Therefore, in order to characterize and specify the location of MR signal irrespective of magnetic field stre ngth, an alternative method is necessary. One method of solving this problem is to report the location of the MR signal in a spectrum relative to a reference signal. Tetramethylsilane (CH3)4Si, usually referred to as TMS has become the reference compound of choice for proton and carbon MR1. Furthermore, to correct these frequency differences for th eir field dependence, they are divided by the spectrometer frequency (example: 63.9 MHz for pr otons in a 1.5 T magnet) This difference in the resonance frequencies from a reference frequency is called chemical shift ( ). The resulting number would be very small, since Hz is divi ded by MHz, and is measured in parts per million (ppm), which is a dimensionless quantity independe nt of field strength. Chemical shift equation is given by: Chemical shift ( ) = 106 [Frequency (sample)-Frequency (reference)] ____________________________________ (3-1) Frequency (operating) 1 http://www.cem.msu.edu/~reusch/Vir tualText/Spectrpy/nmr/nmr1.htm

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86 Note that frequency (sample) is the resonant frequency of the sample signal (example fat), frequency (reference) is the frequency of the refe rence signal (TMS) and frequency (operating) is the frequency of the spectrometer (magnet). When ch emical shift between tissues is expressed in absolute frequency, it is proportional to the magnetic field (Ray H Hashemi 1997). 3.5.5 Correction of Saturation Effects Absolute or rela tive quantification of metabolites using MRS is best obtained after correction of T1 and T2 saturati on effects. This is because spectroscopic sequences employed in obtaining metabolite concentrations from spectral peaks typically use relatively longer echo times and shorter repetition times (less than 6T 1). While the long TE permits T2 decay and phase modulation; the shorter TR times allow for inco mplete saturation of longitudinal magnetization. Collectively, there is a net loss of the acquired MRS echoes that in turn underestimate metabolite concentration. In addition, because of different T1 and T2 relaxation times of metabolites under study, simple metabolite ratios at any one given TE and TR are inaccurate representations of relative metabolite proportions. Ther efore, signal correction is achieved by calculation of precise values of relaxation times and calculating fo r saturation factors for each metabolite. Final metabolite concentrations are obtained after correction with saturation factors. 3.5.6 Proton Spectroscopy (1H-MRS) Hydrogen protons have been used traditionally for spectroscopy because of their natural abundance in organic structures a nd high nuclear magnetic sensitivity. 1H-MRS has been a sensitive and precise tool to quantify gross tissue fat content and derangements in lipid metabolism. Figure 3-10 represents components of a typical proton spectroscopy spectrum from a human skeletal muscle. Muscle creatine cont ains contributions from several metabolites including creatine phosphate. It serves as a reserve for high en ergy phosphates and a buffer for ATP/ADP reservoir (Castillo, Kwock et al. 1996). Interestingly, th e lipid peak ha s contributions

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87 from a variety of lipid components and these components can be reso lved in a spectrum. Decomposition of the fat signal into component s (Figure 3-11) gives specific information regarding intramyocellular lipids (IMCL) and extramyocellular lipids (EMCL). The IMCL is suggested to correspond to lipids with in the myocytes that serve as an important source of energy supply during long duration endurance activ ities. The EMCL corresponds to the extramyocellular lipid pool that constitutes the long term storage depot for lipids (Szczepaniak, Babcock et al. 1999). Intense signals from tissue water and fat swam ps the less conspicuous signals from other metabolites of biomedical interest (muscle metabolit es such as creatine, choline, etc) that are present in much lower concentrations. Successful signals from these metabolites can be obtained by using water and fat suppression techniques duri ng acquisition of spectra or by mathematical elimination of the water peaks from the FIDs during post-processing of raw data (Hope and Moorcraft 1991). 3.5.7 Phosphorus Spectroscopy (31P-MRS) Magnetic properties of the phosphorus nucleus have gained momentum because of the pivotal role of phosphorus cont aining compounds in energy meta bolism of skeletal muscle. Hoult et al published the first obs ervations of metabolites from isol ated rat skeletal muscle using 31P-MRS in 1974 (Hoult, Busby et al. 1974). Since then, 31P-MRS has made considerable impact in studying the bioenergetics of normal and pathological neuromuscular tissues and used expansively to quantify metabolic costs of vari ous physiological processes in skeletal muscle. Almost all muscle metabolic processes involve phosphates and visualizi ng energy metabolism of skeletal muscle with 31P-MRS (both at rest and during exercise) has proven to be valuable. One of the most outstanding features of 31P-MRS is its ability to con tinuously obtain time dependent metabolic information from living tissues. The time resolution of 31P-MRS is around 1ms and

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88 this makes it possible to evaluate and quantify muscle oxidative and glyc ogenolytic energetics in-vivo (Argov and Arnold 2000). In general, seven peaks can be identifie d in a phosphorus spectrum acquired from a skeletal muscle (Figure 3-12). The major peaks correspond to inorganic phosphate (Pi), phosphocreatine (PCr), and three pho sphate groups of adenosine tr iphosphate (ATP). In addition, peaks of phosphomonosterase (PME) and phosphodies terase (PDE) can be identified with greater spectral resolution. Metabolite concentrations are commonly quantif ied from relative sp ectral amplitudes using ATP peaks as an internal standard. Normal Pi concentration is 3-5mM. Pi/PCr ratio is closely related to the phosphorylation potential and reflects the energy state of the muscle (Veech, Lawson et al. 1979; Chance 1984). At rest, PCr/Pi ratios range from 6-12 in healthy human muscles, depending upon the muscle studied. The single Pi peak is actually a combined peak of two molecules (HPO4 2and H2PO4 -) that are in fast exchange w ith each other. These acidic and basic molecules resonate at two different positions in the spectrum. Position of the Pi peak depends upon the relative concentrations of the two molecules and based on frequency shifts of the Pi peak, intracellular pH can be determin ed. Intracellular pH calculated based on the chemical shift difference between PCr and Pi (d ) is rather accurate (up to 0.02pH units) and calculated by the following equation: Intracellular pH = 6.75 + l og [(3.27-d)/(d-5.69)] (3-2) This equation is derived from the more general Henderson-Hesselbach equation that defines the pH value of a sample via a titrati on curve (pH as a function of resonance shift); base concentration Intracellular pH = pKa + log acid concentration (3-3)

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89 Where, pKa is the dissociation constant of Pi (6.9), generally defined as the ability of an acid to give away its H+ protons. Lastly, it is noteworthy that otherwise undetectable metabolites including adenosinediphosphate (ADP) and i onic concentration such as Magnesium (Mg+2) can also be indirectly estimated using 31P-MRS based on measurements of creatine, phosphocreatine, pH and the equilibrium constants. As discussed below, variations in phospha te metabolite concentrations from rest to exercise and during recovery have been used to illustrate muscle energetics in-vivo 3.6 Application of Magnetic Resonance Spectroscopy in Skeletal Muscle Depending upon the nucleus studied, MRS allo ws observation of high-energy phosphates (31P-MRS), glycogen (13C-MRS) or intramyocellular fat (1H-MRS). In 1955, skeletal muscle was the first biological tissue that was studied using MRS (Odeblad and Lindstrom 1955). While initial application of the tec hnique was restricted to cell cu ltures and biological systems in situ MRS is now widely used for a range of in-vivo metabolite measurements in animal and human muscle. These include a) studying different sites of the same muscle and a number of muscles simultaneously, b) yielding metabol ite data from deep muscles that are difficult to access by biopsy, c) directly quantifying muscle metabolites and scrutinizing cellular components of mitochondrial metabolism instead of relying on gl obal and/or invasive measures of oxidative phosphorylation (for example VO2max meas ures) d) determining real time in-vivo biological processes with a temporal resolution on the order of seconds, e) obtaining longitudinal measurements of skeletal muscle bioenerget ics thereby permitting the study of disease progression over time and reliably quantifyi ng effects of therapeutic interventions.

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90 3.6.1 Role of 1H-MRS : Quantify Intramuscular Fat 1H-MRS has the unique ability to measure intr amuscular lipid content free from muscle tissue, thereby overcoming limitations of partial volume filling (seen with MRI). In addition to the total lipid content, 1H-MRS can separately quantify lipid mu scle tissue into its intracellular and extra cellular components. Specifically, 1H-MRS is the only currentl y available non-invasive tool to quantify intramyocellu lar (IMCL) and extramyocellular lip id (EMCL) within skeletal muscle. As discussed in chapter 1, intramyocellu lar lipid (IMCL) conten t has the potential to serve as a non-invasive marker of insulin resi stance, especially in se dentary individuals. Noninvasive measures of IMCL via H-MRS are f ound to correlate well with IMCL quantified by electron microscopy and histochemistry usi ng Oil Red O staining (Hinderling VB 2006). Sensitivity of 1H-MRS to changes in lipid measurements is high and 1H-MRS has proven to be a more valid technique for measuring IMCL than morphometry and histochemical analysis of IMCL (Schrauwen-Hinderling, Hesselink et al 2006). The accuracy and sensitivity of 1H-MRS in measuring IMCL concentrations is also sufficient to measure changes in IMCL (depletion and recovery) after exercise (Boesch, Decombaz et al. 1999). As a result, a variety of studies have utilized spectroscopy to quan tify whole muscle fat and its components and their possible association with insulin resistance (Schick, Ei smann et al. 1993; Mach ann, Haring et al. 2004; Boesch 2007). 3.6.2 Role of 1H-MRS: Assess Muscle T2 Characteristics Chemical shift differences between protons bound to muscle and protons bound to fat yield separate water and lipid p eaks; thereby allowing measuremen ts of T2 relaxation properties of muscle and fat tissues separa tely. Accordingly, muscle tiss ue composition including muscle damage and edema is more reliably measured using 1H-MRS measures. Investigators are using this ability of 1H-MRS to decipher between fatty and dama ged muscle (Walter, Cordier et al.

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91 2005) and to identify muscle degeneration in a va riety of muscle disorders (Bongers, Schick et al. 1992). 3.6.3 Role of 31P-MRS: Quantify Rest ing Muscle Metabolites At rest, most of the muscle energy supplies come from the formation of ATP from ADP and inorganic phosphate (Pi) that takes place in the electron tran sport chain of the mitochondrion (mitochondrial oxidative phosphorylation). Metabolism at rest occurs at a low rate. Accordingly, it is easy to obtain baseline 31P-MRS spectrum. Studies report 31P-MRS measured PCr/Pi ratios as reliable markers of phos phorylation potential (Chance 1984; McCully, Kent et al. 1988). Phosphorylation potential is the energetic potential of the cell The variables [ADP][Pi]/[ATP] describe the effects of energy demand a nd potential for ATP supply by oxidative phosphorylation. Alterations in res ting metabolite content, metabol ite ratios and change in pH reflect disturbances in the meta bolic pathways and/or structural dysfunctions of mitochondria or a diseased state of the muscle as a whole (Hee rschap, Houtman et al. 1999; Mattei, Bendahan et al. 2004). Increase in resting Pi content and P i/PCr ratios of skeletal muscle obtained by 31P-MRS have been observed in a variety of diso rders including primary mitochondrial diseases, myopathies, muscle injury, disuse and dene rvation (McCully, Kent et al. 1988; Argov and Arnold 2000; Tartaglia, Chen et al 2000). In the lower limb muscle disuse model, Pathare et al have reported an increase in ba sal Pi along with concurrent decr ease in the PCr/Pi ratios in the plantarflexor muscles of indivi duals following cast immobilization secondary to ankle fracture (Pathare, Walter et al. 2005; Pa thare, Vandenborne et al. 2007). Moreover, the elevated Pi content and Pi/PCr ratios were found to significantly impact the force generating capacity of the skeletal muscle. Elevated Pi/PCr ratios are al so demonstrated during muscle injury (McCully, Kent et al. 1988; Pathare, Vande nborne et al. 2007). Furthermore, right shift of the Pi peak

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92 relative to the PCr peak in a 31P spectrum implies elevated H+ ions produced by lactate and hence an acidic state of the muscle. Information provided by resting spectra reflects metabolic state of the muscle at rest. Abnormalities in muscle bioenergetics however, are better characterized in dynamic experiments performing 31P-MRS during exercise and recovery. The s ubsequent section describes one of the most popular applications of 31P-MRS measurement of metabolic oxidative capacity of the skeletal muscle. 3.6.4 Role of 31P-MRS: Identify Fiber-type and Muscle Fatigue Three individual Pi peaks have been identified in the 31P-MRS spectrum of the human gastrocnemius muscles following brief periods of muscle contraction (Vandenborne, McCully et al. 1991). Based upon distinct positions of the th ree peaks in the MRS spectrum that correspond to heterogeneity in pH, the authors hypothesized that the three Pi spec tral peaks correspond to slow oxidative, fast twitch oxidative and fast tw itch glycolytic fibers. Similarly, split in the Pi peak has been reported by Kutsuzawa et al in patients with chronic respiratory impairment during moderate exercise intensity of the forearm muscles; implyi ng the contributi on of different fiber types to muscle work. Interpretation of musc le fiber type is also based upon the ratios of phosphate metabolites (Kutsuzawa, Shioya et al. 1992). Resting Pi/PCr ratios are higher in red muscle as compared to white muscles because of baseline higher levels of Pi in red muscle and higher PCr levels in white muscle (Meyer, Br own et al. 1985; Kushme rick, Moerland et al. 1992). Amongst various other sources of peripheral mu scle fatigue, altered levels of muscle metabolites (example excessive Pi levels, H+ and H2PO4-) are purported as a potential source of skeletal muscle fatigue at the myocyte level (K evin K McCully 1994). Fatigue is described as the decreased force generating capacity of the muscle. Metabolic aspects of muscle fatigue can be

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93 evaluated by monitoring concentration of oxidative metabolites and pH of exercising muscle. For example: A strong relationship has been observed in the time frame of fatigue in the dorsiflexor muscles of healthy subjects and the accumulation of H2PO4 (Kent-Braun, Ng et al. 2002). 3.6.5 Role of 31P-MRS: Measure Muscle Oxidative Capacity One of the most valu able contributions of 31P-MRS is the non-invasive measurement of the oxidative capacity of skeletal muscle (McCully, Fielding et al. 1993; Kemp, Sanderson et al. 1996; Argov and Arnold 2000). Measur ing oxidative capacity via 31P-MRS has gained tremendous attention because of pa rticipation of energy rich phospha tes in muscle bioenergetics. One of the most reliable measures of oxidative capacity using 31P-MRS is the rate of PCr resynthesis. PCr recovery rates have been extensively used in both healthy and diseased muscles as estimates of muscle oxidative capacity (M eyer 1988; Paganini, Fole y et al. 1997; McCully, Mancini et al. 1999; Argov and Arnold 2000; Kent-Braun and Ng 2000; Pathare, Vandenborne et al. 2007). Energy released from ATP hydrolysis form s the principal energy source for muscle metabolism during rest and work. The immediate s ource of ATP at onset of exercise is provided by hydrolysis of PCr catalyzed by the enzyme creatine kinase. MgADP+ PCr + H+ MgATP2+ Creatine (3-4) Creatine Kinase The resultant ATP is quickly hydrolyzed into inorganic phosphate (Foley, Jayaraman et al.) and ADP while releasing energy enough to meet energy demands of the cell. PCr + H+ Creatine + Inorganic phosphate (3-5) Noteworthy, homeostatic mechanisms exist within the myocyte that couple overall ATP utilization with ATP synthesis; thereby main taining nearly steady concentrations of ATP (Erecinska and Wilson 1982; Kushmerick 1995). In this respect, one of the most important

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94 contributions is the temporal and spatial buffer role of creatine kina se reaction in maintaining energy homeostasis. High PCr content and creatine kinase enzyme activity in skeletal muscle makes the creatine kinase equilibr ium reaction an extremely efficient enzymatic system that is capable of buffering transient changes in ATP (Kushmerick and Meyer 1985). A closer look at bioenergetics data during mode rate exercise reveals that whil e ATP is held constant, the PCr levels continue to deplete (PCr hydrolysis). The following steps (Be ssman and Geiger 1981) briefly describe the creatine kinase buffer phenomena (Figure 13) : a) During beginning of exercise, cystolic PCr hydrolysis produces ATP b) Subsequent cystolic respiration (aerobic and anaerobic) is controlled through kinetic control of ADP (respir atory control of ADP) (Dudley, Tullson et al. 1987) while the reverse creatine ki nase continues to maintain ATP levels ( temporal buffer role of the creatine kinase reaction by myofibrillar creatine kinase) c) the creatine-kinase reaction at sites of ATPase activity also trig gers the creatine-phosphocreatine shuttle mechanism such that creatine released from sites of contr action diffuses (shuttled) in to the mitochondria d) ATP produced by oxidative phosphorylation and gl ycogenolysis is used by mitochondrial creatine kinase isoenzyme to resynthesize PCr. This PCr is then shuttled back for participation in the CK reaction to release ATP at the sites of muscle contraction. Thus, the mitochondrial creatine kinase serves as an intermediate in the transfer of high energy phosphate from sites of ATP production (mitochondria and glycolytic loci) to ATP consuming locations in the myocyte (Kushmerick 1995; Heerschap, Houtman et al. 1999). Therefore, while muscle creatine kinase controls the backward reaction (PCr + ADP + H+ Creatine + ATP) outside the mitochondria; the forward reaction is controlled by mitochondria l creatine kinase isoenzyme (Sahlin, Harris et al. 1979). In this regard, the cr eatine kinase reaction acts as a spatial buffer and maintains cell ATP homeostasis (Figure 3-13) e) However, the rate of ATP release from oxidative

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95 phosphorylation and glycogenolysis is not as fast as the rate of ATPase reaction thereby accounting for depletion of PCr; which is in proportion to ATP turnover until the end of the workloads. Large declines in PCr are therefore accompanied by small or no changes in ATP and ADP. At steady workloads, the oxidative phosphoryl ation reaches a steady st ate of ATP release, thereby leveling off PCr levels as long as no further demand for ATP arises. [Note that the shuttle mechanism explained above is only one th eory that describes PCr replenishment by ATP in the skeletal muscle. This metabolite homeostas is can also occur by simple diffusion processes (the buffer and spatial roles); which are the altern ate and more modern explanations to describe this homeostatic mechanism (Kushmerick 1995, Kushmerick and Meyer 1985)]. 3.6.6 Relationship between PCr Recovery Ra tes and Muscle Oxidative Capacity During recovery from exercise, ATP breakdow n is minimal and PCr levels return to baseline values. Studies have well established that duri ng recovery from exercise, glycolysis ceases and PCr in the muscle cell is replen ished at the expense of ATP produced via mitochondrial oxidative phosphoryla tion (Taylor, Bore et al. 1983; Meyer 1988; Quistorff, Johansen et al. 1993; Kemp, Roberts et al. 2001; Mattei, Bendahan et al. 2004). Indeed, for steady state, low intensity exercise, where no cha nge in intracellular pH (Phillips, Wiseman et al.) is expected, a constant pos itive relationship exists in the time scales and rates of oxygen consumption in the mitochondria following exerci se and PCr recovery rates (Piiper and Spiller 1970; Meyer 1988; McCully, Iotti et al. 1994; Th ompson, Kemp et al. 1995). These results confer that PCr resynthesis after exercise is mediated via oxidative phosphorylation. Accordingly, rate constant of PCr recovery (PCr recovery rates) is conventionally been recognized as an index of m itochondrial oxidative capacity. PCr resynthesis follows a pseudo first order ki netics and the rate c onstant is described by a monoexponential curve as long as PCr is depl eted by a sufficient amount (~50%) and pH does

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96 not decline below 6.75 units (Taylor, Bore et al. 1983; McCully, Iotti et al. 1994; Boesch 2007). In addition, for the rate constants of PCr recovery to correctly re flect oxidative capacity, several assumptions are made: a) the creatine kinase reaction is in equilibrium b) presence of an intact vascular (hence oxygen) supply to the muscle during recovery c) the PCr resynthesis is predominantly due to ATP released by oxidati ve processes (thus anaerobic ATP production is negligible) (Meyer 1988; Paganini, Foley et al 1997). Furthermore, PCr resynthesis following depletion necessitates the presence of intact bl ood supply and oxygen availability (Sahlin, Harris et al. 1979; Quistorff, Johansen et al. 1993). Importantly, while PC recovery rates can be used as indices of oxidative capacity, absolute estimations of metabolic capacity from PCr recovery data is possible using rigorous theoretical framework (Paganini, Foley et al. 1997). For practical purposes, the rate constant of PCr recovery (k) that reflects muscle oxidative capacity is calculat ed using the following equation (Hartkopp, Harridge et al. 2003; Kitahara, Hamaoka et al. 2003): PCr (t) = PCr0 + PCr (1-e kt) (3-6) Where, PCr is the concentration of PCr at a given time t during post exercise recovery; PCr0 is the PCr concentration at end of exercise and PCr is the change in PCr concentration after recovery from exercise. Note that the rate constant (k) is in fluenced by change in pH such that lower pH decreases the rate constants of PCr recovery (Bendahan, Confort-Gouny et al. 1990; McCully, Iotti et al. 1994; Paganini, Foley et al. 1997; Heerscha p, Houtman et al. 1999). Alterations in pH apparently a lter the equilibrium state of the creatine kinase reaction and/or mitochondrial ATP yield (Bendahan, Confort-Gouny et al. 1990; McCully, Iotti et al. 1994). Accordingly, measurements of oxidative capac ity typically accompa nying pH decrease do not correctly reflect oxidative capacit y. The inverse of the rate cons tant kPCr is called the time

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97 constant of PCr recovery ( PCr,) and is related to peak oxygen uptake (VO2) and maximal oxygen consumption (VO2 max) (Thompson, Kemp et al. 1995). The PCr is reported to be independent of exercise intensity an d end exercise levels of PCr. Another reliable measure, initial post exerci se PCr resynthesis rate (Vmeas), is also demonstrated to reflect mitochondrial oxidative capaci ty and is less affected by the end exercise PCr levels and pH (Meyer 1988; Kemp, Thompson et al. 1994; L odi, Kemp et al. 1997). This initial rate of PCr resynthesis during the first few seconds is es timated from the first two-three data points in recovery and is quantified by: Vmeas = (d[PCr]/dt) (3-7) Also, a more complete estimation of maximum mitochondrial capacity (Qmax) has also been used (Kemp, Thompson et al. 1994); Qmax = k [basal PCr] (3-8) where k is the rate constant of PCr recovery and [PCr] is the basal PCr levels. As mentioned above, increase in ADP levels that follow exercise, tend to return to baseline levels soon after exercise. Faster the recovery of ADP levels, faster is ATP formed and therefore this implies an increased effici ency of mitochondria. Indeed, ADP concentrations in myocytes are known to regulate mitochondri al ATP synthesis. As ATP is used up during exercise, the [ADP] increases and return of [ADP] levels back to normal are indicative of in vivo mitochondrial function or oxidative capacity (Arg ov, De Stefano et al. 1996). Few studies have in fact proven that the rate of decline in ADP is a more sensit ive measure of oxidative capacity than PCr recovery rates because of its robustness to changes in intracellular pH (Kemp and Radda 1994; Argov and Arnold 2000). However, since ADP levels are not directly measurable, it is less commonly used. Also, owing to its re latively lower concentration; [ADP] cannot be

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98 directly measured from a normal 31P spectrum; but can be estimated using 31P-MRS assuming a constant magnesium ion concentration (Kemp, Sa nderson et al. 1996; Duro zard, Gabrielle et al. 2000). 3.7 Summary This chapter reviews the funda mental concep ts of MR with main focus on MRI and MRS applications in skeletal muscle. MR has become the investigative tool of choice because of its non-invasiveness, ability to perform longitudinal studies and obtain a wealth of anatomical and biochemical information of skeletal muscle While MRI more clearly assesses muscle morphology, MRS estimates physiological metabolic pr ocesses in real time. Various applications of MRI in skeletal muscle include assessment of muscle size, identification of muscle damage and quantification of fatty ti ssue infiltration. The most comm on nuclei used in MRS include proton and phosphorus. Through its uni que ability to decipher between protons in water and fat peak in muscles, 1H-MRS has gained widespread applic ation in characterizing tissue T2 relaxation properties and separately quan tifying IMCL and EMCL fat components. 31P-MRS has been utilized to estimate muscle pH and quan tifying energy rich phosphate s that participate in basic physiological processes. The high time resolu tion and the ability to quantify metabolites in real time has made 31P-MRS a unique non-invasive tool to reliably measure muscle oxidative capacity in-vivo

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99 Table 3-1. Longitudinal (T1) relaxation times (in milliseconds) of water and lipid components obtained from human skeletal muscle at different magnetic field strengths. Table 3-2. Transverse (T2) relaxation times (in milliseconds) of water and lipid components obtained from human skeletal muscle at different magnetic field strengths. Reference Muscle Magnetic field strength Water Fat IMCL EMCL Bruhn 1991 Plantar flexors 1.5T n/a 300 n/a n/a Bongers 1992 Plantar flexors 1.5T 1100-1400 280-330 n/a n/a Schick 1993 Soleus 1.5T 1100-1500 270-280 n/a n/a Sinha 2002 Soleus 2.1T 1220-1370 n/a 306-378 221-461 Hwang Jong Hee 2001 Soleus 4T 1300-2300 n/a 340-440 340-440 Reference Muscle Magnetic field strength Water Fat IMCL EMCL Bongers 1992 Plantar flexors 1.5T n/a 75-100 n/a n/a Bruhn 1991 Plantar flexors 1.5T 30 90 n/a n/a Szczepaniak 1999 Soleus 1.5T 37-43 n/a 82-90 66-76 Schick F, 1993 Soleus 1.5T 50 70-85 n/a n/a Sinha R, 2002 Soleus 2.1T 29-34 n/a 70-83 76-86 Hwang Jong Hee 2001 Soleus 4T 21-31 n/a 68-82 58-78

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100 Figure 3-1. Schematic representation of a nucleus a nd its behavior as a bar magnet in an external magnetic field Bo Figure 3-2. Application of a 90o RF pulse.

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101 Figure 3-3. T1 relaxation time Figure 3-4. T2 relaxation time

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102 Figure 3-5. Fourier transf orm of the FID signal. Figure 3-6. Schematic representation of two tissues with different T1 rela xation times. A) Faster T1 relaxation times of fat tissue and relatively slower T1 relaxation times of skeletal muscle tissue. B) Representative T1 wei ghted trans-axial image of a healthy human calf muscle obtained 3T.

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103 Figure 3-7. Schematic representation of two tissues with different T2 rela xation times. A) Faster T2 relaxation times of muscle tissue and re latively slower T2 relaxation times of fat tissue. B) Representative T2 weighted trans-axial image of a healthy human calf muscle obtained at 3T. Figure 3-8. Bandwidth of frequencies excites a specific width of slice in the sample.

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104 Figure 3-9. Frequency and phase encoding gradient effects on spins. A) Spins (arrows depict spin phases) in all the rows of the grid are aligned in the same direction of the main magnetic field before application of a phase encoding gradient (Gy) B) After application of Gy, spins in the upper row experience a hi gher magnetic field and spins in the lowest row experience the least magnetic field. C) After application of frequency encoding gradient, spins in the upper and lower rows change phases while those in the center row experience no change in magnetic field. Figure 3-10. Representative 1H-MR spectrum of a healthy human soleus muscle at 1.5Tesla.

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105 Figure 3-11. Representative 1H-MR spectrum of a human skeletal muscle at 1.5Tesla showing decomposition of the lipid p eak into its IMCL and EMCL components after water suppression (at ppm = 0). The creatine (C r) and choline peaks represent muscle metabolites. Figure 3-12. Representative typical 31P-MR spectrum of a rat calf muscle at 11Tesla (NADH = nicotinamide dehydrogenase; PME= phosphomonoesterase)

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106 Figure 3-13. Schematic representation of the cr eatine-phosphocreatine shu ttle and buffer role of the creatine-kinase reaction muscle.

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107 CHAPTER 4 OUTLINE OF EXPERIMENTS The overall objective of this dissertation was to investig ate the lower extremity skeletal muscle adaptations after incomplete spinal cord injury. An outline of aims and hypotheses related to experiments is given below. 4.1 Experiment One 4.1.1 Specific aim a) To quantify lower extrem ity skeletal muscle size in persons with chronic incomplete spinal cord injury (SCI) and in age-matched healthy individuals b) To determine atrophic response in anti-gravity versus the non anti-gravity muscles c) To examine the impact of ambulatory ability on muscle size. Maximum cross sectional areas (CSA) of the thigh and calf muscles were quantified using high-resolution magnetic res onance imaging (MRI). 4.1.2 Hypotheses a) Chronic incom plete SCI leads to a decline in lower extremity muscle CSA. b) Greater decreases are observed in the lower extremity anti-gravity muscles as compared to the non anti-gravity muscles. c) Persons with incomplete SCI who use a wheelchair as primary means of mobility show a larger atrophic response than persons with SCI who do not use a wheelchair for ambulation. 4.2. Experiment Two 4.2.1 Specific Aim a) To characterize m uscle characteristics via T2 relaxation times of lower extremity muscles in persons with incomplete SCI and co mpare that with age-matched controls. b) To

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108 quantify the intramuscular lipid content along wi th intramyocellular and extramyocellular lipid composition of the soleus muscle after incomplete SCI in humans. Skeletal muscle T2 relaxation properties and soleus muscle lipid content are determined using combinations of MRI and proton spectroscopy (1H-MRS). 4.2.2 Hypotheses a) Persons w ith incomplete SCI show alterati ons in the T2 relaxation times of the lower extremity muscles as compared to healthy individuals. b) Persons with incomplete SCI show elevat ed soleus muscle lipid content along with elevations in both the intramyo cellular (IMCL) and extramyoce llular lipid (EMC L) components as compared to healthy individuals. 4.3 Experiment Three 4.3.1 Specific Aim To assess the im pact of two and nine week s of locomotor training on markers of muscle damage in persons with incomplete SCI. Lower le g skeletal muscle T2 re laxation properties are determined using combinations of MRI and spectroscopy (1H-MRS) measures. 4.3.2 Hypothesis Two and nine weeks of locom otor training alte r the T2 relaxation properties of lower leg muscles of persons with incomplete SCI. 4.4. Experiment Four 4.4.1 Specific Aim a) To assess the im pact of nine weeks of lo comotor training on the lower extremity muscle size of persons with incomplete SCI. b) To assess the impact of nine weeks of locomotor training on the soleus muscle composition of persons with incomplete SCI.

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109 Skeletal muscle CSA and soleus muscle lipid content are determined using combinations of MRI and spectroscopy (1H-MRS). 4.4.2 Hypotheses a) Locom otor training attenuate s the atrophic response and increases the muscle crosssectional area in lower extremity skeletal mu scle size of persons with incomplete SCI. b) Locomotor training alters the lipid content of soleus muscle in persons with incomplete SCI, with changes seen both in the IMCL and EMCL. 4.5. Experiment Five 4.5.1 Specific Aim a) To determ ine the impact of acute spinal contusion (one week) on the content of resting phosphates, and hence the phosphoryl ation potential of rat calf muscle after spinal cord contusion. b) To longitudinally monitor alterati ons in the phosphorylation potential of the calf muscles. Phosphorus magnetic resonance spectroscopy (31P-MRS) at a high magnetic field strength (11T) was used to monitor basal phosphate me tabolites for three weeks after spinal cord contusion. Intracellular quantific ation of phosphate metabolites was achieved using biochemical assays. 4.5.2 Hypotheses a) Imm ediately after spinal cord contusion (one week), there is an alteration in the resting muscle phosphate content; and he nce a change in the phosphorylat ion potential of the paralyzed hind limb muscle. b) Muscle phosphate levels approach control va lues by three weeks after spinal cord contusion.

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110 4.6. Experiment Six 4.6.1 Specific Aim: a) To determ ine the impact of acute spinal contusion (one week) on oxidative capacity of rat hind limb muscle after spinal cord contusion. b) To longitudinally monitor alterations in the skeletal muscle oxidative capacity of the hind limb muscles. Phosphorus magnetic resonance spectroscopy (31P-MRS) was performed on the animal hind limb muscle using an electrical stimulation protocol to quantify in-vivo muscle bioenergetics in real time. Measurements were obtained once weekly for three weeks starting at one week post injury. 4.6.2 Hypotheses a) Imm ediately after spinal cord contusion (one week), there is a decrease in the oxidative capacity of the paralyzed hind limb muscle. b) Muscle oxidative capacity approach contro l values after three weeks of spinal cord contusion.

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111 CHAPTER 5 METHODOLOGY In this dissertation, experim ents one th rough four are focused on studying muscle adaptations in humans with incomplete spinal cord injury. Experiments five and six are conducted in a rat model of spinal contusion injury. While non-invasive MR techniques are employed as the key tool for all experiments; co mbinations of biochemi cal assays, histology and pap smears have been employed as additional methodologies to supplement MR findings in the animal studies. The following sections discuss relevant experimental protocols and data analyses relevant to both human and animal studies. 5.1 Human Studies 5.1.1 Subjects Persons with incom plete SCI and age-matched c ontrols were recruited for this study. Prior to participation in the study, all subjects were informed of the study purpose and all provided written informed consent as approved by the Institutional Review Boards at the University of Florida. Able-bodied controls: Age, weight, height and gender ma tched individuals from the local University of Florida setting were recruited as healthy volunteers for the study. Control subjects were recreationally active, but not engaged in any rigorous exercise pr ogram. Controls were screened for MR compatibility before participation. Individuals with incomple te spinal cord injury: Individuals with SCI were recruited from the local community at the University of Flor ida in Gainesville, FL or at the University of Georgia, Athens, GA. The inclusi on criteria for participants were 1) diagnosis of traumatic SCI at cervical or thoracic levels (C4-T12) resulting in upper mo tor neuron lesions in the lower extremity, 2) history of SCI as defined by the American Spinal Injury Association (ASIA)

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112 Impairment Scale categories C or D, and 3) a me dically stable condition at the time of testing 4) persons who were MR compatible. Participants had varied ambulatory capabilities and accordingly, used a wheelchair, cane, crutches and/or ankle foot orthosis for assistance in mobility. 5.1.2 Clinical Assessments American S pinal cord Injury Association (ASI A) scores for neurological classification of spinal cord injury: The ASIA impairment scale for neurol ogical classification of spinal cord injury is a nominal measure for measuring comp leteness of injury (ASI A 2001). Injury to the spinal cord is considered complete if there is absence of sensory and motor functions in the lowest sacral segments and incomplete if there is preservation of sensory or motor function below the level of injury, including the lowest sa cral segments. Impairments in muscle strength and sensory function are graded by the ASIA impairment scale (ASIA 2001) as follows: ASIA A Complete: No sensory or motor function is preserved in sacral segments S4-S5. ASIA B Incomplete: Sensory, but not motor, function is preserved below the neurological level and extends through sacral segments S4-S5. ASIA C Incomplete: Motor function is preser ved below the neurological level, and most key muscles below the neurological le vel have muscle grade less than 3. ASIA D Incomplete: Motor function is preser ved below the neurological level, and most key muscles below the neurological level have muscle grade greater th an or equal to 3. ASIA E Normal: Sensory and motor functions are normal. The sacral fibers are located more at th e periphery of the cord making them least susceptible to injury after a spin al cord traumatic event. Accord ingly, after a SCI, sacral-sparing is evidence of the physiologic co ntinuity of spinal cord long tract fibers. Indication of the presence of sacral fibers is of significance in defining the co mpleteness of the injury and the

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113 potential for some motor recover y. This finding tends to be repeated and better defined after the period of spinal shock. Lower Extremities Motor Score (LEMS): This is a commonly used measure to define the motor capabilities after SCI. This score uses the manual muscle testing scores of the ASIA key muscles in both lower extremities with a total possible score of 50 (i.e., maximum score of 5 for each key muscle per extremity). Accordingl y, a LEMS score of 20 or less indicates that individuals with incomplete SCI are less likely to be community ambulators. In contrast, a LEMS of 30 or more suggests a better lik elihood for community ambulation (ASIA 2001). Walking Index for Spinal Cord Injury (WISCI): Originally developed by the international study group, the Walking Index for Spin al Cord Injury (WISCI) score is a scale that indicates the ability to walk after SCI. It is a reliable and valid tool that has been found to correlate well with ASIA scores (Burns, Golding et al.). Researchers and clinicians have widely used the WISCI score to assess the walking ability after SCI. The testing incorporates assessing the ability to walk for 10meters with the help of assistive aids, crutch a nd physical assistance of one or two persons. Accordingly, the severity of walking impairment is graded from most to least severe on a scale of 0 to 20. A score of zero implies inability to stand and/or walk at all with assistance and a score of 20 implies the abil ity to ambulate independently for 10m (Ditunno, Ditunno et al. 2000). Note worthily, the scale is no t reflective of the func tional ambulatory status of the individual in the community. 5.1.3 Locomotor Training Locom otor training (LT) consisted of 9 weeks of step training (30 minutes, 5days/week) on the treadmill with body weight support and manual assistance followed by over ground training (20 minutes). Bodyweight support, initially set to 40% of the subjects body weight was adjusted to maintain proper limb kinetics wh ile also maximizing bilateral limb loading. If

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114 necessary, manual assistance was provided by trainers to assist in correct stepping. In addition, assistance was provided in maintaining an upright trunk by stabilizing the pelvis. Subjects were encouraged to swing their arms voluntarily or with the aid of poles. Verbal encouragement by trainers along with visual cues provided by a mirro r placed in front of th em served as additional sensory cues to facilitate a near-normal walki ng pattern. Speed of treadmill stepping was kept in a range consistent with normal walking, optimi zed for each subject (2.02.8 miles/hr). Training sessions were interspersed with adequate rest periods. During each rest break, the participant stood with minimal BWS required to maintain balan ce with minimal assistan ce from the trainers. Initially, treadmill sessions required up to 60 minut es to achieve 30 minutes of stepping, but the amount decreased with improved walking ability and endurance such th at stepping could be completed in 45 minutes or less. Progression of training was achieved by decreasing BWS, altering speed, increasing trunk control, decr easing manual assistance for limb control and increasing the stepping time on the treadmill per bout. All participants recei ved the same number of sessions and spend approximately the same amount of time involved in training, although progression of training paramete rs was individualized. Immediat ely following step training on the treadmill, each participant engaged in 20 minutes of over ground training. Over ground training incorporated the use of assistive devices, but partic ipants were otherwise bearing full weight on their lower extremities. A more detail ed description of the training principles and parameters has been provided (Behrman a nd Harkema 2000; Behrman, Lawless-Dixon et al. 2005). 5.1.4 Proton Magnetic Resonance Imaging (1H-MRI) Proton magnetic resonance imaging (1H-MRI) was used to determine a) maximum muscle CSA and b) T2 relaxation properties of the lower extremity muscles.

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115 A 1.5 Tesla super conducting magnet scanner (Signa; General Electric)2 was used to collect trans-axial images of the leg and thigh. The self-reported more involved leg of the SCI group and the right leg of control subjects was scanned. A standard (20cm long) lower extremity quadrature coil or body coil was employed for imag ing. The extremity coil covered the length of the leg starting from above the lateral malleolus and extending to ~3 cm centimeters proximal to the superior patella. T1 weighted image of the leg with a pulse se quence of TR=300ms, TE= minimum, matrix = 128*128 and FOV = 16-30cm served as a coronal localizer for axial imaging (Figure 5-1). Trans-axial images for measuremen ts of CSA and T2 relaxation properties were obtained using distinct NMR sequences. 5.1.4.1 Muscle cross sectional area: data collection and analysis Spin echo or fat suppressed 3D SPGR i maging sequence was utilized with the following imaging parameters: acquisition matrix size, 256x256 to 256x192; field of view of 16cm to 32cm for the leg and 22cm to 40cm for the thigh; pulse repetition time, 51ms ms; echo time, 10ms-27ms; slice thickness of 5-7mm; slice gap, 0-5mm. The images were transferred to a silicon gr aphics UNIX workstation and fat-free maximal muscle CSA of lower extremity muscles was de termined using a custom-designed interactive computer program as previously de scribed (Elliott, Walter et al. 1997). Briefly, multiple slices (8-20) of each muscle were outlined taking ca re to avoid fascia and blood vessels, manually thresholded to include pixels of similar signal intensity that represent muscle tissue and then segmented to determine the slice with maximum CS A. A unique feature of this software was its calculations for partial volume effects thereby yielding an accurate measure of muscle CSA. Maximum CSA of the quadriceps femoris (QF) and hamstring ( HAMS) muscle groups in the 2 GE Medical Systems global headquarters: Waukesha, Wisconsin

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116 thigh, the soleus (SOL), medial gastrocnemius, lateral gastrocnem ius (LG), tibialis anterior (TA) muscles in the lower leg and ma ximum CSA of the entire posterior compartment (PC) of the lower leg (that includes the tibialis posterior musc le) were calculated (Fig ure 5-2). In addition, maximum CSA of the ankle anti-gravity muscles [plantar flexors (PF)] was considered at the level of the lower leg where the SOL, MG and LG taken together resulted in the largest CSA. 5.1.4.2 T2 relaxation times: data collection and analysis T2 weighted imaging using multiple slice spin echo sequence was performed with following imaging parameters TR = 2000ms; TE = 26, 52, 78 and 108ms; FOV = 16cm, slice thickness = 7mm, matrix of 256x128. The T2 weighted images were transferred to a UNIX workstation and characteristic T2 relaxation time of the muscle and bone marrow were calculated using a custom designed software assuming a singe exponential decay with respect to the four imaging echo times. The T2 relaxation times were represen ted on resultant images called T2 maps (Figure 5-3). Specific regions of interests (ROI) that avoid visible blood vessels in th e SOL, MG, LG and TA muscles were identified in 8-10 T2 map slices and the average T2 of each muscle was subsequently calculated. T2 times of bone marrow were measured as internal references to assess reproducibility of T2 values. 5.1.5 Proton Magnetic Resonance Spectroscopy (1H-MRS) Localized unsuppressed spectra were acquire d from the soleus muscle to measure a) muscle lipid content b) T2 relaxation charac teristics of muscle independent of fat. For precise localization of vol ume of interest, a voxel (35mm thickness) was prescribed over the localized transaxial image of the soleus muscle avoiding visible blood vessels and muscle fascia (Figure 5-4). In order to ascerta in voxel position over the muscle without signal contamination from non-selecte d surrounding tissue, a phantom experiment was performed to

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117 obtain an image of the prescrib ed voxel. Figure 5-5 shows a raw image of the voxel acquired at the 1.5T from a CuSO4 phantom (TR =2000ms, TE = 18, FOV =16cm). Note that the boundaries of the voxel are sharp and its dimensions the sa me as the prescribed voxel dimensions. An unsuppressed water stimulation acquisition m ode spectroscopic se quence STEAM (Bruhn, Frahm et al. 1991) sequence was used with four different echo times (13ms, 30ms, 60ms and 120ms), a TR of 6000ms, 4 scans, 512 points an d 2500 Hz spectral width. In addition, a 32scan average was performed at the 120ms TE to estimat e total lipid content and individual lipid components; intramyocellular (IMCL) a nd extramyocellular lipids (EMCL). 5.1.5.1 Muscle lipid: data analysis A zero order phase correction was perform ed on the 32 scan raw spectrum and water and lipid spectral peaks were quantified using an Advanced Magnetic Resonance (AMARES) timedomain-fitting algorithm using jMRUI (Naressi, Coutur ier et al. 2001). Prior knowledge values were constructed from healthy controls for m easuring water and lipid amplitudes. Whole lipid peak and water peak were identified at 1.5ppm and 4.7ppm (Figure 5-4). Once amplitudes from the water and whole lipid peak were calculated, the water peak was manually suppressed during data analysis. Lipid resonance peak was subs equently deconvoluted to estimate IMCL and EMCL at approximately 1.25pp m and 1.4ppm using the AMAR ES method (Figure 5-6). Thereafter, IMCL and EMCL lipid amplitudes were calculated from their spectral peaks and corrected for T2 relaxation effects using T2 relaxation times of 85ms for IMCL and 75ms for EMCL. T2 relaxation times of IMCL and EMCL in the soleus muscle used in this study match with lipid T2 relaxation values reported in literature (Bruhn, Frahm et al. 1991; Szczepaniak, Babcock et al. 1999). In concurrence with othe r studies, the overall li pid, IMCL and EMCL content in our study were expressed as a ratio us ing the spectral water peak as an internal reference (Sinha, Dufour et al. 2002; White, Ferguson et al. 2006).

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118 Uniqueness of this method to analyze fat was that it exclusively repr esents intramuscular fat. Studies in the past have quantified fat w ithin muscle using imaging techniques. However, this method is plagued by the inclusion of inte r-muscular fat (Elder, Apple et al. 2004) and requires a good amount of fat to adequately qu antify it (Huang, Majumdar et al. 1994). Proton spectroscopy on the other hand, can yield a good lipid peak along with fat components from within the same muscles. 5.1.5.2 Muscle T2 relaxation times: data analysis The T2 relaxation time of soleus muscle independent of fat was calculated assuming a single exponential decay with respect to the four spectral echo times. Mono-exponential decay versus multi-exponential decay curves: In this study, only four echo times were used to acquire both im aging and spectroscopy data, which consequently required assumption of a single exponential deca y to calculate T2 relaxation times from each muscle. This might pose a potential limitation to data collection in our present study. Chances prevail that the single exponential decay curves might in fact be multi-exponential, with contributions from multiple T2 components. Va rious algorithms including the non-negative least squares (NNLS) are commonly used to decompose multiple component curves into its distinct components. However, for decomposition of the signal into its components using NNLS, several TEs are necessitated during data collection. 5.2 Animal Studies 5.2.1 Animals Sixteen adult Sprague D awley female rats (16 weeks of age, 228-260g; Charles River, NJ) either underwent a spinal cord contusion (n=8) or served as controls for wet lab procedures relevant to the study. Briefly, expe rimental rats were tested for outcome measures before injury and were followed up longitudinally for three weeks after injury. Animals were housed in a

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119 temperature controlled room at 21 0C with a 12:12 hour light: dark cycle and were provided rodent chow and water ad libitum. All experimental procedures were performed in accordance with the U.S. Government Principle for the U tilization and Care of Vertebrate Animals by approval of the Institutional Animal Care & Use Committee at the University of Florida. At the end of the study, all animals were euthanized and hind limb muscles were extracted and processed for biochemical assays. 5.2.2 Gender Differences in Animal Models of SCI Most studies pertaining to spinal cord injury research have u sed the female rat model of spinal injury. While stud ying both male and female models for muscle adaptations will assist in teasing out any gender differences in muscle adaptations, we have chos en to utilize female rats in the present work. One of the main reasons for our choice vests in remaining consistent with reports in literature and taking advantage of valid comparisons with published work by other investigators. 5.2.3 Spinal Cord Contusion Injury Spinal cord contusion injury was produced using a NYU (New York University) impactor device. A 10g weight was dropped from a 2.5-cm hei ght onto the T8 segmen t of the spinal cord exposed by laminectomy under sterile conditions. Animals received two doses of Ampicillin per day for 5 days, starting at the day of surger y. Procedures were performed under ketamine (100mg/kg)-xylazine (6.7mg/kg) anesthesia (Reier, Anderson et al. 1992; Thompson, Reier et al. 1992). Subcutaneous lactated Ring ers solution (5 ml) and antibio tic spray were administered after completion of the surgery. The animals were kept under vigila nt postoperative care, including daily examination for signs of distress, weight loss, dehydration, and bladder dysfunction. Manual expression of bladders was performed 2-3 times daily, as required, and animals monitored for the possibility of ur inary tract infection. Animals were housed

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120 individually following surgery. At post-operative day 7, open field locomotion was assessed using the 21 Basso-Beattie-Bresnahan (BBB) loco motor scale (Basso, Beattie et al. 1995) and animals that did not fall within a preset range (0-7) were excluded from the study. After baseline measurements before injury, rats underwent once weekly MRS measurements for three weeks starting at week one after injury. 5.2.4 Experimental Electrical Stimulation Protocol An electrical muscle stimulation protocol was adopted to determine the mitochondrial oxidative capacity of the rat hind limb muscle in-vivo Animals were anesthesized using gaseous isoflurane in oxygen (3% box induction), and maintained at 0.5 %-2.5% during the MR procedures. After shaving the limb and cleaning it with alcohol, an oval surface coil tuned to 31P (190.5 MHz) was placed over the belly of the gastrocnemius muscles. A 1H surface coil was placed underneath the hind limb to perform sh imming. Two needle electrodes were placed subcutaneously one over the region of the th ird lumbar vertebrae and the other land marked over the greater trochanter to stimulate the hind limb plantarfle xor muscles via stimulation of the sciatic nerve (Figure 5-9). Electrical stimulat ion was carried out for four to six minutes to deplete PCr by 30 to 40%. A Grass Stimulator (Quincy MA) with a Grass Model SIU8T stimulation isolation unit (Grass Instruments, West Warwick, RI) was used to deliver a monophasic, rectangular pulse w ith a 1ms pulse duration, 1Hz fr equency and 10V. Following the electrical stimulation, the muscle was allowed to recover for twenty minutes. Conceptually, electrical stimulation depletes phos phocreatine (PCr) from the contr acting muscle an d the rate of PCr recovery is measured to determine skelet al muscle oxidative capac ity. During the entire duration of stimulation and recovery, spectra were collected. No attempts were made to synchronize the radiofrequency pulse with the muscle stimulation. The FIDs were multiplied by

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121 an exponential corresponding to a 25Hz line br oadening. Vital signs of the animal were monitored throughout the experimental procedure. 5.2.5 Phosphorus Magnetic Resonance Spectroscopy (31P-MRS): Data Collection All data was collected using a high magne tic field strength Bruker 11Tesla/470 MHz spectrometer. A 1.5 x 1.7 cm oval surface coil tuned to 31P (190.5 MHz) was placed over the belly of the gastrocnemius muscle. A 3-cm standard 1H surface coil was placed underneath the hind limb to perform shimming and the animal's hind limb was extended such that the calf muscles were centered over the surface coil. For the baseline 31P MRS spectra data, spectra were acquired with a 50 s square pulse, a TR of 2s, spectral width of 10,000 Hz, 150 averages and 8000 complex data points. For the 31P kinetic data, spectra were averaged into 20s bins and acquired at rest (5 min), electr ical stimulation (4-6 min), and r ecovery (20 min); thus amounting to a total of 93 fids. The partially relaxed spectra were then ca librated by comparison with fully relaxed spectra (acquired at TR of 15sec onds) to determine correction factors (CF). 5.2.5.1 31P-MRS spectral analysis at rest Resting spectra yields measures of basal Pi and PCr concentration from the resting gastrocnemius rat hind limb muscles. The spectra were manually phased, and the areas of the ATP, Pi, and PCr peaks determined by area integration. The enzymatically determined ATP concentration in frozen muscle tissue was equated with the integral of the -ATP. Equating tissue measurements of ATP under the -ATP peak is established as valid for skeletal muscle (Hitchins, Cieslar et al. 2001) Pi and PCr concentrations were determined by using -ATP as an internal standard and af ter accounting for correction factors (CF) as follows:

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122 [Metabolite] ( mol/g wet wt) = CFMetabolite x IntegralMetabolite x [ATP] ( mol/g wet wt) Integral -ATP (5-1) ATP concentration in gastrocnemius mu scle obtained by ATP assay was 4.6 0.3 mol/g wet wt and is similar to ATP concentrations in rat gastrocnemius muscle reported in literature (Authier, Albrand et al. 1987; Hitchins, Cieslar et al. 2001 ; Gigli and Bussmann 2002). Similarly, our correction factors for Pi and PCr were 1.69 and 1.40 resp ectively and match well with published values from rat gastrocnemius muscle (Mizobata, Prechek et al. 1995). Intracellular pH were calculated fr om the chemical shift of the Pi peak relative to PCr using the equation, pH = 6.75 + log [( 3.27) / (5.69 )], where is the chemical shift of the Pi peak in ppm. The cystolic phosphorylation potentia l was calculated in reciprocal form as [Pi][ADP]/[ATP] since the phosphorylation potentia l itself is not normally distributed. Free cystolic ADP was calculated from the creatine kinase equilibrium reaction as previously described (Mizobata, Prechek et al. 1995; Thomps on, Kemp et al. 1995; Pathare, Vandenborne et al. 2007): [ADP] = {[free creatine][ATP]}/{[PCr][H+][Keq] (5-2) where the free creatine was quantified by subtracting the PCr content obtained by 31P-MRS from the total creatine content (42.2mM) de termined biochemically. Intracellular Mg concentration and equilibrium constant (Keq) of the creatine kinase reaction were assumed as 1mM and 1.66 x 109 respectively for mammalian skeletal muscle (Veech, Lawson et al. 1979). 5.2.5.2 31P-MRS spectral analysis of elec trical stimulation protocol The electrical stimulation protocol data yields kinetic measurements of PCr. Dynamic changes in PCr levels were measured using complex principal component analysis (Elliott,

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123 Walter et al. 1999). Recovery data were fitted to a single exponential curve, and the pseudo firstorder rate constant for PCr recovery (kPCr) was determined (Meyer 1988). The maximal rate of PCr resynthesis, a measure of mitochondrial oxida tive capacity (Vmax-lin) was calculated based on kPCr and PCr rest (Vmax-lin = kPCr [PCr] rest) (Walter, Vandenborne et al. 1997). Initial rates of PCr recovery (Vmeas mM/min, a direct measure of mitochondria l ATP synthesis) was determined from the first three to four data point s in recovery; depending upon the best linear cu rve fit. The initial rate of PCr resynthesis was also ex trapolated from the product of kPCr and amount of PCr depletion ( PCr). Thus, Vex (mM/min) = kPCr. PCr (Walter, Vandenborne et al. 1997). Rates of PCr depletion at onset of stimulation (Vdep mM/min, a measure of ATP demand) were determined from the first three to nine data points (first thr ough three minutes) of PCr declines during stimulation. The maximum oxidative ATP synthesis rate (Qmax), which is a function of intrinsic mitochondrial content and enzyme activity, oxygen and substrate supply to the mitochondrion, and cytosolic redox state (Kemp, Sanderson et al. 1996), was calcu lated from the known hyperbolic relationship between PCr resynthe sis and cytosolic free [ADP] and from PCr resynthesis and [ ADP][Pi]/[ATP]. Qmax-ADP = Vmeas (1 + Km/[ADP]) mM/min, where Km is the Michaelis constant and is assumed as 50 M for rat leg muscle (Thompson, Kemp et al. 1995). (5-3) Qmax-[ADP][Pi]/[ATP] = Vmeas (1 + Km/[ADP][Pi]/[ATP]) mM/min, where Km is assumed as 0.11 mM. (5-4) 5.2.6 Biochemical Assays Ani mals were sacrificed at the end of the experiments and gastrocnemius muscle excised and snap frozen at -800C for subsequent biochemical quantification.

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124 5.2.6.1 ATP measurements ATP was measured as previously described (Hitchins, Cieslar et al. 2001; Gigli and Buss mann 2002; Pathare, Vandenborne et al. 2007) Frozen gastrocnemius muscle was ground to fine powder using a mortar and pestle under dr y-ice. 100mg of the tissu e was homogenized for 30seconds with a Mini-bead-beater in a plastic Eppendorf tube containing beads and ice-cold 0.9% perchloric acid (5v/w). The sample was then centrifuged at 9000 g for 15 minutes at 40C (~4000rpm). The supernatant wa s extracted and added to 4M KOH (1.125v/w) and centrifuged again for 5 minutes at 40C and 9000 g. The supernatant was frozen at -800C and processed for ATP measurements with an ATP assay kit (Sig ma) using a luminometer (Biotek Instruments, CA). 5.2.6.2 Total creatine measurements Total m uscle creatine content was determined using the diacetyl/ -napthtol assay ((De Saedeleer and Marechal 1984; Va ndenberghe, Gillis et al. 1996; Tarnopolsky and Parise 1999). Approximately 10-15 mg (average 11.55 2.25 mg) of muscle tissue was cut and placed in a microfuge tube, and then placed in a v acuum centrifuge (Savant ISS110 SpeedVacTM Concentrator, Thermo Scientific, Milford, MA) to be spun for 18-24 hours. After sufficient muscle drying, the samples were then placed in an ultra-low freezer at -80C. Dried muscle was powdered by grinding on a porcelain plate with a pestle. Connective tissue was removed and discarded, whereas powdered muscle was placed into pre-weighed microfuge tubes and dry weight determined. Powdered muscle (avera ge 2.27 0.44 mg) was extracted in a 0.5 M perchloric acid/1 mM EDTA so lution at a relative ratio of 800 l per 10 mg powdered muscle on ice for 15-minutes, while periodically vortexi ng. Samples were then spun at 15,000 rpm at 4 C for 5-minutes. The supernatant was transferred in to a microfuge tube and neutralized with 2.1 M KHCO3 / 0.3 M MOPS solution at a ratio of 1:5 a nd then centrifuged again at 15,000 rpm for 5-

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125 minutes. The resulting supernat ant was then stored at -80 C for future use. In order to determine muscle total creatine concentration, 40 l of the supernatant from the above reaction was combined with 140 l ddH2O and 20 l 0.4 N HCl and heated at 65 C for 10-minutes to hydrolyze phosphate groups. The soluti on was then neutralized with 40 l of 2.0 N NaOH and analyzed as described above. TCr and ATP levels were determined in mol (g wet weight)-1 and mmol (l tissue)-1, assuming a muscle density of 1.06 gml-1. These were converted to mmol l1 intracellular water assuming a cellular water fraction of 0.83 in the rat gastrocnemius muscle (Veech, Lawson et al. 1979; Cieslar, Huang et al. 1998).

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126 Figure 5-1. Representative 1H-MRI coronal image of the calf mu scles from a healthy control at 1.5T. Trans-axial images were obtained along the length of the coronal image (represented by horizontal green lines) Figure 5-2. Representative 1H-MRI trans-axial image of the calf muscles from a healthy control at 1.5T. A 3D fast gradient echo imaging sequence was used in a 1.5Tesla magnet. (A= anterior; P=posterior; M= medial; L=lateral)

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127 Figure 5-3. A) Representative 1H-MRI T2 weighed images of the calf muscles obtained at four different echo times. B) Individual pixel signal intensities were fitted to an exponentially decaying curve along four different echo times (TE) C) Representative T2 map of the T2 weighted images acquired in A. Figure 5-4. Representative transaxial image presents a voxel pres cribed over the soleus muscle and a resultant 1H-MR spectrum with water and fat peaks.

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128 Figure 5-5. Decomposition of a lip id peak obtained from a hea lthy soleus muscle into its intramyocellular and extramyocellu lar components using jMRUI. Figure 5-9. Experimental-setup for elect rical stimulation protocol during 31P-MRS data acquisition.

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129 CHAPTER 6 EXPERIMENT ONE LOWER EXTREMIT Y MUSCLE C ROSS-SECTIONAL AREA AFTER INCOMPLETE SPINAL CORD INJURY 6.1 Summary The purpose of this study was to: a) To quan tif y skeletal muscle size in lower extremity muscles in persons after incomplete-SCI, b) to assess differences in muscle size between involved lower limbs, c) to determine the impact of ambulatory status (using wheelchair for community mobility versus not using a wheelchair for community mobility) on muscle size after incomplete-SCI, d) to determine if differen tial atrophy occurs among individual muscles after incomplete-SCI. Seventeen persons with incomplete-SCI and 17 age, gender, weight and height matched non-injured controls participated from the university research setting. Maximum cross sectional area (CSA) of indivi dual lower extremity muscles [soleus, medial gastrocnemius, lateral gastrocnemius, tibialis anterior, quadr iceps femoris and hamstrings] was assessed by Magnetic Resonance Imaging. Overall, subjects with incomplete-SCI dem onstrated significantly smaller (24% 31%) average muscle CSA in aff ected lower extremity muscles as compared to control subjects (P<0.05). Mean differences were highest in the thigh muscles (~31%) compared to the lower leg muscles (~25%). No differences were noted between the self-reported moreand less-involved limbs within the incomplete-SCI group. Dichotomiz ing the incomplete-SCI group revealed significantly lower muscle CSA values in both the wheelchair [range = 21% 39%] and non-wheelchair groups [range = 24% 38%]. In addition, the wheel chair group exhibited significantly greater plantar flexor muscle atrophy compared to th e dorsi-flexors, with maximum atrophy in the medial gastrocnemius muscle (39 %). Our results suggest marked and differential atrophic response of the affect ed lower extremity muscles th at is seemingly impacted by ambulatory status in persons with incomplete-SCI.

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130 6.2 Introduction An e merging trend in the care and treatment of persons after spin al cord injury (SCI) is the increased proportion of persons diagnosed with in complete injuries. Persons with incompleteSCI exhibit variable paralysis a nd paresis of affected muscles, typically resulting in impaired motor performance and varying degrees of func tional limitations (Subbarao 1991; Tang, Tuel et al. 1994; Burns, Golding et al. 1997). Interest ingly, although incomplete-SCI constitutes ~51% of all new spinal injuries, the majority of huma n and animal research related to physiological and morphological adaptations following SCI has fo cused on subjects with complete injuries. As such, a large body of literature exists that describes skeletal muscle adaptations after this type of injury (Baldi, Jackson et al. 1998; Hopman, Du eck et al. 1998) with few data describing adaptations within affected skelet al muscle after incomplete injuries. Similar to persons with complete-SCI, persons with incomplete-SCI exhib it a variety of clinically relevant motor and functional deficits, including local muscle fa tigue, weakness of aff ected muscles (Sloan, Bremner et al. 1994; Johnston, Finson et al. 2003) and diminished capacity to ambulate(Waters, Adkins et al. 1994; Ulkar, Yavu zer et al. 2003). We recently dem onstrated that after chronic upper motor lesions and incomplete-SCI, both knee ex tensor and plantar fle xor skeletal muscles generate ~70% less peak torque (Jayaraman, Gregory et al. 2006). Other studies have shown a significant reduction in ambulatory capacity, with a reduced gait spee d, step frequency and stride length. Despite such obvious motor dysfunction, no studies have documented the extent of muscle atrophy in paralyzed skeletal muscle fo llowing incomplete-SCI in humans. Given that muscle atrophy relates strongly to compromised muscle streng th (Berg, Dudley et al. 1991; Ploutz-Snyder, Tesch et al. 1995; Vandenborne, Elliott et al. 1998; Stevens, Walter et al. 2004) as well as locomotor ability,(Viss er, Kritchevsky et al. 2002; Visse r, Goodpaster et al. 2005) an

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131 in-depth understanding of the exte nt of impairment in this population would be valuable to the field of rehabilitation research. Persons with incomplete-SCI constitute an extremely heterogeneous group. For example, people after this type of injury exhibit a c ontinuum of ambulatory ab ilities ranging from being completely wheelchair dependent to nearly normal walking without the use of assistive devices. Consequently, the mechanical load ing and activation of the affected lower extremity muscles is extremely variable (Melis, Torres-Moreno et al 1999). Given that forced inactivity of lower extremity muscles (i.e. immobilization, limb suspensi on) results in differential skeletal muscle atrophy (Adams, Hather et al 1994; Adams 2002; Alkner and Te sch 2004) one might expect variable patterns of muscle ad aptations in persons af ter incomplete-SCI. Accordingly, we sought to examine the morphological characteristics of lo wer extremity skeletal muscles in persons with incomplete-SCI. Specifically, the purpose of our study was four-fold: 1) to compare lower extremity muscle maximum cross sectional area (CSA) in persons with incomplete-SCI to a group of age, gender, height and weight matched controls 2) to make comparisons of maximum muscle CSA between the self-reported more a nd less involved limbs within a group of persons with incomplete-SCI 3) to evaluate whether ambulatory status (i.e using wheelchair for community mobility versus not using a wheelchai r for community mobility) influences lower extremity skeletal muscle CSA after incomplete SCI 4) to compare the magnitude of atrophic response a) between the flexor and extensor muscles about the knee a nd ankle, b) between proximal and distal anti-gravity extensor muscle s, and c) among individual ankle plantar flexor muscles.

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132 6.3 Methods We perfor med a case-control study in which th e lower extremity maximum muscle CSA of persons with incomplete-SCI was compared with maximum muscle CSA of age, gender, weight and height matched non-injured cont rols. To address the impact of ambulatory status on skeletal muscle size, we dichotomized our incomplete-SCI subjects into those who did not have upright mobility in the community, but demonstrated ambulatory ability through use of a wheelchair (W/C group) and those who did not use a wheelchair for community mobility (non-W/C group). 6.3.1 Subjects Persons w ith incomplete-SCI: A convenient sample of seve nteen persons (2 women, 15 men; 13 months post-inju ry) with incomplete-SCI (39 yr, 76 kg, 178 cm) volunteered to participate in the study. Of these, 10 subjects were studied at the University of Florida, Gainesville, Florida; and 7 subjects were studied at the Univers ity of Georgia, Athens, Georgia. All participants 1) had a diagnosis of traumatic SCI at ce rvical or thoracic levels (C4T12) resulting in upper motor neuron lesions in the lower extremity, 2) had a history of SCI as defined by the American Spinal Injury Associati on (ASIA) Impairment Scale categories C or D, and 3) had a medically stable condition at the time of testing. Seven of the persons with incomplete-SCI used a wheelchair, two subjects used forearm crutches, and six used a singlepoint cane for community ambulation (Table 6-1). Controls: Seventeen persons (2 women, 15 men; 39 12 yr, 78 12 kg, 178 cm) volunteered to serve as control subjects. Thes e subjects were matched to incomplete-SCI subjects on the basis of age, gender, and height and body mass. Though large demographic variability existed among subjec ts in both the incomplete-SCI and control groups, each control person was closely matched (age 7 yr, he ight 10 cm, and body mass 8 kg) to the corresponding person with incomple te-SCI. Control subjects were recreationally active, but not

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133 engaged in any rigorous exercise program, a nd were recruited from the Gainesville, FL community. 6.3.2 Maximum Muscle Cross-sectional Area Proton m agnetic resonance imaging (MRI) wa s used to determine maximum muscle CSA of the lower extremity. MRI for a ll subjects was performed specifi cally for the study. Details of the MRI procedures are described in Chap ter 5 (section 5.1.4.1). Figure 6-1 illustrates representative trans-axial prot on magnetic resonance images of patient and control subjects obtained at 1.5Tesla magnetic field. 6.3.3 Data Analysis Independent sam ples t-tests were employed to determine if differences existed in the demographic characteristics (age height and body weight) and to compare the maximum muscle CSA of the muscles of interest in the pooled in complete-SCI (n=17) and control groups (n=17). For skewed data, distribution-free Mann Whitney tests were used to compare muscle CSA between controls and persons with incomplete-SCI. The self reported moreand less-involved limbs of persons with incomplete-SCI (n=7) were compared using a two-related sample Wilcoxon test. To determine the impact of ambulatory status on maximum muscle CSA we compared the mean CSA of the above-mentione d muscles in the W/C (n=7) and non-W/C groups (n=10) with their corresponding matched controls using Mann Wh itney test. In all analyses involving comparisons for incomplete-SCI with controls, a un idirectional hypothesis (incomplete-SCI CSA < control CSA) was tested Further, relative differences between the extensor and flexor muscles about the knee a nd ankle were determined by intra-compartment (PF:TA, QF:HAMS) ratios. In a ddition, relative differences between proximal and distal antigravity muscles were compared using inter-compartmen