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Adaptations in Skeletal Muscle following Spinal Cord Injury and Locomotor Training

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PAGE 1

ADAPTATIONS IN SKELETAL MUSCLE FOLLOWING SPINAL CORD INJURY AND LOCOMOTOR TRAINING By MIN LIU 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 2006

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Copyright 2006 by Min Liu

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This document is dedicated to my pa rents, wife, sister and daughter.

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iv ACKNOWLEDGMENTS The completion of this project would not have been possibl e without the support and guidance of many people. At the moment when I am finishing my journey of graduate study at UF, I am extremely grateful to all of those invol ved in my education, research, and life. I would first like to express my sincer e thanks to my advisor, Dr. Krista Vandenborne, who welcomed me as a researcher into her laboratory and permitted me to pursue the area of research most compelling to me. Her excellent guidance and constant encouragement were invaluable throughout my entire doctoral studies. I am very grateful to Dr. Glenn Walter for his remarkable wis dom and invaluable ideas. His passions on science and sharp comments will always be my model in the future. I thank Dr. Floyd Thompson for his broad knowledge. I am blessed to have learned so much about science from him. I thank Dr. Prodip Bose for his immeasurable support and assistance during my doctoral program. I also would like to e xpress my appreciation to Dr. Orit Shechtman and Dr. David Fuller for serving my dissert ation committee and for their incredible scientific advice. It has been a pleasure and honor to work in Dr. Vandenbornes lab. My deepest thanks go to the members in the lab. I am grateful for the assistance of Dr. Jennifer Stevens. This project would not have been completed without he r support. I owe great thanks to Ye Li. Her assistance for deve loping new assay was instrumental in the completion of this project. Special thanks go to Chris, Tiffany, Neeti, Arun, Prithvi,

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v Gabe, Fan, and Shiv. I am grateful to all for their help and friendship. Due to the presence of all these wonderful people, wo rk is more than enjoyable. Finally and most importantly, I would like to express my deepest gratitude to my family members. It is with much love th at I acknowledge and deeply appreciate the sacrifices and difficulties that my loving wi fe, Qing Yang, patiently endured. I also thank my lovely daughter, Melodie, who made my gr aduate study colorful. I am indebted to my father and sister for their relentle ss support, care and encouragement.

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vi TABLE OF CONTENTS page ACKNOWLEDGMENTS.................................................................................................iv LIST OF TABLES...............................................................................................................x LIST OF FIGURES...........................................................................................................xi ABSTRACT.....................................................................................................................xiii CHAPTER 1 INTRODUCTION........................................................................................................1 2 REVIEW OF RELATED WORK................................................................................3 2.1 Spinal Cord Injury Animal Models........................................................................3 2.1.1 Spinal Cord Transection Model....................................................................3 2.1.2 Spinal Cord Isolation Model........................................................................4 2.1.3 Spinal Cord Hemisection Model..................................................................5 2.1.4 Spinal Cord Contusion Model......................................................................6 2.1.4.1 Early contusion models......................................................................6 2.1.4.2 Rat spinal contusion models...............................................................7 2.1.4.3 The New York university impactor....................................................8 2.1.5 Summary.......................................................................................................9 2.2 Muscular Adaptations Following Spinal Cord Injury..........................................10 2.2.1 Muscle Atrophic Response Following SCI................................................10 2.2.2 Force Mechanical Properties Following SCI.............................................10 2.2.3 Myosin Heavy Chain Expression Following SCI......................................12 2.3 Treadmill Locomotor Training Following Spinal Cord Injury.............................16 2.3.1 The Recovery of Walking Ability Following SCI and Locomotor Training............................................................................................................17 2.3.1.1 Locomotor recovery following SCI in animals................................17 2.3.1.2 The recovery of walking abil ity in individuals after SCI.................18 2.3.2 The Impact of Locomotor (Treadmill or Cycling) Training on Skeletal Muscle Following SCI.....................................................................................20 2.4 IGF-1 Signaling and Muscle Plasticity.................................................................22 2.4.1 IGF-I and Its Related Recep tor and Binding Proteins................................22 2.4.2 Protein Synthesis Induced by IGF-I/PI3K/Akt Pathway............................25 2.4.3 IGF-I/PI3K/Akt Pathway and Protein Degradation...................................26

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vii 2.4.4 Role of IGF-I in Satellite Cell Proliferation...............................................27 3 OUTLINE OF EXPERIMENTS................................................................................29 3.1 Experiment 1.........................................................................................................29 3.1.1 Specific Aim...............................................................................................29 3.1.2 Hypotheses.................................................................................................29 3.2 Experiment 2.........................................................................................................30 3.2.1 Specific Aim...............................................................................................30 3.2.2 Hypotheses.................................................................................................30 3.3 Experiment 3.........................................................................................................31 3.3.1 Specific Aim...............................................................................................31 3.3.2 Hypotheses.................................................................................................31 3.4 Experiment 4.........................................................................................................32 3.4.1 Specific Aim...............................................................................................32 3.4.2 Hypotheses.................................................................................................32 4 A LONGITUDINAL STUDY OF SKELETAL MUSCLE FOLLOWING SPINAL CORD INJURY AND LOCOMOTOR TRAINING...................................33 4.1 Abstract.................................................................................................................33 4.2 Introduction...........................................................................................................34 4.3 Materials and Methods.........................................................................................36 4.3.1 Experimental Animals................................................................................36 4.3.2 Spinal Cord Contusion Injury.....................................................................36 4.3.3 Treadmill and Cycling Locomotor Training..............................................37 4.3.4 Magnetic Resonance Imaging....................................................................38 4.3.5 Open Field Locomotor Function................................................................39 4.3.6 Statistical Procedures..................................................................................40 4.4 Results...................................................................................................................40 4.4.1 Muscle Size After Spinal Cord Contusion Injury.......................................40 4.4.2 Effect of Locomotor Training on Muscle Size...........................................42 4.4.3 Relationship Between Hindlimb Muscle Size and Locomotor Function...44 4.5 Discussion.............................................................................................................46 5 CHANGES IN SOLEUS MUSCLE FUNC TION AND FIBER MORPHOLOGY WITH ONE WEEK OF LOCOMOTOR TRAINING IN SPINAL CORD CONTUSION INJURED RATS................................................................................52 5.1 Abstract.................................................................................................................52 5.2 Introduction...........................................................................................................53 5.3 Materials and Methods.........................................................................................55 5.3.1 Experimental Animals................................................................................55 5.3.2 Open Field Locomotor Function................................................................56 5.3.3 Spinal Cord Contusion Injury.....................................................................56 5.3.4 Treadmill Locomotor Training...................................................................57 5.3.5 In situ Soleus Force Measurements............................................................58

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viii 5.3.6 Tissue Harvest............................................................................................59 5.3.7 Immunohistolochemistry............................................................................60 5.3.8 Data Analysis..............................................................................................60 5.4 Results...................................................................................................................61 5.4.1 BBB Open Field Locomotor Scores...........................................................61 5.4.2 Soleus Contractile Properties.....................................................................62 5.4.3 Soleus Muscle Fiber Cross Sectional Area................................................66 5.5 Discussion.............................................................................................................67 6 CHANGES IN SOLEUS FIBER TYPE CO MPOSITION WITH ONE WEEK OF TREADMILL LOCOMOTOR TRAINING IN SPINAL CORD CONTUSION INJURED RATS........................................................................................................73 6.1 Abstract.................................................................................................................73 6.2 Introduction...........................................................................................................74 6.3 Materials and Methods.........................................................................................75 6.3.1 Experimental Animals................................................................................75 6.3.2 Spinal Cord Contusion Injury.....................................................................75 6.3.3 Treadmill Locomotor Training...................................................................76 6.3.4 Immunohistolochemistry............................................................................77 6.3.5 Data Analysis..............................................................................................78 6.4 Results...................................................................................................................78 6.4.1 Soleus fiber type composition....................................................................78 6.4.2 Soleus fiber cross-sectional area................................................................80 6.5 Discussion.............................................................................................................81 7 EFFECTS OF TREADMILL TRAINING ON IGF-I EXPRESSION IN RAT SOLEUS MUSCLE FOLLOWING SPINAL CORD INJURY.................................85 7.1 Introduction...........................................................................................................85 7.2 Materials and Methods.........................................................................................86 7.2.1 Experimental Animals................................................................................86 7.2.2 Spinal Cord Contusion Injury.....................................................................86 7.2.3 Treadmill Locomotor Training...................................................................87 7.2.4 Tissue Harvest............................................................................................88 7.2.5 RT-PCR Measurement...............................................................................88 7.2.6 Immunohistolochemistry............................................................................90 7.2.7 Data Analysis..............................................................................................90 7.3 Results...................................................................................................................91 7.3.1 mRNA Expression of IGF-I and Its Receptor and Binding Proteins.........91 7.3.2 Embryonic Myosin.....................................................................................94 7.3.3 Central Nuclei.............................................................................................94 7.4 Discussion.............................................................................................................95 8 CONCLUSION...........................................................................................................99

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ix LIST OF REFERENCES.................................................................................................101 BIOGRAPHICAL SKETCH...........................................................................................122

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x LIST OF TABLES Table page 4-1. Maximal cross-sectiona l area of individual muscles................................................51 6-1. Monoclonal antibody specificity..............................................................................78

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xi LIST OF FIGURES Figure page 4-1. A representative trans-axial MR image of the rat lower hindlimb...........................39 4-2. Relative changes in the CSAmax of th e tibialis anterior, triceps surae, extensor digitorum, and flexor digitorum muscles following SCI.........................................41 4-3. Relative change in total CSAmax in the no training group, cycling training group and treadmill training group.....................................................................................42 4-4. Relative change in the CSAmax of the triceps surae in the no training group, cycling training group and treadmill training group................................................44 4-5. Relative change in the CSAmax of the tibialis anterior in the no training group, cycling training group and treadmill training group................................................45 4-6. Relationship of rat locomotor function and total lower hindlimb CSA...................45 5-1. Average BBB locomotor scores for the SCI + TM and SCI no TM group at 1 and 2 weeks post SCI...............................................................................................61 5-2. Soleus muscle peak tetanic force..............................................................................62 5-3. Soleus muscle specific force.....................................................................................63 5-4. Peak soleus tetanic force vs BBB score....................................................................64 5-5. Soleus muscle fatigue...............................................................................................64 5-6. Soleus muscle force-time integral during a fatiguing protocol................................65 5-7. Soleus muscle peak twitch force..............................................................................65 5-8. Soleus muscle time to peak......................................................................................66 5-9. Soleus muscle relaxation time..............................................................................66 5-10. Average soleus muscle fiber CSA............................................................................67 6-1. Serial cross sections of a contro l soleus stained with monoclonal antibodies directly against spec ific MHC isoforms...................................................................79

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xii 6-2. Serial cross sections of a SCI no TM soleus stained with monoclonal antibodies directly against spec ific MHC isoforms...................................................................79 6-3. Serial cross sections of a SCI + TM soleus stained with monoclonal antibodies directly against spec ific MHC isoforms...................................................................80 6-4. MHC based fiber type percentage composition.......................................................81 6-5. Soleus muscle fiber type specific CSA.....................................................................81 7-1. IGF-I mRNA expression relati ve to 18s in soleus muscle.......................................92 7-2. MGF mRNA expression relative to 18s in soleus muscle........................................92 7-3. IGF-R mRNA expression relati ve to 18s in soleus muscle......................................93 7-4. IGF-BP4 mRNA expression relati ve to 18s in soleus muscle..................................93 7-5. IGF-BP5 mRNA expression relati ve to 18s in soleus muscle..................................93 7-6. Cross-section of soleus muscle stained with monoclonal antibody against embryonic myosin isoform.......................................................................................94 7-7. Cross-section (hemotoxilin & eo sin stained) of soleus muscle................................95

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xiii 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 ADAPTATIONS IN SKELETAL MUSCLE FOLLOWING SPINAL CORD INJURY AND LOCOMOTOR TRAINING By Min Liu May 2006 Chair: Krista Vandenborne Major Department: Rehabilitation Science Spinal cord injury (SCI) is one of the most devastating human afflictions, which leaves its victims paralyzed. Skeletal muscle s distal to the injury site experience significant muscle atrophy and loss of function, leading to impaired walking and motor function. Recently, novel intervention therapies, focusing on repetitive locomotor training, have shown great promise in promoti ng spinal plasticity and recovery in motor function following SCI. Recovery of motor function after SCI likely requires both neural and muscular adaptations. The major goal of th is study was to invest igate adaptations in skeletal muscles following SCI and locomotor training. A combination of MR imaging, in situ functional measurements, imm unohistochemical assays and RT-PCR was performed in an animal model of incomple te SCI. Our findings demonstrate that SCI results in significant atrophy in all rat hindlim b muscles, and that locomotor training halts the atrophic process and accelerates the rate of recovery. Additionally, our data suggest that early therapeutic intervention using tread mill training significantly increases animal

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xiv locomotor function and soleus muscle size and function. Compared to untrained SCI animals, one week of treadmill training results in a 32% improvement in BBB scores, a 38% increase in peak soleus tetanic force, a 9% decrease in muscle fatigue, larger muscle fiber CSA (23%), and a reduced shift toward faster fiber types. Finally, our findings demonstrate that treadmill training significantly increases mRNA levels for IGF-I, MGF, IGFR, IGFBP4 and decreases IGFBP5 mRNA expression in the soleus muscle. In addition, immunohistochemical analysis shows the increased presence of small fibers expressing embryonic myosin, a hallmark of muscle regeneration, following treadmill training. Taken together, the findings from the present work demonstrate that early therapeutic intervention promotes muscular pl asticity following SCI. We anticipate that this study will provide essential feedback fo r the development of early rehabilitation interventions in SCI individuals.

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1 CHAPTER 1 INTRODUCTION Spinal cord injury (SCI) is one of the most devastating human afflictions, which leaves its victims paralyzed or with imp aired motor control (57, 67, 107, 132, 245). It is estimated that there are approximately 200,000 persons with SCI living in the United States, with roughly 11,000 new cases occurr ing each year, making SCI a leading cause of disability (200). Skeletal mu scles distal to the injury si te experience significant muscle atrophy and loss of function, lead ing to impaired walking an d motor function. The neural responses to spinal cord injury vary from patient to patient, depending on the severity of the injury. As a result of new developments in the acute management of spinal cord injury, the majority of spinal cord injuri es sustained are clin ically incomplete. Traditionally, orthotic and assistive devices have been used as compensatory strategies to counter muscle weakness in SCI patients in an attempt to facilitate functional walking. Successful mobility is often depe ndent on learning a new behavior requiring either a wheelchair and/or bracing with assistive devices. More recently, locomotor training has emerged as an alternative modality for retraining walking after incomplete SCI and has revealed encouragi ng breakthroughs (27, 62, 64, 65, 105, 106, 234). Improved gait speed, improved balance, less re liance on assistive devices and orthoses, less physical assistance from caregivers, a nd improved functional performance have all been documented with locomotor training (63, 235). The purpose of this study was to inves tigate the impact of locomotor training on skeletal muscle plasticity and muscle recovery as well as growth factors known to play a

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2 role in muscle regeneration in a rat contusion SCI model. In addition, we set out to determine the effect of locomotor training on muscle function in contusion spinal cord injured animals and to perform a preliminar y investigation of the relationship between alterations in muscle size/func tion and locomotor behavior.

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3 CHAPTER 2 REVIEW OF RELATED WORK 2.1 Spinal Cord Injury Animal Models Animal models of SCI can be used to characterize the lesion development, study the mechanism of recovery, develop therapeuti c interventions, and al so to study skeletal muscle adaptations with decrease d use (7, 18, 44, 59, 72, 73, 76, 79, 91, 98, 117, 118, 137, 138, 152, 155, 161, 170, 194, 200, 222, 248). Currently, the experimental SCI animal models include the tr ansection, isolation, hemisecti on, and contusion model. Due to differences in limb loading, neural activa tion and behavioral outcomes resulting from each of these methods, it is important to comp are the similarities and differences across these models. 2.1.1 Spinal Cord Transection Model In the transection spinal cord injury m odel, the transmission of descending and ascending information between th e caudal cord and the brain is mechanically eliminated (83). In this case, SCI is crea ted by an incision into the spinal cord. The spinal cord can be either completely transected and left in place or a small section of the spinal cord can be removed (83). Following transection injury, th ere is an initial flaccid paraplegia stage in which animals drag their limbs (135, 187). The animals are able to move using their forelimbs, and have no difficulty reaching f ood and water. At approximately 3 to 4 weeks, the paralyzed hind limbs of the an imals change from flaccid to spastic. After spasticity develops, the limbs are almost alwa ys held in extension and no recovery of voluntary activity is observed (135, 187) EMG recordings monitored over 24-hour

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4 periods show a 75% decrease in the total inte grated EMG and a 66% decrease in the total duration of muscle activity in the SOL musc le, 5 to 6 months after transection when compared to control (7). Thus, in the spinal transection model hind limb muscles experience a significant reduction in both electrical activation and loading. The complete transection model has been used extensively to evaluate the effectiveness of interventions with regard to both axona l regeneration and functional recovery (83). The advantage of this model is a relative stabilization of pathological changes and subsequent neurol ogical outcomes (210). Theref ore, the effectiveness of particular strategies can be readily assessed (201). However, the transected spinal cord model also has some disadvantages. First, du e to the natural tension present in spinal cords, the two ends of a cut cord will separate Such a gap is rarely present in human SCI. In addition, in order to cut th e spinal cord, the dura has to be opened, allowing invasion by external cells. Therefore, the spinal cord fails to demonstrate spontaneous spinal regeneration (210). Finally, due in part to the advanced emergency care, the number of spinal cord injuries classified as inco mplete has risen dramatically (http://Refwww.spinalcord.uab.edu/). Thus, the transect ion model, while valuable for certain applications, may not be the best model to study the potential of rehabilitation interventions to promote neuromuscular plasticity. 2.1.2 Spinal Cord Isolation Model Spinal cord isolation has been referred to as the classic silent preparation, which was first described in dogs by Tower (228). In this preparation, the lumbar region of the spinal cord is functionally isolated via complete spinal cord transections at two sites. In addition, all the dorsal roots ar e cut bilaterally be tween the two transe ction sites (190). Thus, this model eliminates supraspinal, in fraspinal, and periphera l afferent input to

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5 motoneurons located in the isolated cord se gments while leaving the motoneuron skeletal muscle fiber connections intact The motoneurons within the islated segment of the spinal cord do not receive sensory input from the dorsa l roots, or neural si gnals originating from either above or below the two transec tion sites. Based on 48 hours of continuous intramuscular EMG recordings from a represen tative ankle extensor and a representative ankle flexor, the muscles innervated by these motoneuron pools are essentially electrically silent, even during passive manipulation ( 190). Acute recordings, during tactile stimulation of the legs or feet, also showed virtually no EMG activity in a variety of hindlimb muscles (75, 76, 209). Moreover, based on observations during cage activity, it can be assumed that minimal forces are generated in these paralyzed muscles (187). Therefore, spinal cord isolation represents one model that has been successfully used to study the effects of a comple te elimination of neurom uscular activity on muscle properties (221). 2.1.3 Spinal Cord Hemisection Model In partial transection models, an attempt is made to cut tracts of the spinal cord selectively. Depending on the severity of the lesion, the resulting neurologic deficit can be relatively mild, thus making the postopera tive animal care fairly easy, particularly with regard to bladder functi on. Partial injury models also may allow for comparison of the regenerative response in a particular tract with its uninjured partner on the contralateral side (92). Most hemisection injuries are performed on the cervical spinal cord, interrupting the descending respiratory pathways and causing respiratory muscle paresis or paralysis. Thus, this model has long been used to unde rstand the mechanisms related to plasticity and recovery of the respirator y pathways after spinal cord injury. Unfortunately, partial

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6 injury models also suffer from difficulti es determining whether observed functional improvement is due to true regeneration of th e injured tract or to functional compensation from other systems that are spared. For this reason, this model is not commonly used to study hind limb muscle adaptations. 2.1.4 Spinal Cord Contusion Model Most human spinal cord injuries result fr om fracture and dislocation of the spinal cord column (88). Although penetrating wounds of the spinal cord can result from a knife or gunshot, most human spinal cord injuries are caused by transient compression or contusion of the spinal cord (39, 119, 125, 223). Even in the setting of complete paraplegia after blunt injury, th e cord rarely is completely transected, but rather leaves some residual, normal-appearing cord parenchy ma peripherally at the injury zone (39). The first observation of human SCI neuropath ology begins with an early phase of spreading hemorrhagic necrosis and edema. Then it reaches an in termediate phase of tissue repair and reorganizati on. Finally it ends up with a chronic phase characterized by the formation of cystic cavities (108). Thes e injury patterns are well simulated by spinal cord contusion injury (37, 99, 100). Therefore, scientists ha ve long used animal spinal cord contusion models to study the pat hophysiology of spinal cord injury and regeneration (23, 35, 37, 44, 87, 99-101, 121, 131, 147, 166, 167, 182, 190, 235, 253). In our investigation we utilize th e contusion injury model and in the section below a brief overview of the history of this model is provided. 2.1.4.1 Early contusion models In 1911, Reginald Allen described a spinal cord injury model where he dropped a weight onto dog cords exposed by laminectom y (9). In 1914 (199), he reported that midline myelotomy reduced progressive tissu e damage in the contused spinal cord.

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7 Unfortunately, Allen died in World War I a nd his work was discontinued for nearly 50 years. In 1968, Albin and colleagues (8) revived the contusion model when they used a primate spinal cord contusion model to assess the efficacy of hypothermic therapy following SCI. After that, several investigators started using the canine spinal cord contusion model again. Parker and coll eagues (168, 169) assessed the effects of dexamethasone and chlorpromazine on edema in contused dog spinal cords. At the same time, Koozekanani and colleagues (128) examined the causes of variability in this model, while Collmans and others (52) measured edema, blood flow and histopathological changes in the contused dog spinal cord. In 1971, Osterholm and Mathews proposed that catecholamine accumulation explains the progressive central hemorrhagic necrosis in the contused cat spinal cord (165). Although subs equent studies did not confirm the predicted spinal cord catecholamine changes, this was th e first excitotoxic theory of neural injury (109). 2.1.4.2 Rat spinal contusion models In 1985, the Wrathall group (85, 163, 242) de scribed morphologica l and behavioral changes in a rat weight-drop contusion model. The weight-drop device was similar to that used for the feline spinal cord contusion model, i.e., a weight dropped dorsally onto thoracic spinal cord exposed by laminect omy. In addition, Wrathall and colleagues developed a combined behavioral score to asse ss motor, sensory, and reflex recovery in spinal injured rats, co rrelating these scores with quanti tative axonal counts, neuronal and glial loss, and evoked poten tials (175). In 1987, Somers on and stokes described a feedback controlled electromechanical device th at indented the spinal cord at a defined force, duration, and extent (206). This device has two levels of inde ntations that cause

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8 mild or moderate injury, producing consiste nt 3D morphological and locomotor changes. The device was subsequently used to assess the neuroprotective effects of MP and other drugs (84). 2.1.4.3 The New York university impactor The most commonly used rodent spinal cord contusion model is the New York University (NYU) impactor, developed at the NYU Neurosurgery Laboratory and first described by Gruner (98) in 1992. This model us es a weight-drop devi ce that differs from previous devices in several resp ects. First, the impactor dropp ed a steel rod directly onto the spinal cord exposed by a laminectomy, achieving more consistent contusions. Second, the device used digital optical potentiometers to measure the trajectory of the falling rod with a precision of 20 um and 20 us, providing accurate measurements of the delivered trauma. Third, the impactor measured movement of the spinal column at the impact site, subtracting this movement from the cord movement. Fourth, the device was designed to deliver three differe nt levels of injury to ra t spinal cord by dropping a 10-g rod 12.5, 25.0, or 50.0 mm onto the spinal cord, respectively producing mild, moderate, and severe injuries (98). In 1993, NIH funded a Multicenter Animal Spin al Cord Injury tr ial (MASCIS), in which a group of eight spinal cord injury laboratories validated and standardized the Impact model (246). The group demonstrated that the impactor produced consistent spinal cord injuries, reflected in a variety of measurements. This device allows for the precise measurement of a number of biomech anical parameters including the impact velocity of the rod, the distance of cord compression, the cord compression rate, and the dynamic force applied to the cord (23). In addition, the MASCIS group standardized the ages of the rats (77 1 day), anesthesia, a nd injury timing (60 5 min after anesthesia).

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9 Using these guidelines, this device apparently can produce extremely consistent injuries in terms of the resulting neuropathology. Fi nally, MASCIS validat ed the Basso-BeattieBresnahan (BBB) locomotor score, a 21-point ordinal behavioral sc ale that linearly predicts spinal cord hi stological changes (246). The BBB locomotor rating scale was describe d by Basso, Beattie, and Bresnahan in 1995 (24) as a measure of motor performance in rats. With this 21-point scale, animals achieving scores in the lower third (1 to 8) are capable of hindlimb joint movements without weight support. Those rating in the mi ddle part of the scale (9 to 13) demonstrate varying degrees of hindlimb weight suppor t and forelimbhindlimb coordination, and those achieving scores in the upper one third (14 to 21) show improvements in paw and tail position, toe clearance, and trunk stability during a fu lly supported and coordinated gait. The BBB scale has been shown to be a relatively reliable measure of locomotor function and a sensitive reflection of the de gree of tissue injury after spinal cord contusion (23). Its fairly widespread use has been valuable for allowing the communication and standardized comparison of results from different institutions (43, 142, 145). 2.1.5 Summary Animal models have proved to be invaluable for the development of experimental therapies, and undoubtedly will continue to play an essential role in the field of spinal cord injury research. Models in which the spinal cord is sharply transected, either completely or partially, ar e useful for studying the anat omic regeneration of axons, whereas the contusion models better simulate the biomech anics and neuropathology of human spinal cord injury. In order to study the effect of locomotor training on muscle

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10 plasticity and recovery we se lected to use a moderate midthoracic rat contusion injury model. 2.2 Muscular Adaptations Following Spinal Cord Injury 2.2.1 Muscle Atrophy Following SCI Following SCI, due to the reduced musc le activity and limb unloading, skeletal muscles show a significant reduction in their size and mass (45, 46, 59, 65, 70, 72-74, 118, 137, 141, 150, 155, 174, 178, 194, 200, 201, 210, 212, 218, 248). In addition, muscle atrophy is more pronounced in paraly zed muscles that normally bear weight, especially those that cros s single joints (148, 186, 237). Th ese muscles often contain a large proportion of slow fatigue-resistant musc le fibers and are largely responsible for maintaining posture and bearing weight (112, 186). For example, the soleus muscle, a postural muscle that extends the ankle, undergoes significant mu scle atrophy following SCI. In contrast, the tibialis anterior mu scle, which flexes the ankle and does not normally contract against a high load, atrophies considerably le ss in a number of species, including humans (148). The medial gastro cnemius muscle, which crosses both the knee and ankle joints, also under goes less atrophy than the soleus muscle even though it serves as a synergist to the soleus musc le during plantar flexion (186). 2.2.2 Force Mechanical Prop erties Following SCI Neural activity is very important to de termine the mechanical properties of a skeletal muscle. In 1960, Buller et al. (38) firs t reported that cross-re innervation of the cat slow soleus muscle with th e nerve of the fast flexor digitorum longus resulted in incomplete conversion of the isometric twitch properties from slow to fast. Similarly, cross-reinnervation of the fast flexor digito rum longus muscle with the nerve of the slow soleus muscle led to incomplete conversion of the isometric twitch properties from fast to

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11 slow (38). These findings demonstrate th at the pattern or amount of motoneuron activation strongly influences the muscle properties. Following SCI, animal hindlimb muscle s show pronounced decrease in maximal force-generating capacity and the acquisition of faster mechanical properties (185, 191193, 215, 217, 220, 221). In these studies, muscle force mechanical properties were determined using an in situ set up. Briefly, the animal leg was securely positioned in an apparatus such that the muscle was in a near-horizontal plane. The muscle distal tendon was cut and then attached to a force transducer. The muscles were stimulated via bipolar silver electrodes plac ed on the transected tibial nerve. They f ound that the maximum isometric twitch and tetanic tension of the cat soleus muscle were reduced by 38 and 39%, respectively, ~10 mo after spinal cord transection (185, 191). In these same cats, the time to peak tension and half-relaxation times were 41 and 50% shorter, when compared to control cats (185). The changes in force generating capacity and contractile speed were accompanied by a rightward shift in the force-frequency relationship and a small, but significant, decrease in fatigue resistance (193). The adaptations in the mechanical properties of the cat medial gastro cnemius, a fast ankle extensor, were less pronounced than that in the soleus, in both young and adult animals (185, 191). Presently, rat is the most prevalent animal model used to assess the effects of SCI and to determine the potential beneficial infl uence of rehabilitative procedures in the recovery of function after SCI (60, 69-71, 115). In general, the adaptations in the mechanical properties of the rat soleus after a complete spinal transection are relatively similar to those reported for cat muscles, a lthough the magnitude of the effects appears somewhat greater in cats than rats (194, 214) Talmadge and his colleagues (214) showed

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12 that in rats maximal tetanic force is redu ced by ~44% at 3 months post spinal cord transection. In addition, spinal cord transec tion resulted in faster twitch properties as evidenced by a shorter time to peak tension (~45%) and half-relaxa tion time (~55%). In addition, a significant reduction in fatigue resistance of the soleus was observed (214). The adaptations in the mechanical properties of muscle following SCI are similar to those observed in other models of reduced ne uromuscular activity. For example, chronic muscle unloading, via either hindlimb suspen sion or actual space flight, results in significant reductions in tetanic force, and twitc h contraction times of the rat soleus (41, 42, 219). Most unloading and spaceflight studies have also reported a decrease in halfrelaxation time and an increase in V max (41, 42, 239). The similar ity in the adaptations in SCI and chronic unloading models furthe r indicates the role of loading and neuromuscular activity in mediating muscle ad aptations following spin al cord injury. 2.2.3 Myosin Heavy Chain Expression Following SCI Myosin is one of the molecules that regulates contractile speed in mammalian skeletal muscle and is highly correlated with the myofibrillar ATPase histochemical identification of specific muscle fiber types (216, 217). It is a hexa mer composed of two heavy and four light chains (197). To date four different myosin heavy chain (MHC) isoforms have been identified in varying proportions in the hindlimb muscles of rats. These have been identified as a slow isoform called MHC-I a nd three fast isoforms called MHC-IIa, MHC-IIx, and MHC-IIb (224). A numb er of studies have closely linked the MHC isoform composition of the individual mu scle fibers with their velocities of unloaded shortening, such that there is a grad ation in the contractile speed of fibers containing a given isoform in the order of (fas test to slowest) IIb > IIx > IIa > I (36).

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13 The complement of MHC isoforms expressed by mammalian hindlimb muscles can be regulated by conditions that alter the levels of neuromus cular activation (defined as electrical activation and lo ad-bearing) of a muscle ( 97, 170, 187, 193, 217). Interventions that result in increased neuromuscular activat ion, such as chronic electrical stimulation, functional overload and endurance exercise, re sult in MHC shifts to wards the slower isoforms. In contrast, factors that reduce neuromuscular activ ation, including space flight, hindlimb suspension, and spinal cord injury, result in MHC shifts towards the faster isoforms (222). With a spinal cord injury there is no dire ct damage to the muscle, and innervation of muscles is not physically disr upted; therefore, th e interruption of tran sfer of electrical activity through the motoneurons likely stimul ates the changes that are observed (136, 171). Spinal motoneurons caudal to a lesion site lose neural input from descending brain stem pathways, which influences their tonic and phasic firing patterns and consequently affects muscle contractile and metabolic activ ity. Studies involving cross-reinnervation of muscles have further defined the influence of motoneuron input on the structural and functional characteristic s of a muscle (188). Myosin type adaptations following spinal cord transection. Almost all muscles studied show an increase in the percentage of fast fibers and a decrease in the percentage of slow fibers following spinal co rd transection (15, 114, 121, 137, 147, 166, 213, 217, 222). In cat soleus muscle, as many as 50% of the fibers react with only a fast MHC, and a small percentage of the fibers react with both a fa st and a slow MHC antibody following transection (121, 122). In contrast, co ntrol adult soleus muscle does not appear to express the fast MHC (121, 122, 215). The fast fibers post-SCI tended to be found

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14 disproportionately at the boundari es of the fascicles, sugge sting that myogenic spatial factors had influenced the ATPase conversion of the fibers. Roy et al (186, 192) also showed that cat fast muscle (tibialis anteri or) also shows an increase in the fast fiber proportion and MHC-IIx expression after 6 mont hs of spinal cord transection, but the increases are less prominent than in the soleus. Rodents show a higher degree of MHC isoform transformation after spinal transection than cats. The propor tion of MHC-I in the rat sole us is reduced from ~90% in controls to ~25% only 3 months followi ng a complete mid-thoracic spinal cord transection (222). The MHC-IIx, which is norma lly not found in the rat soleus, increased to nearly 50% and that of MHC-IIa to ~30% 6 months after spinal transection (217, 222). Myosin type adaptations following spinal cord isolation. In cats, spinal isolation also has been shown to result in an increas e in the percentage of fibers labeled by antibodies specific for fast MHC in the cats soleus and tibialis anterior (93). Based on mATPase staining at an alkali ne preincubation, Graham (93) found 64% fast fibers in regions sampled in the soleus of control cat s and 100% in similar re gions in the spinalisolated cats. In addition, esse ntially all fibers in the MG of the spinal isolated cats reacted exclusively with a fa st MHC antibody (93). These fibe r type data are consistent with the observation that the myosin ATPase act ivities in spinal isolated cats are 85% and 30% higher in the SOL and MG, respectively, 6 months after spinal isolation (123). In rats, spinal isolation also resu lted in a slow-to-fast shift in the myosin isozyme pattern (97). Similar to spinal transection, the MHC is oform that is primarily up-regulated at the expense of MHC-I is the MHC-IIx isoform (97).

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15 In addition, the reduction in the proportion of fibers c ontaining MHC-I after spinal isolation has been shown to be greater than th at observed for spinal transection alone. For example, 6 months following spinal transect ion the cat soleus contains only 67% MHC-I fibers (215) compared to 48% after spinal isolation (93). Thus, the magnitude of the adaptations observed following sp inal isolation is greater than that observed after spinal transection. This suggests that the residual amount of electrical act ivation in the cat soleus after spinal tran section plays a role in maintaini ng the levels of MHC-I expression. Myosin type adaptation following spinal cord contusion injury. Hutchinson (116) found that there was no change in the relative percent of MHC expression at 1 week post spinal cord contusion injury. Howe ver, at the 3 week time point, there was an upregulation of the transitional IIx heavy ch ain (116). Although the increased expression of IIx MHC after SCI was not statistically signifi cant, it appears to have been sufficient to induce effects of biological significance (spe eding up of RT). That such a modest increase in IIx MHC profiles could produ ce significant physiological differences indicates the tight regulat ory control MHC phenotype im parts on contractile speed. Skeletal muscle MHC expressi on in individuals with SCI. A few studies have directly assessed the effect s of SCI on MHC isoform expression. In 1999, Castro (45) reported that there is very little adaptati on in MHC isofrom expr ession in the vastus lateralis muscle during the first several months after complete SCI in patients. This study investigated vastus lateralis bi opsy samples in 12 patients as soon as they were clinically stable (45). No significant slow-to-fast changes in MHC isoform composition of the vastus lateralis were found at any time poi nt (6, 11, 24 weeks post SCI), and the mean proportions of each of the MHC isoforms we re comparable to non-disabled control

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16 subjects. However, there was an apparent conversion among type II fibers, with a significant MHC-IIa to MHC-IIx sh ift, in the vastus lateralis at 24 weeks post SCI. Thus, at 24-week after injury there was an elevati on in the proportion of MHC-IIx, the fastest of the human limb MHC isoforms. These data are consistent with a previous study showing that in long term SCI subjects (3-20 years post SCI), the vastus lateralis muscle contained a higher percentage of MH C-IIx or in combinati on with MHC-IIa (11). Histochemical data support the hypothesi s that slow to fast MHC isoform transformations occur in human muscle after SCI, even though to less of an extent than observed in animal models. For instance, the proportion of type IIb fibers was elevated and type I fibers decreased in the tibialis anterior and VL of SCI subjects (95, 148). The decrease in the proportion of histochemically identified type I fibers appeared to be related to the durati on of time following the SCI injury. In fact, Round (184) found that there was no decrease in the proportion of type I fibers in 2 individuals who had suffered a SCI less than 15 months prior to biopsy of the VL. In contra st, in those individuals that had suffered a SCI 3 years or more prior to biopsy, the proportion of type I fibers decreased relative to non-injured control valu es (184). These data al so highlight that the change in fiber type compos ition in human skeletal muscle occurs slower, a phenomina that has also been observed in ot her models of unloading (184). 2.3 Treadmill Locomotor Training Following Spinal Cord Injury Motor recovery following spinal cord in jury can be enhanced or accelerated by repetitive locomotor training ( 26, 27, 64, 86, 174, 233-235, 240, 241). The most frequently used locomotor training is tread mill training. Treadmill training is performed using a body weight support and manual assi stance for stepping. The underlying premise of locomotor training relates to the hypothesi s that rhythmic loading of the limbs and

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17 force feedback from the hindlimb muscles i nduces task appropriate activity-dependent plasticity. The following several paragraphs review the effects of locomotor training on functional recovery in both SCI animals and subjects. 2.3.1 The Recovery of Walking Ability Fo llowing SCI and Locomotor Training 2.3.1.1 Locomotor recovery following SCI in animals Experimental studies have shown that locomotor training of SCI animals can effectively improve the ability to step on a treadmill in cats following complete SCI (18, 54, 58, 59, 113, 140). Over the last ten years or so, the Edgerton group has published several papers to address this issue. For exam ple, de leon (58) charac terized the effects of training by comparing groups of untrained with trained spinal cats and reported that steptrained spinal cord cats can reach considerab ly higher speeds and make more consecutive steps. Treadmill training also improved inte rlimb coupling, overall limb excursion and joint excursion significantly (59). In addi tion, Edgertons group demonstrated a certain task-specificity in the improvement of locomo tor function. Therefore, spinal cats trained to stand were not able to walk. More importa ntly, they claimed that the spinal cord can learn or store memories of simple motor responses that are acquired through conditioning (59). For instance, the acquire d motor function can last up to six weeks followed by a graduate decay of locomotor abil ity (59). This reduction can be reinstated within one week of training. However, studies using rats with incomp lete SCI are scarcer and have provided contradictory results even if it appears c linically and pathophysi ologically relevant. Fouad et al. (81) found no beneficial effect on free locomotor activit y after a very partial lesion of the cord, whereas Thota et al. (227) reported improved functional rec overy after 7 weeks treadmill training in rats with partial spinal cord lesion. Ho wever, Fouad (81) proposed a

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18 possible explanation for the ab sence of observed benefits in their study. They noted that animals in their study had a high level of moto r function and were very mobile after SCI, limiting the potential beneficial effects of the treadmill training. Recently, a group in Belgium (158) studied the effect of treadmill tr aining on motor recovery in adult rats after severe contusion injury induced by balloon in flation. Multon et al. (158) re ported that early treadmill training improved locomotor recovery a nd showed that the beneficial effect was observable after only 2 week, with a maximal e ffect at 4 weeks. As a result, severely contused injured rats could support their body weight after 12 week s of treadmill training, while non-trained rats could not (158). 2.3.1.2 The recovery of walking abilit y in individuals after SCI. Based on animal research, the interactive approach using treadmill and body weight support system for locomotor training has been extended to human individuals and several studies have yielded positiv e reports (18, 26, 27, 64, 80, 89, 110, 233-235). In 1987, Barbeau and colleagues (18) first reporte d suspending a human over a treadmill to assess the feasibility for walking retraini ng. During locomotor training, SCI individuals were suspended over a treadmill in a harn ess and an overhead support system. In addition, Behrman (27) emphasized the follo wing training princi ples: 1) generating stepping speeds approximating normal walki ng speeds (0.75-1.25 m/s); 2) providing the maximum sustainable load on the stance limb; 3) maintaining an upright and extended trunk and head; 4) approximating normal hip, kn ee, and ankle kinematics for walking; 5) synchronizing timing of extension of the hi p in stance and unloading of limb with simultaneous loading of the contralateral lim b; 6) avoiding weight bearing on the arms and facilitating reciprocal arm swing; 7) f acilitating symmetrical interlimb coordination; and 8) minimizing sensory stimulation that would conflict with sensory information

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19 associated with locomotion. Each of these pr inciples is designed to maximize sensory stimulation that matches the kinetic and kine matic properties associated with phases of stepping. Therefore, this system provides an environment in which one can facilitate balance control and manually assist trunk and leg movement during weight-bearing stepping. In 1992, Wernig and Muller repo rted that patients with va rying degrees of paralysis increased their overground speed following treadmill training with BWS (233). In addition, subjects with unilateral limb paraly sis were able to walk a short distance without using knee-stabi lizing braces and could negotiate st airs with the help of cane and handrails. In 1993, Barbeau and colleagues (17) reported an increase of overground walking speed and endurance in nine subjects with incomplete SCI after walking on the treadmill with BWS. Moreover, subjects walk ed with a more normal gait pattern and lower limb muscle EMG profiles (17). Subsequently, Wernig (234) did a co mprehensive study of the efficacy of locomotor training in 44 chronic and 45 acute clinically incomplete SCI subjects. In addition, those subjects were compared with 64 patients (24 chronic and 40 acute) treated conventionally. Following locomotor training, 25 subjects learned to walk independently and 7 patients could walk with assistance. In addition, those patients who could already walk before therapy improved their walking speed and endurance. By comparison, there were much fewer people improving their walk ing ability in the c onventionally treated group. In a follow-up study, it was reported that all subjects retained and in some cases further improved their ability to walk over gr ound (236). Since then, this intervention has

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20 been widely applied in the incomple te SCI subjects (18, 26, 27, 64, 80, 89, 110, 233235). Unfortunately, to date, there has not b een a similar training effect on overground walking in humans with clinically comp lete SCI. However, treadmill training can improve several aspects of walking on a trea dmill. For example, Dietz et al. (36, 62) reported that, after several week s of treadmill training, the levels of weight bearing significantly increased during training. In addition, when stepping on a treadmill with BWS, rhythmic leg muscle activation patterns can be elicited in clinically complete subjects who are otherwise unable to voluntarily produce muscle activity in th eir legs (144). Another study has demonstrated that the leve ls of leg extensor muscle act ivity recorded in clinically complete SCI subjects significantly improved over the course of se veral weeks of step training (240). Thus clinically complete SC I subjects can improve their stepping ability by locomotor training, but the level of improve ment has not reached a level that allows them to walk without assistance. 2.3.2 The Impact of Locomotor (Treadmill or Cycling) Training on Skeletal Muscle Following SCI Recovery of locomotor function after spin al cord injury lik ely requires neural plasticity as well as the maintenance or re storation of skeletal muscle. Some of the adaptations observed in the muscles follo wing SCI are effectively ameliorated by inducing full weight-bearing in spinalized animals while th ey are stepping on a treadmill (112, 173, 189). In spinal cats trained to step, th e atrophic response of the muscles with a high proportion of slow fibers, i.e., the SOL, is significantl y reduced (186). The largest effect has been shown in the soleus muscle, the extensors at the ankle and knee which is expected to be highly recruited dur ing treadmill exercise (112, 173, 189).

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21 Compared with treadmill training, however, cy cling exercise is less frequently used and studied. The cycling training consists of a circular pedaling mo tion, which flexes one limb while extending the other. The animal is suspended in the harness, and the hindlimb feet are strapped onto the pedals of the moto rized cycle, which is controlled by a speed controller (115, 203). Houle (115) studied the hind limbs of spinalized rats by daily cycling exercise for relatively long duration (60 min) at lo w intensity (45 rotations per minutes, 30 minutes per trials, two trials per da y, 5 days per week); th at is, there was no apparent load on the muscle. He found that 3 months cycling exercise diminished the extent of atrophy that was obser ved without exercise (115). In addition, treadmill training has been dem onstrated to prevent conversion of SOL myofibers from slow oxidative to fast glyc olytic properties in spinalized cats. For example, Roy et al. (192) trained the adult spinal cord transected cats on a treadmill for around 5 months and found that step trained cats showed 100% t ype I MHC in soleus. In contrast, cycling did not show a similar effect. Houle et al. (115) re ported that cycling exercise did not prevent or reverse the observed change s in muscle fiber type following SCI. The expression of primarily fast MHC isoforms pers isted in soleus muscle, and in fact, there was an exaggerated decrease in the expre ssion of type I MHC following 3 months of cycling exercise. Treadmill training has also been shown to have a positive effect on muscle function. Lovely and his colleagues (141) found that 5 months treadmill training significantly improve hindlimb muscle tetanic force and specific tension. The magnitude of the forces produced at the soleus tendon wh ile stepping on the treadmill during the last 2 weeks of experiment were close to normal levels (141). In addition, Roy et al. (192) studied

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22 the mechanical and biochemical response of a slow extensor (SOL) and fast extensor muscle (MG) in the adult cat hindlimb follo wing a complete low-thoracic spinal cord transection, with and without a daily shor t bout of stepping training on treadmill. He found that step training had no effect on the ad aptations in the cont ractile properties of the MG associated with chronic spinal transe ction. However, step training had a positive effect in maintaining force in the soleus muscle. 2.4 IGF-1 Signaling and Muscle Plasticity IGF-I has been shown to play an important role in muscle regeneration following injury (4, 5, 16, 127, 154, 195, 204), muscle hype rtrophy (51, 90, 160). Systemic release of IGF-I may contribute to an increase in protein content and a reduction in protein degradation in skeletal muscle (247). Incuba tion of muscle cells with IGF-I (1 MicroM) for 3-7 day stimulates both cell hyperplasia and myofiber hypertrophy (230). In addition, over-expression of IGF-I using a muscle spec ific promoter has been associated with myofiber hypertrophy in transg enic mice (51), and local in fusion of IGF-I has been shown to contribute to skeletal muscle hypert rophy (1), as well as block the aging-related loss of muscle mass in mice (19). Moreover, numerous in vivo activity models, such as increased loading, stretch and eccentric contra ction are known to result in increases of IGF-I peptide and IGF-I mR NA expression in skeletal muscle (4, 5, 16, 127, 195, 204). Resistance training also has been associated with increased IGF-I mRNA expression in both animal models (3) and individuals with complete spinal cord injuries (29). 2.4.1 IGF-I and Its Related Recep tor and Binding Proteins IGF-I : mature IGF-I is a 70-amino acid si ngle-chained polypeptide with many three-dimensional structural similarities to the proinsulin (see Review (202)). IGF-I is synthesized in the liver as a consequence of growth hormone. Systemic IGF-I produced

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23 in the liver promotes cell division and is generally responsible for normal growth and development (157). More recently, it has been recognized that IGF-I is also produced locally by skeletal muscle in a growth hormone independent manner (244). There is significant evidence that locally produced, auto crine and/or paracrine IGF-I, is important for muscle regeneration and hypertrophy ( 4, 5, 16, 127, 195, 204). Specifically, Jennische (120) showed IGF-I immunoreactv ity in the cytoplasm of my oblasts and myotubes and in satellite cells during muscle regeneration. LeFaucheur (134) further showed that antibodies that neutralized IGF-I reduced the number and size of regenerating myofibers after muscle damage. Experimental manipula tion of muscle of IG F-1 levels, in the absence of changes in loading state, also has been shown to induce muscle hypertrophy. For example, transgenic mice in which IGF-I is over-expressed using a muscle specific promoter undergo hypertrophy (51, 160). In a ddition, direct infusion (1) of IGF-I in muscle results in hypertrophy, whereas inhibi tion of IGF-I function can prevent this response (32). Over-expression of IGF-I in musc le has also been shown to prevent some of the age-related decline in muscle mass (19). Muscle specific isoforms and mechano-growth factor (MGF): IGF-I has different isoforms by alternative splicing. One of them is only detect able following injury and/or mechanical activity. Yang (244) cloned the c DNA of a splice variant of IGF-I that is produced by active muscle and th at seems to be the factor that controls local repair, maintenance, and remodeling. This protein has been called mechano growth factor (MGF). Because of a reading-frame shift, MGF has a different 3 sequence and a different mode of action compared with syst emic or liver IGF-I. Although MGF has been called a growth factor, it may be regulated as a local repair fact or (244). In addition,

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24 MGF has been shown to be significantly upregul ated in response to stretch and increased loading (151, 167). Yang (244) showed that MGF was markedly upregulated in rabbit extensor digitorum longus muscle, which had been subjected to acute stretch by immobilizing the hindlimb in the extended position. This work was further supported in a study in which mRNA expression of the mu scle-specific isoforms of IGF-I was upregulated in rabbit muscle after electrical stimulation at 10 Hz for 4 days (151). It appears that the expression of both systemic and autocrine IGF-I in muscle provides an interesting link between the mechanical si gnal and tissue remodeling and repair. IGF-I receptor (IGF-IR) : the cellular effects of IGF-I are mediated by the activation of a specific receptor, which has two binding alpha subunits and two transmembrane beta subunits (101). It is known that the biologi cal activity of growth factor depends on the concentration of the receptor and the affinity of its intera ction (238). It has been shown that there is a change in IGF-IR mRNA e xpression during muscle inactivity (102, 103) and resistance exercise (101). W illis (238) also reported an in crease of IGF-IR density in aged skeletal muscle, suggesting that an imals retain plasticity for IGF-IR. IGF binding protein (IGFBP): the interaction between IGFs and their binding proteins represents prereceptor regulation. So far, six IGFBP were identified. Their functions are: 1) stabilize and transport IGFs from the circul ation to peripheral tissues, 2) maintain a reservoir of IGFs in the circulat ion, 3) potentiate or i nhibit IGF function, and 4) mediate IGF-independent biological effects. Of the six systemic -type binding proteins, IGFBP-3 is primarily responsible for mainta ining IGF-1 levels in the circulation in conjunction with another prot ein called acid labile subunit (82, 207). In contrast, IGFBP4 and IGFBP-5 are located in skeletal musc le (207). There is some evidence that

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25 alterations in IGFBP expression occur during unloading or overload ing and that IGFBPs play a role in skeletal mu scle adaptation (14, 101-103). After binding with the receptor, the IGFR subsequently recruits the insulin receptor substrate, which results in the activation of two signaling pathways: the Ras-Raf-MEKERK pathway and the PI3K-Akt pathway (53). The Ras-Raf-MEK-ERK pathway is crucial for cell proliferation and cell survial in mitosis-competent cells (152). However, in adult skeletal muscle, the function of the Ras-Raf-MEK-ER K pathway is less clear. By contrast, activation of PI3K is thought to pl ay an important role in inducing skeletal muscle hypertrophy (159). 2.4.2 Protein Synthesis Induced by IGF-I/PI3K/Akt Pathway The binding of IGF-I to its receptor indu ces a conformational ch ange in the IGF-I receptor tyrosine kinase, resulting in the activation of several intracellular kinases, including phosphatidylinositol3-kinase (PI3K) (180). PI3K is a lipid kinase, which phosphorylates phosphatidylinositol-4-5-bispho sphate to phosphatid ylinositol -3-4-5trisphosphate (PtdIns(3,4,5)P3). PtdI ns(3,4,5)P3 provides a binding site for serine/threonine kinase Akt. After translocation to the me mbrane, Akt is phosphorylated and activated by phosphoinositide-depe ndent protein kinase (PDK). Akt plays a very important role in me diating muscle hypertrophy. Recently, it has been demonstrated that activation of Akt is sufficient to induce hypertrophy in vivo (131). Lai (131) showed that acute activation of Akt induces dramatic increases (> 2 folds) in the size of skeletal muscle in a tr ansgenic mouse in which a constitutively active form of Akt can be inducibly expressed in a dult skeletal muscle. In addition, skeletal muscle atrophy is coupled with a decreased ability to activ ate the Akt pathway in burned

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26 rats (211) and expression of ac tive form of Akt in skeletal muscle cells is sufficient to cause hypertrophy in normal mouse gastrocnemius muscle (212). Akt can phosphorylate mammalian target of rapamycin (mTOR) directly and indirectly by inhibition of Tsc1-Tsc2 comple x (117). In turn, activation of mTOR results in an increase in protein translation by two mechanisms: first, mTOR activates p70S6k (153, 164), a positive regulator of protein tr anslation; second, mTOR inhibits the activation of PHAS-1, a negativ e regulator of the protein in itiation factor eIF-4E. In addition, glycogen synthesis kina se (GSK) is another substrate of Akt that has been shown to play an important role in me diating hypertrophy. GSK is inhibited following phosphorylation of Akt and i nhibition of GSK is suffici ent to stimulate myogenic differentiation (56). In addition, expression of a kinase-inactive form of GSK induces dramatic hypertrophy in skeletal myotubes (180 ) and blocks protein translation initiated by the eIF2B protein (104). 2.4.3 IGF-I/PI3K/Akt Pathway and Protein Degradation Even though it is difficult to identify th e muscle specific mediators of atrophy, a search for markers of the atrophy suggests that two genes are up-regulated in multiple models of skeletal muscle atrophy. They ar e called MuRF1 (32) and MAFbx (91). Both genes encode ubiquitin ligases (E3), proteins that mediate ubiquitination of specific substrates. MuRF1 has recently been shown to be able to induce the ubiquitination of the cardiac form of TroponinI and possibly the myof ibrillar protein titin at the M line (126). MAFbx is suggested to bind to substrates including MyoD and calcineurin (91). Recently, studies have shown that the upregulation of MAFbx and MuRF1 is antagonized by treatment of IG F-I (196). The mechanism is focused on the function of FOXO. The phosphorylation of Akt can inhib it the FOXO (133), whose activation is

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27 required for the upregulation of MuRF1 and MAFbx (196). Thus, the activation of Akt can block the upregulation of MuRF1 and MAFbx by inhibition of FOXO. 2.4.4 Role of IGF-I in Sate llite Cell Proliferation. Satellite cells are quiescent muscle precursor cell in adul t skeletal muscle. They are located under the basal lamina of the muscle fiber, but sepa rate from the muscle fiber itself. Satellite cells are the main, if not only, cell type that co ntributes to muscle regeneration (30, 31). During muscle hypertrophy, there appear s to be a myogenic response where satellite cell-derived myoblas ts are thought to fuse with existing myofibers (49, 205). The importance of this response stems from the observations that mature mammalian skeletal muscle fibers appear to maintain a relatively finite relationship be tween the size of the myofiber and the number of myonuclei pres ent in a given myofiber (10, 111, 150, 219). However, mammalian myofibers become perman ently differentiated shortly after birth and cannot undergo mitotic division or direct ly increase their myonuc lear number (47). The requirement for additional nuclei to s upport hypertrophy appears to be met via the proliferation, differentiation, and finally the fusion of muscle satellite cells or their progeny with the enlarging myofibers, pr oviding the new myonuclei needed to support the hypertrophy process (10, 172, 181, 182). Am ong the well-characterized growth factors, IGF-I is the only one that has been c onsistently reported to facilitate each of these processes. Increasing evidence indicates that IGF-I st imulates both myoblast proliferation and differentiation (78). IGF-I acts via the mitogen-activated protein (MAP) kinase pathway, activating the expression of the cell cycle prog ression markers, such as cycling D, cdk4, c-fos, and c-jun (78, 124). Af ter withdrawal of the Akt pathway, IGF-I subsequently

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28 modulates expression of terminal muscle differentiation markers, such as p21, MyoD, myocyte enhancer factor 2 (MEF2) and myogenin (161). However, although it is not clear what role the PI3K pathway plays in differentiation, recent evidence demonstrates a key role for the PI3K pathway in primary satellite cell proliferation (46). Chakravart hy et al. (46) demonstrated that IGF-Istimulated proliferation of primary satell ite cells isolated from transgenic mice overexpressing IGF-1 is associated with th e activation of the PI3K/Akt signalling pathway and the downregulation of the cellcycle inhibitor p27Kip1 (46). In addition, ectopic expression of p27Kip1 has been shown to block the IGF-I-induced increase in satellite cell proliferation (46). Furthermore, Machida et al. (143) recently reported that IGF-I represses p27Kip1 transc riptional activity through phos phorylation of Akt. Thus, p27Kip1 has been proposed to be a key regulator y factor, particularly in its ability to regulate satellite ce ll cycle progression.

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29 CHAPTER 3 OUTLINE OF EXPERIMENTS The purpose of this study was to inve stigate locomotor training impacts on muscular adaptations following incomplete SC I. An outline of the experiments performed is provided in the section below: 3.1 Experiment 1 3.1.1 Specific Aim To quantify changes in rat hindlimb mu scle cross-sectional area following moderate T8 spinal cord contusion injury and 3 months of locomotor training (treadmill vs. cycling). Animals (n=8/group) were assigned to either a treadmill training group, cycle training group or a SCI (no trai ning) group. Moderate spinal co rd contusion injuries were produced using a standard NYU (New York Un iversity) impactor. Animals assigned to either training group were trai ned continuously for 3 months (5 days/week, 2 trials/day, 20 minutes/trial), starting on pos t-operative day 8. Non-invasi ve 3D magnetic resonance (MR) images were collected from the lower hi ndlimb muscle at pre-injury as well as at 1, 2, 4, 8, and 12 weeks post injury. Based on the MR images, the in vivo maximal muscle cross-sectional area of the tibialis anterior, tr iceps surae, extensor digitorums and flexor digitorums were determined. 3.1.2 Hypotheses a) Following moderate T8 spinal cord c ontusion injury, rat hindlimb muscles show an acute decrease in maximal CSA, followed by spontaneous recovery.

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30 b) Following moderate T8 spinal cord c ontusion injury, hindlimb extensor muscles show a greater decrease in maximal CS A than hindlimb flexor muscles. c) Both cycling and treadmill locomotor training attenuate the decrease of muscle CSA following T8 spinal cord contusion injury and facilitate the recovery of muscle CSA. 3.2 Experiment 2 3.2.1 Specific Aim a) To determine the impact of moderate T8 spinal cord contusion injury on the rat soleus morphology (fiber CSA) and in situ co ntractile properties. b) To determine the effect of 1-week treadmill locomotor traini ng on the rat soleus morphology (fiber CSA) and in situ contractile properties follow ing T8 spinal cord contusion injury. Adult female rats (n=8 rats; 16 muscle s/group) were assigned to either a SCItreadmill training group, a SCI-no training group or a control group. Animals assigned to the training group were traine d continuously for 1week (5 days/week, 2 trials/day, 20 minutes/trial), starting on post-operative day 8. Morphological and contractile properties of the predominantly slow twitch soleus musc le were assessed at 2 weeks post-injury. Specifically, we measured the soleus fiber CSA, in situ isometric force, twitch properties and fatigability. 3.2.2 Hypotheses a) Two weeks after moderate T8 spinal co rd contusion injury, rat soleus muscle experiences a significant decrease in musc le fiber CSA and in situ isometric force production, compared to the normal control rat soleus. In addition, the rat soleus muscle is more fatigable compared to the normal control soleus.

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31 b) One-week treadmill locomotor training attenuates the decrease in rat soleus fiber CSA and loss in in situ is ometric force production observed following moderate T8 spinal cord contusion injury. In addition, the rat soleus muscle is less fatigable following 1-week treadmill training, compared to the soleus muscle in the SCI-no training group. 3.3 Experiment 3 3.3.1 Specific Aim a) To determine the impact of moderate T8 spinal cord contusion injury on the rat soleus fiber type composition. b) To determin e the effect of 1-week treadmill locomotor training on rat soleus fiber t ype composition following moderate T8 spinal cord contusion injury. Adult female rats (n=12 rats; 24 muscles/ group) were assigned to either a SCItreadmill training group, a SCI-no training group or a control group. Animals assigned to the training group were traine d continuously for 1week (5 days/week, 2 trials/day, 20 minutes/trial), starting on post-operative day 8. Fiber type composition of the predominantly slow twitch soleus muscle was assessed at 2 weeks post-injury. Specifically, the soleus fiber type composition was determined using immunofluorescence techniques with monocl onal antibodies (BA-D5, SC-71, BF-F3, and BF-35). 3.3.2 Hypotheses a) Two weeks after moderate T8 spinal cord contusion injury, the rat soleus muscle will show a fiber type shift from MHC-I to wards MHC-II compared to the normal control soleus. b) One-week treadmill locomotor training will attenuate the rat soleus fiber type shift observed following T8 spinal cord contusion injury.

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32 3.4 Experiment 4 3.4.1 Specific Aim a) To determine the impact of moderate T8 spinal cord contusion injury on mRNA expression of IGF-I and its related receptor and binding proteins in rat skeletal muscle. b) To determine the impact of 1-week treadmill locomotor training on mRNA expression of IGF-I and its related receptor and binding proteins in rat skeletal muscle following T8 spinal cord contusion injury. Adult female rats (n=6 animals, 12 muscles/group/time point) were assigned to either a SCI-treadmill training group, a SCI-no training group, or a control group. Treadmill training started on post-operative day 8. mRNA expression levels of IGF-I were assessed at both 36 hour s (4-6 hrs after last (3rd) bout of training) and 1 week posttreadmill training. Specifically, a semi-quan titative RT-PCR was used to quantify mRNA expression of IGF-I, MGF, IG F-R, IGFBP4, and IGFBP5. 3.4.2 Hypotheses a) Two weeks after moderate T8 spinal co rd contusion injury, rat soleus IGF-I and MGF mRNA expression levels will not be si gnificantly different than that of control animals. However, mRNA expression of IGF-R, IGFBP4, and IGFBP5 will be significantly altered after spin al cord contusion injury. b) One week of locomotor training in creases rat soleus IGF-I and MGF mRNA expression levels in the spinal cord contused injured rat, compared to non-trained SCI animals. In addition, 1-week treadmill loco motor training attenuates the alterations observed in soleus mRNA expression of IGF-R, IGFBP4, and IGFBP5 following moderated thoracic contusion injury.

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33 CHAPTER 4 A LONGITUDINAL STUDY OF SKELET AL MUSCLE FOLLOWING SPINAL CORD INJURY AND LOCOMOTOR TRAINING 4.1 Abstract Spinal cord injury (SCI) results in loss of muscle mass and motor function. Recently, novel intervention therapies, focusing on repetitive locomotor training, have shown great promise in promoting spinal pl asticity and recovery in motor function following SCI. Recovery of motor function af ter spinal cord injury likely requires both neural and muscular adaptations. The objective of this study was to implement magnetic resonance imaging to characterize the longit udinal changes in rat lower hindlimb muscle morphology following contusion SCI and to dete rmine the therapeutic potential of two modes of locomotor training. After modera te midthoracic contusion SCI, Sprague Dawley rats were assigned to either tread mill training, cycle training or an untrained group. Lower hindlimb muscle size was examined at pre, 1-, 2-, 4-, 8-, and 12-weeks post injury. Following SCI, we observed significan t atrophy in all rat hi ndlimb muscles. The greatest amount of atrophy (11.1 -26.3%) wa s measured at 2-week post-injury and spontaneous recovery in musc le size was observed by 4 w eeks post SCI. Both cycling and treadmill training halted the atrophic pr ocess and accelerated the rate of recovery. The therapeutic influence of both training interventions was observed within 1 week of training. Finally, a significan t positive correlation wa s found between locomotor functional scores and hindlimb muscle size following SCI.

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34 4.2 Introduction Spinal cord injury (SCI) is one of the most devastating human afflictions, leaving its victims paralyzed or with impaired motor control (107, 132). Animal models have long been used to characterize spinal cord lesions, study the mechanisms of recovery, and develop effective therapeutic interventions. Among SCI animal models, contusion injury is the most clinically relevant since it reproduces the histpa thological features observed in approximately 40% of human trauma cases ( 39). Mid-thoracic spinal cord contusion injury of moderate severity disrupts connectivity in the lumbar spinal cord, but allows some communication between th e supraspinal centers and ca udal regions of the spinal cord(24). Novel intervention therapies, focusing on repetitive locomotor training, have shown promise in promoting spinal plasticity and r ecovery in motor function after spinal cord injury in animal models (18, 58, 73, 74, 140) Clinical research has also shown encouraging results for treadmill locomotor training in incomplete paraplegic patients(27, 62, 106). The underlying premise of locomotor treadmill training relates to the hypothesis that rhythmic loading of the limbs and force feedback from the hindlimb muscles induces task appropriate activity-depe ndent plasticity (63, 74, 105). Depending upon the requirements of each subject, treadmill training benefits from the use of partial body weight support and a team of expe rienced gait facilitators (27). A less equipment and personnel intensive alternative training intervention is cycling locomotor training. Cycle locomotor training is accomplished by the simp le circular movement of the hind limbs on a bicycle-type device, driven by a motori zed belt (203). Since there are significant practical differences in the equipment and pe rsonnel requirements for the performance of

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35 treadmill versus cycle locomotor training, there are important questions to be addressed regarding the relative efficacy of each type of training modality. Recovery of locomotor function after spin al cord injury lik ely requires neural plasticity as well as the maintenance or rest oration of skeletal mu scle. Patients with chronic complete spinal cord injury disp lay extensive muscle atrophy and associated secondary health related complications (44, 45). In addition, a la rge number of studies have shown a rapid loss in muscle mass follo wing spinal cord tr ansection and spinal isolation in animals (70, 137, 149, 178, 186). Both treadmill locomotor training and cycling training have demonstrat ed a positive influence on musc le size after spinal cord transection. Spinal cord tran sected cats showed a signifi cant reduction in the atrophic response of muscles with a high proportion of slow twitch muscle fibers after about 6 months of treadmill training. The largest effect of treadmill training was observed in the soleus, a muscle containing approximately 90 % slow fibers (112). A similar benefit has been reported in spinalized rats following cycling training. Interestingly, the impact of either cycling or treadmill locomotor training on skeletal muscle following contusion SCI has not been explored, even though th e remaining communication between the supraspinal input and periphera l skeletal muscles may create a better target for activity dependent plasticity. In addition, only a limite d amount of studies ha ve investigated the atrophic response in skeletal musc le following contusion injury (116). The purpose of this study was to utilize magnetic resonance (MR) imaging to investigate the longitudinal changes in muscle morphology in the rat lower hindlimb muscles following midthoracic (T8) contusi on SCI. A second aim of this study was to

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36 determine the potential of treadmill versus cycling locomotor training to ameliorate muscle atrophy and to induce mu scle plasticity following spinal cord contusion injury. 4.3 Materials and Methods 4.3.1 Experimental Animals Twenty-four Sprague Dawley rats (12 week, 228-260g; Charles River, NJ) were studied before SCI, and at early (1, 2 week s), intermediate (4 weeks), and late (8, 12 weeks) time points following contusion SCI. The rats were housed in a temperaturecontrolled room at 21 0C with a 12:12 hours light:dark cycle and were provided rodent chow and water ad libitum. All procedures were performed in accordance with the U.S. Government Principle for the Utilization a nd Care of Vertebrate Animals and were approved by the Institutional Animal Care & Us e Committee at the University of Florida. 4.3.2 Spinal Cord Contusion Injury Spinal cord contusion injuries were pr oduced 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 unde r sterile conditions. Animals received two doses of Ampicillin per day for 5 days, starti ng at the day of surgery. Procedures were performed under ketamine (100mg/kg)-xylazin e (6.7mg/kg) anesthesia (details in (179, 226)). Subcutaneous lactated Ringers so lution (5 ml) and antibiotic spray were administered after completion of the surg ery. The animals were kept under vigilant postoperative care, including daily examina tion for signs of distress, weight loss, dehydration, and bladder dysfunction. Manual expression of bladders was performed 2-3 times daily, as required, and animals were mon itored for the possibility of urinary tract infection. Animals were housed in pairs with the exception of the first few hours following surgery. At post-operative day 7, open field locomotion was assessed using the

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37 Basso-Beattie-Bresnahan (BBB) locomotor scale (24) and animals that did not fall within a preset range (0-7) were excluded from the study. Animals were subsequently divided (randomly) into three groups (n=8/group). Tw o groups were assigned to either treadmill or cycling locomotor training, and the third group did not receive training. 4.3.3 Treadmill and Cycling Locomotor Training In both training paradigms animals were trained continuously for 3 months (5 days/week, 2 trials/day, 20 minutes/trial), starting on post-operat ive day 8. Animals assigned to the treadmill training group were given five minutes to explore the treadmill on the first training day and then encouraged to walk on the moving treadmill (11 mpm) (130) for a series of four, five-minute bout s. A minimum of five minutes rest was provided between bouts. On the second day of training, animals completed two bouts of ten minutes each, twice a day. Starting on da y 3, animals trained continuously for 20 minutes with a minimum interval between trials of 2 hours. Tr aining consisted of quadrapedal treadmill stepping. Body weight support was provided manually by the trainer. The level of body weight support was ad justed to make sure that rats could bear their weight and there was no collapse of their hindlimbs. Typically, the rats started stepping when they experienced some small load on their hindlimbs. In addition, during the first week of training, when all rats had profound paraplegia, assistance was provided to place the rat hind paws in plantar st epping position during training. In general, following 3-4 weeks of training, rats were independent in stepping with occasional assistance. The design of the cycle trainer used in these studies was adapted from the one developed by Houle and Skinner (115). The rat bicycle is composed of a direct drive gear box, adjustable foot pedals, and a support ha rness, as described previously (34). The

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38 animal was suspended in the harness, and th e hindlimb feet were strapped onto the pedals of the motorized cycle, which is controll ed by a speed controller and an on/off switch. The exercise consisted of a circular pe daling motion, which flexes one limb while extending the other. Care was taken not to overstretch either one of the limbs. The pedaling rate was set at 31 rpm as it approxi mates the self-selected cadence at which animals walk on the treadmill. During the firs t week of training, the rat tail was attached to an aluminum support beam with surgical tape to maintain the trunk stability during exercise. Gradually the load was increas ed by positioning the body harness towards the chest, so that the hind portion of the body falls over the pedal. A similar exercise method has been used in spinalized rabbits (231). The timing and duration of bicycle training was equivalent to the treadmill training protocol. 4.3.4 Magnetic Resonance Imaging All imaging procedures were performe d in a horizontal, 4.7 Tesla magnet with Paravision 2.1.1 software (Bruker Medical, Ettlingen, Germany). The spectrometer was equipped with the Bruker S116 actively shielded gradient coil and a custom-built, 5-cm long, 3.3-cm inner diameter birdcage extremity co il. Rats were initially anesthetized with 4% gaseous isoflourane in oxygen (1 L/min flow rate) and maintained with 1-2% isoflourane in oxygen for the duration of the MR experiment. Animals were placed on a cradle in the prone position with the right le g secured in the center of the coil covering the region from mid-thigh to ankle. Pulse rate, blood oxygen saturati on, respiration rate, and body temperature were monitored continuously. 3D proton MR images were obtained at pr e-injury as well as at 1, 2, 4, 8, and 12 weeks post injury, using a fast gradient ec ho imaging sequence. The data were acquired with an encoding matrix of 516 x 256 x 64, field of view of 2.5 x 2.5 x 4 cm, pulse

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39 repetition time of 100 ms, and an echo time of 6.4 ms. The total re gion imaged extended from the mid-thigh to the calcaneus. Chemi cally selective fat suppression was used to enhance the definition between muscle groups. The CSA of the tibiali s anterior, triceps surae, extensor digitorums a nd flexor digitorums was determined for each slice and the maximal CSA (CSAmax) was recorded (Fig 1). Image analysis was performed using a custom-designed interactive computer program, EXTRACTOR (77). The total CSAmax was defined as the sum of the individual CSAmaxs. Figure 4-1. Representative trans-axial MR image of the rat lower hindlimb. A) Data were acquired with a slice thickness of 1 mm and field of view of 2.5 x 2.5 cm. B) Outline of tibialis anterior, tr iceps surae, extensor digitorum, and flexor digitorum muscles. 4.3.5 Open Field Locomotor Function In order to determine the relationship between changes in muscle size and locomotor function, a standardized test fo r open field locomotion known as the Basso, Beattie, Bresnahan (BBB) Locomotor Rating Scale was implemented (24). Rat locomotor function was evaluated at preinjury as well as at 1, 4, 8, and 12 weeks post injury. The animal was placed in a test apparatus, obser ved for 4-minute, and scored in real time by A B

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40 2-blinded observers (24). All open field loco motor testing was video-taped for further analysis and review. 4.3.6 Statistical Procedures All statistical analysis was perfor med using SPSS for Windows (Version 10.0). Results were expressed as mean standard error of mean (S.E.M). Research hypotheses were tested at an alpha level of 0.05. On e-way analysis of variance (ANOVA) for repeated measurements was performed to test CSAmax changes following contusion SCI. In addition, one-way ANOVA was used to compare individual muscles at each experimental time point. Traini ng effects on the muscle CSAmax of the hindlimb muscles were assessed using two-way repeated m easures ANOVA (group x time). Post hoc tests were performed using Bonferroni-Dunn proced ure for multiple pairwise comparisons. In addition, Regression analysis was performe d to assess the correlation between BBB locomotor rating scale and hindlimb total CSAmax in all rats studied. 4.4 Results 4.4.1 Muscle Size After Spinal Cord Contusion Injury. MR images acquired before and after sp inal cord contusion injury showed significant atrophy in all hindlim b muscles studied (Table 41). The amount of atrophy was both muscleand time-dependent. The rate of atrophy was greate st during the first week after SCI across all muscles. At 1w-SCI, the CSAmax of the extensor digitorums, flexor digitorums, tibialis anterior, and triceps surae was reduced to 90.6 1.9%, 86.8 2.4%, 88.3 1.9%, and 83.4 2.2% of pre-injury values, respectively (Figure 4-2). The most extensive amount of atrophy was observe d at 2w-SCI, and varied across muscles. The triceps surae demonstrated th e greatest amount of atrophy (73.7 4.1% of pre-injury

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41 values), the extensor digitorums the least (88.9 2.6%), followed by the tibialis anterior (79.5 3.2%) and flexor digitorums (84.6 7.5%) muscles (Figure 4-2). Figure 4-2. Relative changes in the CSAmax of the tibialis anterior (TA), triceps surae (TS), extensor digitorum (ED), and fle xor digitorum (FD) muscles at 1, 2, 4, 8, and12 weeks following SCI. Data are expressed as a percentage of the CSAmax measured at pre-injury. Statistically significant differences between ED and TS (P < 0.05). Statisti cally significant differences between FD and TS (P < 0.05). Starting at the fourth week post SCI, a steady increase in the muscle CSAmax was observed. The rate of increase in muscle CSAmax ranged from 0.48 to 6.26 mm2/week. By 12 weeks post-SCI, the CSAmax of all muscles studied had re covered to values that were no longer statistically different from pre-injury control values. At 12w-SCI, the CSAmax was 102.2 1.6% in the extensor digitorums, 102.1 4.4% in the flexor digitorums, 98.3 3.4% in the tibiali s anterior and 95.4 2.1% in the triceps surae. As a result, the total CSAmax for all muscles combined was 98.3 1.6% of pre in jury values.

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42 Figure 4-3. Relative change in total CSAmax in the no tr aining group, cycling training group and treadmill training group. Data are expressed as a percentage of the CSAmax measured at pre-injury. Trai ning started at 1 week post injury (dashed line). Statistically significa nt differences betw een no training group and treadmill training group (P < 0.05). St atistically significant differences between no training group and cycling training group (P < 0.05). 4.4.2 Effect of Locomotor Trai ning on Muscle Size. Both cycling and treadmill training decreased the rate and magnitude of atrophy in the lower hindlimb muscles following spinal co rd contusion injury and accelerated the rate of recovery in muscle CSAmax. The influence of locomotor bicycle and treadmill training on CSAmax was observed as early as 1 week af ter the onset of training (Figure 43). The control untrained triceps surae mu scles showed a significant decrease (.6 4.0 mm2/week) in muscle CSAmax from 1 week to 2 weeks post-SCI. In contrast, the triceps surae muscle in both training gr oups revealed significant muscle hypertrophy, +3.6 1.4 mm2/week in the bicycle training and +4.7 0.9 mm2/week in the treadmill training group. As a result, at 2 weeks post-SCI the CSAmax of the triceps surae was 73.4

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43 4.1% of pre-injury values in the non-training SCI group and 88.8 1.4% and 91.2 0.9% in the cycling and treadmill training groups respectively (Figure 4-4). As shown in Figure 4-4, a similar acute response to trai ning was observed in the tibialis anterior muscle as well as the extensor digitorum a nd flexor digitorum muscles (data not shown). Consequently, at 2 week s post-SCI the total CSAmax of the lower hindlimb muscles was on average 10.5% and 12.4% higher in the cyc ling trained and treadmill trained animals, respectively, compared to the non-trained group (Figure 4-3). The therapeutic effect of locomotor trai ning was observed not only at the early time point but also throughout the remaining tr aining period (Figure 4-3). The total CSAmax in both cycling and treadmill locomotor training animals was significantly greater than that of non-trained SCI animals. By 12w-SCI, both cycle and locomotor training groups demonstrated a full recovery in the total CSAmax, with values of 101.3 0.8% and 101.8 1.3%, respectively compared to pre-injury control valu es. In addition, a direct comparison between the cycling and treadm ill trained animals showed no significant difference between the two locomotor traini ng interventions for all muscles studied, except the tibialis anterior muscle. The CSAmax of the tibialis anterior muscle was fully recovered at 4-week post SCI in the tread mill training group, whereas in the cycling training group pre injury values were only reached at 8-week post SCI (Figure 4-4).

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44 Figure 4-4. Relative change in the CSAmax of the Triceps surae (TS) in the no training group, cycling training group and treadm ill training group. Data are expressed as a percentage of the CSAmax measured at pre-injury. Tr aining started at 1 week post injury (dashed line). Statis tically significant differences between no training group and treadmill training group (P < 0.05). Statistically significant differences between no trai ning group and cycling training group (P < 0.05). 4.4.3 Relationship Between Hindlimb Muscle Size and Locomotor Function. Our animals participated in a parallel study evaluating the influence of locomotor training on several functional measures after spin al cord injury (34). In the present study, we examined the relationship between the hindlimb muscle CSA and locomotor function following SCI. Our data demonstrated a sign ificant positive correlation between scores on the BBB locomotor rating s cale and hindlimb total CSAmax ( r =0.71, P < 0.001). Figure 4-5 provides the results of the regression analysis.

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45 Figure 4-5. Relative change in the CSAmax of the Tibialis anterior (TA) in the no training group, cycling training group and treadmill training group. Data are expressed as a percentage of the CSAm ax measured at pre-injury. Training started at 1 week post injury (dashe d line). Statistically significant differences between no training group and treadmill training group (P < 0.05). Statistically significant differences between no training group and cycling training group (P < 0.05). Figure 4-6. Relationship of rat locomotor func tion (BBB scale) and total lower hindlimb CSAmax (r =0.71, P < 0.001). Data of all the rats at each time point were pooled together. Note that the line is dr awn for visual purposes only and not to indicate a linear function.

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46 4.5 Discussion Following midthoracic spinal cord contus ion injury, we observed significant atrophy in the rat hindlimb muscles. The de gree of atrophy appeared to be musclespecific, with the anti-gravity extensor mu scles showing greater atrophy than the flexor muscles. In the non-trained animals, the greatest amount of atrophy (11.1 -26.3%) was measured at two weeks postinjury and spontaneous rec overy in muscle size was observed by 4 weeks post SCI. Both cycli ng and treadmill training halted the atrophic process and significantly accelerated the rate of recovery. The therapeutic influence of both treadmill and bicycle training was observe d within 1 week of training. Finally, a direct comparison between the cycling and treadmill trained animals showed no significant difference between the two locomotor training interventions for all muscles studied, except the tibia lis anterior muscle. Previous studies have examined the atr ophic response of skel etal muscle after complete and incomplete SCI in humans (44, 4 5, 96) as well as in a variety of animal models (70, 136, 149, 178, 186). A large number of studies have shown a rapid loss of muscle mass in spinal cord transected and spinal isolated animals, with more extensive atrophy in slow twitch muscles compared to fa st twitch muscles. However, only a limited amount of studies have investigated the e ffect of contusion SCI on skeletal muscle morphology. Specifically, Hutchins on et al (116) found a signi ficant decrease in muscle wet weight (20-25%) in all lower hindlimb mu scles, except the EDL, compared to agematched controls at 1week following moderate contusion injury. They also reported that muscle atrophy occurred in flexor as well as extensor muscle groups and that the severity was similar in fast and slow muscles. In c ontrast, a pattern of di fferential decrease was noted following contusion injury in the pr esent study. At 2 weeks post spinal cord

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47 contusion injury, when muscle CSA measures revealed the greatest degree of atrophy, MR quantitative assessments showed the follo wing atrophic hierarchy: triceps surae > tibialis anterior > flexor digitorums > exte nsor digitorums. This differential muscle response may be attributed to the higher ne uromuscular activity in extensor muscles relative to flexor muscles (173) The greater degree of atrophy in the extensor vs. flexor muscles may also be influenced by the a nkle joint position post-SCI. The paralyzed hindlimbs of spinalized or contusion-injured rats are maintained in an extended position and dragged by the forelimbs during cage wa lking (23, 187, 213). Larger muscle loss has also been observed when muscles are immobilized in a shortened position, compared to a neutral or stretched position (13). Muscle atrophy following midthoracic contus ion injury of mode rate severity was transient and restoration of muscle mor phology mirrored the spontan eous recovery in locomotor function. In the non-traine d SCI-animals increases in CSAmax (or hypertrophy) were first noted at the 4 week post-SCI tim e point. The rate of spontaneous hypertrophy ranged from 0.1 to 6.3 mm2/week, with the largest gains in muscle size at the intermediate time point (4 weeks). As a re sult, by 12 weeks post-SCI, the total CSAmax for all muscles combined was no longer statis tically different from pre-injury control values. In comparison, Hutchi nson and colleagues (116) repo rted that muscle atrophy, assessed by wet weights, was attenuated starti ng at 3-week post spinal cord contusion injury. By 10 weeks post-injury muscle wet weights were no l onger different from control values, except for the medial and la teral gastrocnemius. In contrast, spinal transection animal models, in which comm unication between the s upraspinal centers and

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48 caudal region of the spinal cord is eliminat ed, have shown little spontaneous recovery in muscle size (97). The restoration in muscle size in the mode rate contusion injury model appears to mirror the spontaneous improvement in locomoto r function. Basso et al. (23) showed that after a moderate thoracic spinal cord c ontusion injury, animal s demonstrate hindlimb paralysis until 7 days post injury, which is followed by a progressive recovery in locomotor function over the next 5 weeks. In the present study, we observed a significant positive correlation between scores on the BBB locomotor rating scale and hindlimb total CSAmax. The consistency between th e muscle morphometry and behavioral data supports the contention that neuromuscular activity is an important determinant of skeletal muscle size and vice versa. One of the primary goals of this study was to determine the effect of both treadmill and cycle training on the posteri or and anterior hindlimb musc les of moderate contusion spinal cord injured animals. The impact of treadmill training on skeletal muscle has been studied in a variety of animal models. Roy et al. (186) demonstrated that in spinal cats trained to step, the atrophic response of the muscles with a high proportion of slow fibers is significantly reduced. The largest effect of training was observed in the soleus muscle, followed by the extensors at the ankle and knee; muscles which are expected to be highly recruited during treadmill exercise (112). In contrast, treadmill stepping appeared to have little effect on a larg e number of muscles with a high proportion of fast twitch fibers, such as the tibialis anterior (186). In this study we found that treadmill training effectively facilitated muscle hypertrophy in both the tibialis anterior and triceps surae muscles of contusion injured animals. Thr oughout the 12 weeks of training, the tibialis

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49 anterior and triceps muscles were significan tly larger than the co rresponding muscles in the untrained animals. The largest difference between the treadmill trained and untrained animals was noted at the early and intermediate time points post-SCI. Depending upon the access and availability of treadmill locomotor training, cycle training may provide a practical alterna tive training strategy, contingent upon the continued demonstration of safety, feasibility, and efficacy of this approach. In the present study, we performed a direct of comparison between cycle and treadmill locomotor training and showed that both locomotor interventi ons induce significant muscle plasticity in all hindlimb muscles, as early as 1 week after initiation of training. While the treadmill training protocol used in this study was designed to maximize loading on the hindlimb muscles, cycling exer cise uses a different strategy and is accomplished by simple circular movements of the hindlimbs on a bicycle-type device, driven by a motorized belt (34, 203). EMG data acquired in previous studies show that during cycling locomotor training the left and right hind limb muscles are stretched in an alternating pattern, which results in alternat ing bursts of muscle activity (115). Thus, cycling training also initiates se nsory input to the spinal cord and subsequently influences the firing pattern of the mot oneurons that innervate the hi nd-limb muscles. Houle (115) studied the hind limbs of spin alized rats following cycli ng training (45 rotations per minutes, 30 minutes per trials, two trials per day, 5 days per week) and found that cycling locomotor training ameliorates muscles atrophy in spinalized rats. Similarly, in this study we found that in contusion-injured rats cyc ling training effectivel y halts the atrophic process and accelerates muscle recovery. While untrained triceps surae muscles demonstrated a significant loss in muscle size from 1 week to 2 weeks post-SCI (.6

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50 4.0 mm2/week), 1 week of bicycle training i nduced significant muscle hypertrophy (+3.6 1.4 mm2/week). However, a direct comparison between cycling and treadmill training showed that while cycling training was equally effective in promoting muscle hypertrophy in the triceps su rae muscles throughout the 12 weeks of training, treadmill training induced a larger hypertrophic response in the tibialis anterior muscle compared to cycling at the intermediate and late time points. One explanation for this discrepancy may be that during cycling revolutions the an kle was kept in a dor siflexed (shortened) position on the pedal, minimizing the stretch reflex in the tibialis anterior muscle. A large number of investigators are studying the mechanisms responsible for neural repair, neuroplasticity and muscle hypertr ophy (69). Based on our data and the wellestablished role of loading for muscle growth, we speculate that the training itself is not of sufficient duration to e ffectively induce muscle hypertr ophy. Consequently we propose that repetitive muscle activation, even if induced by passive motion (e.g. during motordriven bicycling), promotes the restorati on of the neuromuscula r interface and the recovery of functional motor units. The activ ation of these functi onal motor units under loaded conditions, such as is observed during cage reambulation, may provide the appropriate stimulus to induce muscle fibe r regeneration and hypertrophy. This in turn may result in improvements in motor function, increased ambulatory activity and usedependent neuroplasticity and muscle hypertrophy. In conclusion, this study demonstrates that rats after spinal co rd contusion injury suffer significant atrophy in th e lower hindlimb muscles, with the greatest amount of atrophy noted 2 weeks followi ng injury. Starting at 4 w eeks post-SCI, rat hindlimb muscles show spontaneous recovery resulti ng in near normal values at 12 weeks post-

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51 SCI. This study also demonstrates that bot h treadmill and cycle training diminish the extent of atrophy and facilitate muscle plasticity after contusion injury. The therapeutic influence of training on skeletal muscle was observed within the first week of training. Since muscle atrophy is associated with a myriad of secondary health problems, the potential of maintaining muscle mass with re petitive locomotor training is exciting and warrants further research. In addition, the re covery of locomotor function after spinal cord injury likely requires both neural and mu scle plasticity, as well as recovery of the neuromuscular interface. Table 4-1. Maximal cross-sectional area (mm2) of individual muscle. Values are means SME. Significantly different w ith pre injury value (p< 0.05) pre injury1 week 2 week 4 week 8 week 12 week Extensor digitorums 14.5 0.1 13.2 0.3*12.9 0.4*13.9 0.414.5 0.3 14.8 0.3 Flexor digitorums 32.1 0.7 27.8 0.7*26.9 1.1*28.6 1.131.1 0.1 32.3 0.9 Tibialis anterior 41.8 0.9 36.9 1.4*33.3 1.7*35.9 1.8*38.8 1.4 40.3 1.5 Triceps surae 106.4 3.388.7 3.5*78.1 4.4*91.5 2.6*98.5 3.3* 101.3 3.5

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52 CHAPTER 5 CHANGES IN SOLEUS MUSCLE FUNCTI ON AND FIBER MORPHOLOGY WITH ONE WEEK OF LOCOMOTOR TRAINI NG IN SPINAL CORD CONTUSION INJURED RATS 5.1 Abstract Recently, a new approach in the treatm ent of individuals with SCI involves rehabilitation to maximize residual function using locomotor training. The purpose of this study was to examine the influence of midt horacic contusion SCI on skeletal muscle function and to evaluate the therapeutic influence of early treadmill locomotor training on soleus muscle function (peak force, fatigabilit y, contractile properties), size (fiber area), as well as overall locomotor function (BBB) af ter SCI in rats. Thirty five adult Sprague Dawley rats (female, 16-20 weeks, wei ghing 250-290g) were st udied. Histological measurements of muscle fiber size were made on all animals. Twenty four animals were designated for muscle contractile measurements (8 controls and 16 receiving a moderate T8 spinal cord contusion injury). Eight of the SCI rats received treadmill locomotor training (TM) starting 1 week after SCI fo r 5 consecutive days, 20 minutes/trial, 2 trials/day. The additional eight injured rats received no exercise intervention (no TM). Locomotor training resulted in a significant improvement in BBB scores (32%), muscle fiber size and function. Compared to untrain ed injured animals, injured animals that trained for one week exhibited 38% greater p eak soleus tetanic forces, a 9% decrease in muscle fatigue, and 23% larger muscle fi ber CSA. In addition, there was a strong correlation between BBB scores of injured animals and peak soleus muscle force (r=0.704). Collectively, these results indicate that early therapeutic intervention using

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53 treadmill locomotor training can significantly improve functional locomotor recovery following SCI. The magnitude of these changes is remarkable considering the relatively short training interval, and clearly illustrates the potential that early exercise intervention has for countering some of the early f unctional deficits resulting from SCI. 5.2 Introduction Spinal cord injury (SCI) is a devastati ng human condition that results in paralysis or impairments in motor control that lead to significant disability (62, 73, 229). Currently, a major therapeutic problem centers ar ound the profound dysfunction of the primary locomotor skeletal muscles following SCI (176). Recently, a new approach in the treatment of individuals with SCI involves rehabilitation to ma ximize residual function using locomotor training (63, 176). Such locomo tor training uses prin ciples derived from animal and human studies showing that st epping can be generated by virtue of the neuromuscular systems responsiveness to phasic, peripheral sensory information associated with locomotion (18, 27, 58, 63, 106, 140). Although these locomotor training programs may promote functional recovery, the particular contributi ons of this therapy for addressing primary locomotor skeletal muscle dysfunctions are not well understood and investigation rega rding these important issues is needed. Animal models of SCI can offer a practical approach to efficiently evaluating the safety, feasibility, and efficacy of therapeuti c procedures on skeletal muscle adaptations during recovery (21, 74). Current animal mode ls of SCI include tr ansection, compression, and contusion (24, 183). The tran section model has been widely used to study limb disuse because it allows for a reproducible complete SCI (23, 69, 70, 166, 218), yet the contusion model most closely parallels the mech anism of injury of the majority of human SCIs (incomplete injuri es) (116, 183) and has been shown to reproduce the

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54 histopathologic sequence of SCI observed in about 40% of human trauma cases (39). In contrast to the transection model where anim als experience a complete loss of locomotor capabilities (23, 87), animals with a moderate contusion injury regain some locomotor function without specific training (2, 22, 23, 28, 183, 225). A lthough contusion injuries have been performed in a variety of animal species, electrophysiologic, behavioral, and imaging studies have indicated that SCI in rats can reasonably model events occurring after human SCI (94, 146). While much attention has been focused on neural damage and recovery after SCI (35, 72, 74, 87, 155), less attention has focuse d on skeletal muscle adaptations after injury, especially following a contusion injur y. One of the few studies to closely examine muscle adaptations after a contusion injury in rats found that, soleus and extensor digitorum longus muscle phenotype and contract ile properties were significantly affected within the first 1-3 weeks after SCI (116). With the r ecovery of weight supported hindlimb stepping over the course of 10 weeks, there was a concurrent return to baseline levels for contractile prop erties and muscle morphology, which suggests that weight bearing plays a critical role in maintaini ng normal muscle physiology. In addition, since changes occur within weeks after SCI, early ex ercise interventions may offer the greatest potential to counter these muscle adaptati ons and restore normal function. In Chapter 4 we showed that exercise training (bicycle or treadmill) after a contusion SCI results in faster recovery of muscle size (anterio r and posterior compartment muscles) and preservation of normal muscle morphology after 3 months of training. In this investigation, we also found th at the greatest differences in muscle size between trained and untrained rats occurred within the first w eeks of training (chapter 4). The time course

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55 of muscle adaptations after SC I in the aforementioned study ar e consistent with those of Hutchinson et al. (2001). Therefor e, we were interested in investigating the short term benefits of early locomotor training in reducing muscle atrophy and improving muscle function. It has been reported that the greater disuse adap tations occur in slow twitch than in fast twitch muscle fibers and in pos tural (extensor) muscles more than flexor muscle groups (2, 28, 225). Accordingly, we chose to study adaptations of the soleus muscle because it is composed of slow twitc h muscle fibers and f unctions as a postural muscle, which makes it more susceptible to muscle atrophy. The purpose of this study was to examine the influence of SCI on skeletal muscle function and to evaluate the therapeutic influence of early treadmill locomotor training on soleus muscle function (peak force, fatigability, contractile properties), size (fiber area), as well as overall locomotor function (BBB) after SCI in rats. Our hypothesis was that early locomotor training would attenuate some of the functional changes in muscle seen early after injury by improving muscle for ce and contractile properties, decreasing muscle fatigue, and preserving normal muscle morphology. 5.3 Materials and Methods 5.3.1 Experimental Animals Thirty five adult Sprague Dawley rats (female, 16-20 weeks, weighing 250-290g) were studied. Histological measurements were made on 35 animals (11 controls, 12 treadmill-SCI, and 12 no treadmill-SCI). Twenty four animals were designated for muscle contractile measurements, with eight serving as controls and sixteen receiving a moderate T8 spinal cord contusion injury using a standard NYU impactor (23). Eight of the injured rats received treadmill locomotor training (TM) starting 1 week after SCI for 5 consecutive days, 20 minutes/t rial, 2 trials/day. The addi tional eight injured rats

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56 received no exercise intervention (no TM). All rats were housed in a temperaturecontrolled room at 21C with a 12:12 hour s light:dark cycle and were provided unrestricted access to food and wa ter. All procedures were pe rformed in accordance with the U.S. Government Principle for the Utili zation and Care of Vertebrate Animals and were approved by the Institutional Animal Care & Use Committee at the University of Florida. 5.3.2 Open Field Locomotor Function One of the most common and reproducib le measures of locomotor function following SCI contusion injury employs a st andardized test for open field locomotion known as the BBB Locomotor Rating Scale ( 24, 25). The BBB rates behavior ranging from individual joint movements of the hi ndlimb to plantar ste pping and coordinated walking, progressing further to finer elements of locomotion, such as trunk stability, paw position, and tail position. Rats were evaluated for locomotion prior to surgery, 1 week after SCI, and 2 weeks after SCI. Movement was evaluated for 4 minutes by 2 examiners using the Basso, Beattie, Bresnahan (BBB) Locomotor Rating Scale (24, 25). 5.3.3 Spinal Cord Contusion Injury Spinal cord contusion injuries were produced using a MA SCIS (Multicenter Animal Spinal Cord Injury Study) impactor and protocol (23). Brie fly, 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 Baytril (10mg/kg) per day for 5 days, starting the day of surgery. Procedures were performed under ketamine (100mg/kg)-xylazine (6.7mg/kg) an esthesia (179, 226). Subcutaneous lactated Ringers solution (5 ml) was administered afte r completion of the surgery. Animals were given Buprenorphine (0.05mg/Kg IM) and Ketoprofen (5.0 mg/Kg SC) for pain and

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57 inflammation over the first 36hrs after SCI. The animals were kept under vigilant postoperative care, including daily examina tion for signs of distress, weight loss, dehydration, and bladder dysfunction. Manual expression of bladders was performed 2-3 times daily, as required, and animals were mon itored for the possibility of urinary tract infection. After SCI, animals were housed individually. On postoperative day 7, open field locomotion was assessed using the Basso -Beattie-Bresnahan (BBB) locomotor scale (24, 25) and animals that did not fall within a preset range (BBB, 4-8) were excluded from the study. Injured animals were subseque ntly divided into two groups (n=12/group) and were randomly assigned to either the treadmill locomotor training or no training groups. The third group consisted of noninjured control ra ts (n=12/group). 5.3.4 Treadmill Locomotor Training Animals that received locomotor treadmill training were trained for 5 consecutive days (hereafter defined as 1 week of traini ng), 2 trials/day, 20 minutes/trial, starting on post-operative day 8. Training consisted of a qua drapedal treadmill stepping. On the first day of training, animals were given five minutes to explore the treadmill and then encouraged to walk on the moving treadmill ( 11 mpm) (130) for a se ries of four, fiveminute bouts. A minimum of five minutes re st was provided between bouts. Body weight support was provided manually by the traine r. The level of body weight support was adjusted to make sure that rats could bear their weight and there was no collapse of their hindlimbs. Typically, the rats started stepping when they experienced some small load on their hindlimbs. In addition, during the first we ek of training, when all rats had profound paraplegia, assistance was provided to place rat hindlimbs appropriately for plantar stepping during training. On the second da y of training, animals completed two 10 minute bouts, twice a day. Starting on day 3, animals trained continuously for 20 minutes

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58 with a minimum interval between trials of 6 hours. Bodyweight support through the trunk and the base of the tail was provided as necessary and gradually removed as locomotor capability improved. 5.3.5 In situ Soleus Force Measurements In situ soleus force measurements were performed in control animals and 2 weeks after SCI (following 1 week of training). Anim als were anesthetized with isoflurane (4% for induction, 1-2% for maintenance). A sma ll dorsal, midline incision was made along the posterior lower hindlimb to expose the gastrocnemius-soleus complex. The gastrocnemius and soleus muscles were di ssected free from each other and surrounding connective tissue with care not to disrupt the blood supply. A non-compliant steel wire suture (5/0 AESCULAP Inc.) was attached to the tendon at the distal end of the soleus muscle. A stimulating bipolar electrode cuff wa s placed around the tibial nerve, proximal to its innervation of the soleus muscle. The sciatic nerve was crushed proximal to the site of the bipolar electrode cuff and the cutaneus tibial branch was severed to ensure that all electrical stimuli were transmitted directly to the soleus muscle. The animal was then placed in a supine position in a specially fabr icated experimental set up that allowed the animal to be secured in a reproducible posit ion over a circulating warm water bath to maintain body temperature (37oC). The left leg was secured in place by a pair of screwdriven pins at the condyles of the femur, wh ile the foot was securely clamped such that soleus muscle was oriented in the horizontal plane. The distal end of the soleus tendon was attached to the variable range force tr ansducer (Biopac Syst ems Inc, TSD105A) via the wire suture. The room temperature was set at 28C and monitored throughout testing. A mineral oil drip (30oC) was used to maintain a cons istent muscle temperature and prevent drying of the muscle during testing.

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59 Testing began by stimulating the soleus muscle using supramaximal (~7V, 0.2ms duration) unidirectional s quare-wave pulses via an S88 Grass stimulator (GrassTelefactor, RI). The transducer force output was amplified and digitized using a Biopac MP100 System (BIOPAC Systems, Inc. Goleta CA) and Acqknowledge 3.7 computer software. The physiological tests were performe d on the soleus adjusted to the isometric optimum length, determined by measuring maximal isometric forces generated at graded muscle lengths. Supramaximal stimulus intensity was verified for each animal by progressively increasing the intensity until a plateau in twitch force was achieved. A force frequency relationship was generated for each animal using supramaximal 1500ms stimulating trains ranging from 1-120Hz to determine the optimal frequency for maximal tetanic force production. This optimal frequenc y (70-80Hz) was used for all subsequent testing. Using the optimal stimulation fre quency, maximal isometric tetanic force was repeated 3 separate times using 1500ms trains with a 5 minute rest in terval between each stimulation. The maximum tetanic force of 3 attempts was recorded. In addition, three twitch stimulations were evoked for maxima l twitch tension and contractile property measurements. Soleus specific tension was calculated by maximal titanic force / muscle weight (N/g). A fatigue test was also perf ormed and consisted of a modified Burkes fatigue test with 300ms trains delivered every second for 2 minutes at the predetermined optimal frequency. Force time integral was ca lculated as the area under the force-time curve during muscle contraction as a measure of muscle isometric work. 5.3.6 Tissue Harvest At the conclusion of the contractile measurements, the soleus muscles were removed from both hindlimbs of the animal. The muscles were subsequently rapidly

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60 frozen in isopentane precooled in liquid nitrogen (s torage at -80 C) for the following histological measurements. 5.3.7 Immunohistolochemistry Cryostat sections (10 m) in a transverse plane were prepared from the central portion of each muscle taken from the both le gs and mounted serially on gelatin-coated glass slides. Immunocytochemical reactions were performed on each cryostat section with anti-laminin to outline the muscle fibe rs for cross sectional area quantification. The soleus fiber CSAs were analyzed usi ng The NIH image program (version 1.62). The pixels setting used for conversion of pi xels to micrometer were 1.50 pixels1 um2 for a 10 X objective. The average fiber CSA of all th e fibers in each fiber type was determined. 5.3.8 Data Analysis One way ANOVAs with Bonferroni-Dunn pos t hoc testing were used to compare results across groups (controls, trained and unt rained animals) for peak soleus tetanic and twitch forces, peak fatigue, average fiber CSA, time to peak tension, relaxation time. A p value of <0.05 was considered significan t. The force decrease during fatigue was calculated as (initial-final force)/initial for ce. Independent t-tests were used to compare differences in BBB scores between groups befo re training (1 week post SCI) to ensure that there were no differences between groups prior to training. Independent t-tests were also used to compare differences in BBB scor es after 1 week of tr aining/no training (2 weeks post SCI). A Pearsons correlation was used to examine the relationship between the soleus peak force and BBB scores for the left leg across both injured groups 2 weeks after SCI.

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61 5.4 Results 5.4.1 BBB Open Field Locomotor Scores Intact control animals consistently dem onstrated normal locomotor behavior on the BBB scale (21 point scale). One week post SCI, injured animals displayed no significant differences in BBB scores between groups desi gnated for TM traini ng (5.44 1.61) vs no training (5.79 1.98) (p>0.05) (Figure 5-1). At two weeks after SCI, and after one week of training for the TM group, BBB scoring was repeated and scores were significantly different between untrained (6.31 2.8) and trained (10.63 1.67) animals (p<0.05) (Figure 5-1). The significant di fference between these scores indicated that one week of training had significantly improve d open field locomotion scores in the trained compared with the untrained animals. Figure 5-1. Average BBB locomotor scores for the SCI + TM and SCI no TM group at 1 and 2 weeks post SCI (n=8/group). *Significantly different between SCI no TM and SCI + TM at 2wks post SCI (p <0.05). SCI + TM received 5 days of TM locomotor training beginning 1 wk post SCI (mean+SEM).

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62 5.4.2 Soleus Contractile Properties At two weeks following SCI, the mean peak soleus muscle force measured in the untrained injured animals was 117 29mN. Howe ver, the mean peak soleus muscle force measured in the injured TM group was 178 19mN. This difference was significantly greater (p<0.05) than that r ecorded in the untrained inju red group, but not significantly different from the mean peak force of 201 14mN recorded in the intact control group (Figure 5-2). Specific tension was signifi cantly lower in untrained injured animals compared control rats (p<0.05). Although a tr end existed, no stat istically significant differences were measured between muscle sp ecific tension in the treadmill trained SCI rats and untrained SCI rats (p>0.05) (Figure 5-3). Figure 5-2. Soleus muscle peak tetanic for ce for CON, SCI no TM, and SCI + TM groups at 2 weeks post SCI (n=8/group). *Si gnificantly lower force for SCI no TM compared to SCI + TM and CON, p<0.05.

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63 Figure 5-3. Soleus muscle specific force fo r CON, SCI no TM, and SCI + TM groups at 2 weeks post SCI (n=8/group). *Significan tly lower specific force for SCI no TM compared to CON, p<0.05. The possibility of a relationship between open field locomotor score and force production in skeletal muscle was tested by plot ting the peak soleus muscle forces for all animals vs. their respective BBB scores (Figur e 5-4). Least squares linear regression of these points revealed a correlation coe fficient of r=0.704, suggesting a predictable relationship between BBB scores and peak soleus muscle force following SCI. In addition, injured rats that received trai ning demonstrated comparable fatigue to control animals (27 0.04% and 26 0.09% respectively) while SCI animals without training were more fatigable (36 0.1 %) (p<0.05) (Figure 5-5). Accordingly, the isometric work (force time integral) ge nerated during fatigue contractions was significantly lower in the untrained injured ra ts than control animals and treadmill trained rats (Figure 5-6). However, no significant differences were f ound in peak twitch force, as well as time to peak tension and half rela xation time for controls, trained SCI, and untrained SCI (p<0.05) (Figure 5-7, 5-8, 5-9).

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64 Figure 5-4. Peak soleus tetanic force vs BBB score for the left legs of SCI + TM and SCI no TM combined (n=16). Pearson correlation coefficient=0.704 (p<0.05). Note: controls are not in cluded in this figure. Figure 5-5. Soleus muscle fatigue (Initial -Final Force/Initial Force) (n=8/group). *Significantly greater fatigue in SCI no TM compared to CON and SCI + TM groups, p<0.05.

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65 Figure 5-6. Soleus muscle force-time integral during a fatiguing pr otocol (n=8/group). *Significantly lower force-time integral in SCI no TM compared to CON and SCI + TM groups, p<0.05. Figure 5-7. Soleus muscle peak twitch for ce for CON, SCI no TM, and SCI + TM groups at 2 weeks post SCI (n=8/group).

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66 Figure 5-8. Soleus muscle time to peak fo r CON, SCI no TM, and SCI + TM groups at 2 weeks post SCI (n=8/group). Figure 5-9. Soleus muscle relaxation time for CON, SCI no TM, and SCI + TM groups at 2 weeks post SCI (n=8/group). 5.4.3 Soleus Muscle Fiber Cross Sectional Area The mean soleus muscle fiber cross sectio nal area (CSA) observed in the untrained animals at two weeks following SCI was 1999 541m2. By comparison, the mean CSA observed in the trained inju red animals was 2654 359m2, which was significantly

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67 greater (p<0.05) than that of the untrained injured group, but not significantly different from the mean peak fiber CSA of uninjured control animals (2467 497m2) (p>0.05) (Figure 5-10). Figure 5-10. Average soleus muscle fibe r CSA for CON, SCI no TM, and SCI + TM groups at 2 weeks post SCI (n=8/gr oup). *Significantly smaller average muscle fiber CSA in SCI no TM compared to CON and SCI + TM groups, p<0.05. 5.5 Discussion Collectively, these results indicate th at early therapeutic intervention using treadmill locomotor training significantly increases locomotor recovery and soleus muscle size and strength fo llowing SCI. Compared to untrained SCI animals, one week of training resulted in a 32% improvement in BBB scores, a 38% greater improvement in peak soleus tetanic force, and larger muscle fiber CSA (23%). The magnitude of some of these changes is remarkable considering the relatively short training interval, and clearly illustrates the potential that early exercise intervention may have on countering some of the early functional deficits resulting from SCI.

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68 Changes in limb loading and neural activation produced by repetitive motor training early after SCI provide critical activity in remaining spinal cord circuits that facilitate neuroplastic ity and muscular plasticity fo r better functional recovery. The specific mechanisms by which locomotor tr aining facilitates plasticity are poorly understood, although it has been proposed that plasticity is guided in an activitydependent manner via biochemical changes at the cellular level of the nervous system including altered synaptic connections, altered sensitivities of neurotransmitter receptors, and altered production of neurotransmitters a nd growth factors (69, 74, 156). One theory that is receiving increasing attention is the ro le that exercise may play in increasing the release of myotrophic and neurot rophic factors in the spinal cord as well as skeletal muscle (69). Another possibl e explanation to account for improvements with locomotor training after SCI is that training may fac ilitate reorganization of undamaged neural pathways (58). It has been suggested that m oving paralyzed limbs activates muscle, joint, and skin afferents which provides critical gui dance to existing circuits to form new patterns of motor output (87, 106) The results of the present study indicate that muscular plasticity occurs via a combination of changes in muscle morphol ogy and physiological contractile properties. Very limited information exists regarding musc ular plasticity early after a contusion SCI (116, 198), or early adaptive changes in mu scle after locomotor training following a contusion SCI. Hutchinson et al. (2001) e xplored muscle adaptations in rats over 10 weeks of recovery from a moderate cont usion SCI without loco motor training. They found that 3 weeks after inju ry, there were significant changes in muscle morphology (wet weight) and phenotype (MHC compos ition) that corresponde d to changes in

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69 contractile properties. Even as early as 1 week after SCI, muscle wet weights were different compared to controls even if no significant differen ces in contractile properties were noted. Our present study examined soleus muscle properties at an intermediate time point to that of Huchinson et al. (2 week s post SCI) and found that there were significant differences in soleus tetanic muscle force between injured and control animals. These findings are consistent with those of our untra ined SCI rats at 2 weeks after injury, and going one step further, we have found that ev en as little as one week of locomotor training prevents the phenotypi c changes towards faster musc le seen within weeks after injury. Using magnetic resonance imaging to longitudinally quantify changes in muscle size after SCI, we have recently found that the rate of hindlimb muscle atrophy without locomotor training was greatest during the firs t week after a modera te contusion SCI in rats, although the greatest absolute amount of atrophy in untrained animals occurred 2 weeks after SCI (chapter 4). Of the hindlimb muscles studied, the triceps surae (soleus and gastrocnemius muscles combined) dem onstrated the greatest amount of atrophy (26.3%), which is consistent with other stud ies of muscle disuse that have described greater atrophy of the antigr avity extensor muscles than the flexor muscles across a variety of animal species. In addition, the aforementioned study (chapter 4) found that one week of locomotor training reduced atrophy of the triceps surae by over 15% compared to untrained, injured animals. The present study provide s additional evidence for the merits of early intervention in the fo rm of not only whole muscle cross sectional area measurements, but via measurements of muscle contractile function combined with muscle fiber size measurements.

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70 Additional studies that have investigated the impact of early locomotor training after a contusion SCI have not included muscle -specific measures of adaptations, yet they provide additional support for functional improvements with ear ly exercise intervention (79, 158). Engesser-Cesar et al. (2005) found that mice with a contusion SCI that participated in one week of flat-surface wheel running starti ng one week after injury had significantly better BBB scores than injured mi ce without training. Mult on et al. (2003) began a 12 week locomotor training st udy with weekly BBB measurements after inducing a spinal cord compressi on injury in rats with a s ubdurally inflated microballoon. Training began within a week of injury and resulted in differences in BBB scores by 2 weeks post injury, consistent with the locomo tor behavior results of the present study. In the present study, we found a good relationship between BBB scores and soleus tetanic force production, even though the soleus only repr esents one of many lower extremity muscles that are involved in hindlimb function. Nevertheless, these results suggest that while the so leus may be more susceptible to disuse with injury (2, 28, 225), its force production appear s to be fairly representa tive of collective hindlimb functional deficits as measured by the BBB Locomotor Rating Scale. The BBB scale has been shown to be a valid and predictive m easure of locomotor recovery and allows for comparisons across different injuries to eval uate treatment efficacy after SCI (24, 25). In fact, our BBB scores 1 week after injury were similar to those reported by Hutchinson et. al. (2001) and therefore allow for more direct comparisons across these 2 investigations of muscle adaptations after a moderate contusion SCI. As little as five days of locomotor treadmill training appeared to decrease soleus muscle fiber atrophy, with no differences in fiber CSA between control and trained SCI

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71 animals. On the other hand, SCI animals w ithout training responded similarly to other models of limb disuse (70, 116), such that fiber CSA without trai ning was significantly different from control and trained animals af ter only two weeks after SCI. In addition, the transformation towards faster, more fatig able fiber types in the untrained, SCI group was evident from our electrically elicited fatigue test with the soleus muscle of untrained animals demonstrating ~27% more fatigue than trained, SCI animals or control animals. At the same time, twitch contraction propert ies (peak force, time to peak tension, relaxation time) were not different across gr oups, despite evidence for initial fiber type transformations in injured animals. Quite pos sibly, measurements of twitch contractile properties were not sensitive enough to de tect small changes in fiber type MHC composition with a single electrically elicited co ntraction early after injury. In contrast to single isolated twitches, fatigue testing consisted of a series of consecutive contractions delivered over 2 minutes, the cumulative effect s of which might allow for better detection of early differences in phenotypic transfor mations of fiber types such as increased fatigability. It is not clear from the present study wh ether locomotor training early after injury prevents changes in contractile function and ma intains normal muscle morphology or actually reverses any changes that have occurr ed within the first week after injury. But it is clear that early exercise intervention with force hindlim b loading counters the disuse atrophy present in the limbs of injured anim als, which are flaccid and unloaded. A study by Burnham et al. (1997) supports our findings that early ch anges in MHC after SCI are present in the absence of training and suggest s that interventions aimed at preventing or minimizing the transformation would need to be implemented within weeks after SCI to

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72 prevent changes (40). Timing appears to be critical because waiti ng longer after SCI injury to initiate an exercise training program has been shown to have less benefit on the soleus muscle of rats injured by transect ion (71). Although compar isons across different SCI methods are not always generalizable, th is study suggests that waiting as long as 4 weeks after injury may not offer the protective benefits that early exercise interventions may offer. Although there are limitations in using animal m odels to understand human SCI recovery with locomotor training, animal m odels allow for a better understanding of the functional properties of complex systems. The results of the present suggest that starting some form of exercise traini ng after SCI as early as medi cal stabilization of patients allows may offer the greatest protective be nefits for maximizing functional recovery.

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73 CHAPTER 6 CHANGES IN SOLEUS FIBER TYPE CO MPOSITION WITH ONE WEEK OF TREADMILL LOCOMOTOR TRAINING IN SPINAL CORD CONTUSION INJURED RATS 6.1 Abstract Neuromuscular activity plays a very im portant role in specifying muscle phenotypic properties. The objective of this st udy was to determine the impact of one week of treadmill training on the soleus fiber type composition following spinal cord contusion injury. Thirty five adult Sprague Dawley rats (female, 16-20 weeks, weighing 250-290g) were studied. Twenty four animal s received a moderate T8 spinal cord contusion injury. Twelve of the SCI rats received treadmill locomotor training (TM), starting 1 week after SCI for 5 consecutiv e days, 20 minutes/trial, 2 trials/day. The additional twelve injured rats received no exercise interv ention (no TM). The soleus muscle fiber type composition was determ ined using immunofluorescence techniques with monoclonal antibodies (BA-D5, SC-71, BF-F3, and BF-35). Two weeks following spinal cord contusion injury, the proportion of type I fibers decr eased to 75.1 3.1% (vs 86.1% in the uninjured animal). In addition, the soleus contained 8.8 2.0% of fibers that were co-labeled for MHC-I and MHC-IIa and 1. 4 .1% of fibers that reacted with both IIa and IIx. Compared to the SCI soleus, 1week of locomotor tr aining resulted in a significant increase in the soleus muscle type I fibers (81.3 1.7%) and a reduction in the proportion of fibers that were stained positiv ely with both type I and IIa (3.4 1.2%). There were no fibers that reacted with both type IIa and IIx in the SCI-TM soleus. In addition, training paradigms significantly increa sed the soleus fiber cross-sectional area

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74 (CSA) across the different fiber types, w ith an average type I fiber CSA of 2170m2 and type IIa of 1402m2 in the no TM group, 2884m2 and 1781m2 in the TM group, respectively. Collectively, these results indica te that early therapeu tic intervention using treadmill locomotor training can effectively reverse both the shift in fiber type composition and decrease in fiber type specific CSA following SCI. 6.2 Introduction Neuromuscular activity (inclu ding electrical activation a nd limb loading) plays a very important role in specifying muscle phenotypic properties (see review (213)). Following SCI, skeletal muscle shows an increas e in the percentage of fast fibers and a decrease in the percentage of slow fibers in both human subjects and animal models (15, 114, 121, 122, 137). Unfortunately, to date, very lit tle data exists describing muscle fiber type changes after incomplete SCI. Interesti ngly, the innate plasti city associated with incomplete SCI furnishes the potential to pos sess greater plasticity than complete SCI (162, 232). In a pilot study, we found that 3 mont hs after spinal cord contusion injury the rat soleus muscle displays a much higher proportion of fibers expressing the fast MHC isoforms compared to control soleus muscle s, with 22% of the total number of fibers being MHC-I compared to ~10% in control mu scles (138). However, limited studies have investigated the fiber type composition of sk eletal muscle early after contusion spinal cord injury. In addition, little information is available in re gard to the fiber type specific changes in fiber CSA. Interventions that result in increased ne uromuscular activation, such as electrical stimulation (170), functional overload and endurance exercise, have been shown to induce shifts in MHC towards the slower isoforms (6). For example, treadmill training of spinalized cats has been demonstrated to prevent conversion of so leus myofibers from

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75 slow oxidative to fast glycolytic (193). R oy et al. (193) trained adult spinal cord transected cats on a treadmill for around 5 months and found that step trained cats showed 100% type I MHC in the soleus, comp ared to 82% untrained cast. Despite these promising findings, less atten tion has focused on how skeletal muscle responds to acute training interventions of shorter duration. Therefore the purpose of this study was to determine the impact of moderate T8 spinal cord contusion injury on acute cha nges in rat soleus fi ber type composition. A second aim of this study was to determine the effect of 1-week treadmill locomotor training on rat soleus fiber t ype composition following moderate T8 spinal cord contusion injury. 6.3 Materials and Methods 6.3.1 Experimental Animals Thirty five adult Sprague Dawley rats (female, 16-20 weeks, weighing 250-290g) were studied. The rats were housed in a temperature-controlled room at 21 0C with a 12:12 hours light:dark cycle and were provided rodent chow and water ad libitum. All procedures were performed in accordance with the U.S. Government Principle for the Utilization and Care of Vertebrate Animal s and were approved by the Institutional Animal Care & Use Committee at the University of Florida. 6.3.2 Spinal Cord Contusion Injury Spinal cord contusion injuries were produ ced in all rats except controls using a NYU (New York University) impactor. Brie fly, 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 Ba ytril (10mg/kg) per da y for 5 days, starting the day of surgery. Procedures were perf ormed under ketamine (100mg/kg)-xylazine

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76 (6.7mg/kg) anesthesia (details in (179, 226)). S ubcutaneous lactated Ringers solution (5 ml) was administered after completion of the surgery. Animals were given Buprenorphine (0.05mg/Kg IM) and Ket oprofen (5.0 mg/Kg SC) for pain and inflammation over the first 36hrs after SCI. The animals were kept under vigilant postoperative care, including daily examina tion for signs of distress, weight loss, dehydration, and bladder dysfunction. Manual expression of bladders was performed 2-3 times daily, as required, and animals were mon itored for the possibility of urinary tract infection. After SCI, animals were housed individually. On postoperative day 7, open field locomotion was assessed using the Basso -Beattie-Bresnahan (BBB) locomotor scale (24) and animals that did not fall within a preset cutoff (BBB<8) were excluded from the study. Animals were subsequently randomly assigned to either SCI-treadmill training group (SCI-TM, n=12) and SCI no training co ntrol group (SCI, n=12). In addition, the rats that did not receive SCI were served as control group (CON, n=11). 6.3.3 Treadmill Locomotor Training Animals in training paradigms animals we re trained continuously for 5 Day (2 trials/day, 20 minutes/trial), starting on pos t-operative day 8. Animals assigned to the treadmill training group were given five minutes to explore the treadmill on the first training day and then encouraged to walk on the moving treadmill (11 mpm) (130) for a series of four, five-minute bouts. A minimum of five minutes rest was provided between bouts. On the second day of training, anim als completed two bouts of ten minutes each, twice a day. Starting on day 3, animals tr ained continuously for 20 minutes with a minimum interval between trials of 2 hours. Body weight support was provided manually by the trainer. The level of body weight support was adjusted to make sure that rats could bear their weight and there wa s no collapse of their hindlimbs. Typically, the rats started

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77 stepping when they experienced some small load on their hindlimbs. In addition, during the first week of training, when all rats had profound paraplegia, assistance was provided to place rat hindlimbs appr opriately for plantar steppi ng position during training. 6.3.4 Immunohistolochemistry At the end of the experiment, the soleus muscles were removed from both hindlimbs. The muscles were subsequently rapi dly frozen at resting length in isopentane, precooled in liquid nitrogen (storage at -80 C). Tranverse cryostat sections (10 m) were prepared from the central portion of each musc le and mounted serially on gelatin-coated glass slides. Immunocytochemical reactions were performed on serial cryostat sections with anti-laminin and anti-MHC antibody at various dilutions. Rabbit anti-laminin (Neomarker, Labvision, Fremont, CA) was used to outline the muscle fibers for cross sectional area quantification. Four anti-MH C Mabs (BA-D5, SC-71, BF-F3, and BF-35) were selected on the basis of their reactivity toward adult MHC (Table 6-1). Sections were incubated with rabbit anti-laminin and one of the anti-MHC antibodies (40C over night), followed by incubation with rhoda mine-conjugated anti-rabbit IgG and Fitcconjugated anti-mouse IgG (Nordic Immunological Laboratories). Stained sections were mounted in mounting medium for fluorescen ce (Vector Laboratories, Burlingame, CA) and kept at 40C to diminish fading. Stained cro ss sections were photographed (10X magnification) by using a Leica fluorescence mi croscope with a digital camera. A region of the stained serial sections from each muscle were randomly selected for MHC composition analysis. The proportions of each fi ber type were determined from a sample of 150-250 fiber across the entire section of each muscle. In addition, the soleus fiber CSAs were analyzed using NIH image (v ersion 1.62). The pixels setting used for

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78 conversion of pixels to micrometer were 1.50 pixels1 m2 for a 10 X objective. The average fiber CSA of all the fibers in each fiber type was determined. Table 6-1. Monoclonal antibody specificity IIIaIIxIIb BA-D5+---SC-71-+--BF-F3---+BF-35++-+MHC isoforms MAb 6.3.5 Data Analysis One way ANOVAs with Bonferroni-Dunn pos t hoc testing were used to compare results across groups (controls, trained and untrained animals) for soleus muscle fiber type composition and average CSA. A p va lue of <0.05 was considered significant. 6.4 Results 6.4.1 Soleus fiber type composition As shown in Figure 6-1 & 6-4, the control soleus muscleprimarily contained fibers reacting exclusively with type I mAb (86.1 2.2%) and a small percentage of fibers (13.9 2.2%) reacting with type IIa mAb exclusively. Two w eeks following spinal cord contusion injury, the proportion of type I fibers decreased to 75.1 3.1%. In addition, the soleus contained 8.8 2.0% of fibers that were co-labeled for MHC-I and MHC-IIa and 1.4 .1% of fibers that reacted with both IIa and IIx (Figure 6-2, 6-4). Compared to the SCI soleus, 1-week of locomoto r training resulted in a significant increase in the soleus muscle type I fibers (81.3 1.7%). There was no significant difference in fibers that were stained only with type IIa mAb among different groups. In addition, the proportion of fibers that were stained positively with bot h type I and IIa deceased to 3.4 1.2% during

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79 the first week of treadmill training. There were no fibers that reacted with both type IIa and IIx in the SCI-TM soleus (Figure 6-3, 6-4). Figure 6-1. Serial cross secti ons of a control soleus stained with monoclonal antibodies directly against specific MHC isofor ms. A) BA-D5 (anti-MHC-I). B) SC71(anti-MHC-IIa). C) BF-35(anti-all MHCs except IIx). D) BF-F3 (antiMHC-IIb). Figure 6-2. Serial cross sect ions of a SCI no TM soleus stained with monoclonal antibodies directly against specific MHC isoforms. A) BA-D5 (anti-MHC-I). B) SC-71(anti-MHC-IIa). C) BF-35(anti-all MHCs except IIx). D) BF-F3 (anti-MHC-IIb). AB CD AB C D

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80 Figure 6-3. Serial cross sect ions of a SCI + TM soleus stained with monoclonal antibodies directly against specific MHC isoforms. A) BA-D5 (anti-MHC-I). B) SC-71(anti-MHC-IIa). C) BF-35(anti-all MHCs except IIx). D) BF-F3 (anti-MHC-IIb). 6.4.2 Soleus fiber cross-sectional area Compared with the control soleus muscle, sp inal cord contusion injury resulted in a significant decrease in soleus fiber CSA, across the different fiber types. At 2w-SCI, the CSA of type I and IIa fiber was reduced by 20.5% and 17.0% respecti vely (Figure 6-5). 1-week treadmill training effectively prevente d the atrophic response of skeletal muscle observed following SCI. There were no significant differences in type I and IIa fiber areas between control and treadmill trained so leus. In addition, the fiber CSA of hybrid fibers (type I + type IIa) was significantly higher when compar ed with SCI soleus (Figure 6-5). AB CD

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81 Figure 6-4. MHC based fiber type per centage composition, as determined by immunohistochemistry, of rat soleus mu scle form control, SCI no TM and SCI + TM group. Significant differen ce compared with the SCI no training group. Figure 6-5. Soleus muscle fibe r type specific CSA in contro l, SCI no TM, and SCI + TM groups at 2 weeks post SCI. Significan tly less average muscle fiber CSA in SCI no TM compared to control and SCI + TM groups. 6.5 Discussion Two weeks following midthoracic spinal co rd contusion injury, we observed a significant shift in fiber type composition from slow to fast in the rat soleus muscle, as

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82 well as a significant decrease in fiber type sp ecific soleus fiber CSA. However, 1-week treadmill training effectively attenuated the soleus fiber type shift as well as the fiber type specific decrease in fiber CSA. SCI results in measurable reductions in the amount of electrical activation in mammalian muscles. For example, in cats, a complete SCI results in a 75% reduction in daily integrated electromyographi c activity of the soleus muscle (7). As a result, complete spinal cord transection has been shown to i nduce a large increase in the expression of fast MHC isoforms (121, 122, 215, 217, 222). Talma dge et al. (222) showed that the proportion of MHC-I in the rat soleus is reduc ed from ~90% in control to ~25% only 3 months following a complete mid-thoracic SC I. One of the few studies to closely examine muscle adaptations afte r a contusion injury in rats showed an upregulation of the transitional type IIx in the soleus and extens or digitorum longus muscle within the first 3 weeks after SCI (116). In additi on, we previously showed in a pilot study that the soleus MHC isoforms were shifted from MHC-I to wards MHC-II 3 months after SCI, with approx. 77.9% (vs 85.1% in the uninjured an imal), 19.9% (vs 15.0% in the uninjured animal), and 4.3% (vs 0% in the uninjured an imal) of the total fibers containing MHC-I, MHC-IIa and MHC-IIx, respectiv ely (138). In the present st udy, we observed a shift in the fiber type composition towards type II as early as 2 week post SCI, providing additional evidence to support the role of ne uromuscular activity in specifying muscle phenotypic properties. In this study we also found an increase in the presence of hybrid fibers following contusion SCI. 2 weeks after contusion SCI near ly 8.2% of the soleus muscle fibers in the untrained SCI rats were hybrid fibers. Our findings are consistent with observations

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83 previously reported (222). For example, Talmadge (222) found that there were high proportions of type I/IIa hybrid s in the soleus muscle at 15 days after spinal cord transection. The mechanism behi nd the presence of hybrid fiber is not clear. Originally, it was thought that muscle was in a transitiona l state of fiber transformation. However, studies have found that hybrid fibers could exist for up to one year after SCI, suggesting that hybrid fibers could be a stable phenotype under a partic ular condition (222). In the present study, we found there were no pure type IIx fibers. All fibers that contained IIx were mixed with IIa. The unique findings of the present study were that only 1 week of daily step training emphasizing weight s upport on a treadmill could eff ectively ameliorate, although not reverse the shift in muscle fiber t ype composition observed following mid-thoracic contusion injury. The soleus muscle of th e 1-week trained SCI animals showed 81.3% type I fibers, whereas the soleus muscle in the untrained SCI animals displayed 75% type I fibers. Numerous studies have shown an incr eased proportion of type I fibers in skeletal muscle after different exercise and traini ng programs (6, 61, 193). For example, Demirel et al. (61) found that treadmill training at all different durations (30, 60, or 90 min/day) resulted in a reduction in the percentage of MHCIIb and an increase in the percentage of MHCIIa in the plantaris muscle. Unfortunate ly, to date, the regulatory mechanism for changes in muscle fiber type composition is still under debate. Recent studies suggested that calcineurin plays a very important role in the regulation of fiber phenotypic transformations (48, 208, 243). It has been hypot hesized that calcineur in acts as a calcium server to transform mechanical stimulis to muscle signaling pathways (208). Thus increased muscle activation and limb loading during treadmill training may elevate

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84 cytosolic calcium, and as such facilitate the fiber type transformation mediated by calcineurin (see review (129)). Collectively, the data presented here clea rly demonstrate that 2 weeks of SCI result in a fiber type shift in the rat soleus muscle towards fa ster fiber types. In addition, the increased coexpression of MHC isoforms in all injured animals may point to a dynamic process of fiber type transformation in th e early weeks following contusion SCI. More importantly, as little as five days of locomotor treadmill training appeared to attenuate the shift in MHC composition towards faster isof orms and ameliorate fiber type specific changes in fiber CSA. Future work should be directed towards explaining mechanisms by which physical activity changes skeletal mu scle phenotype and influences muscle plasticity after SCI.

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85 CHAPTER 7 EFFECTS OF TREADMILL TRAINING ON IGF-I EXPRESSION IN RAT SOLEUS MUSCLE FOLLOWING SPINAL CORD INJURY 7.1 Introduction One of the primary consequences of spinal cord injury (SCI) is pronounced muscle atrophy and loss of muscle function distal to the site of injury (116, 136, 137, 178). Recently, locomotor training programs have sh own to help maintain muscle mass and strength and thereby promote functional re covery (63, 176). In a previous study, we showed that locomotor treadmill training amelio rates the loss in muscle size in the rat hind limb muscles following midthoracic spinal cord contusion in jury (139). Although the effect of training was prevalent for up to 3 months, the largest therapeutic impact of locomotor training was observed within the fi rst week of training. In addition, we showed that following 1 week of locomotor training mu scle strength and fiber specific muscle CSA in the postural slow twitch soleus muscle of trained spinal cord injured rats was significantly higher than that of non-trained SCI rats (Chapter 5, 6). Although the exact mechanisms for how loco motor training potentially confers its benefits are not well understood, a number of signaling pathways have been proposed to potentially regulate cellular and molecular processes involved in skeletal muscle remodeling (33, 53, 68, 118). Insulin-like growth factor I (IGF-I) has been shown to play a particularly important role in mediating protein synthesis, pr otein degradation and satellite cell mediated repair (1). Systemic re lease of IGF-I may contribute to an increase in protein content and a reduc tion in protein degradation in skeletal muscle (247). In

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86 addition, overexpression of IGF-I has been associated with myofiber hypertrophy in transgenic mice (51), and local infusion of IGF -I has been shown to c ontribute to skeletal muscle hypertrophy (1), as well as block the ag ing-related loss of muscle mass in mice (19). Moreover, resistance training has been associated with increased IGF-I mRNA expression in both animal models (3) and indivi duals with complete spinal cord injuries (29). Although, the effect of treadmill loco motor training on the muscle IGF-I signaling pathway has not been studied, such informa tion may help explain some of the positive therapeutic benefits of locomotor training. The purpose of this study was to determine the impact of treadmill training on mRNA expression of IGF-I and its related rece ptor (R) and binding pr oteins (BP) in rat soleus muscle following moderate T8 spinal cord contusion injury. 7.2 Materials and Methods 7.2.1 Experimental Animals Thirty adult Sprague Dawley rats (fem ale, 16-20 weeks, weighing 250-290g) were studied. The rats were housed in a temp erature-controlled room at 210C with a 12:12 hours light:dark cycle and were provided r odent chow and water ad libitum. All procedures were performed in accordance with the U.S. Government Principle for the Utilization and Care of Vertebrate Animal s and were approved by the Institutional Animal Care & Use Committee at the University of Florida. 7.2.2 Spinal Cord Contusion Injury Spinal cord contusion injuries were produ ced in all rats except controls using a NYU (New York University) impactor. Brie fly, a 10g weight was dropped from a 2.5-cm height onto the T8 segment of the spinal cord ex posed by laminectomy under sterile conditions. Animals received two doses of Ba ytril (10mg/kg) per da y for 5 days, starting

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87 the day of surgery. Procedures were perf ormed under ketamine (100mg/kg)-xylazine (6.7mg/kg) anesthesia (details in (179, 226)). S ubcutaneous lactated Ringers solution (5 ml) was administered after completion of the surgery. Animals were given Buprenorphine (0.05mg/kg) and Ketoprofen ( 5.0 mg/kg) for pain and inflammation over the first 36hrs after SCI. The animals were kept under vigilant postoperative care, including daily examination fo r signs of distress, weight loss, dehydration, and bladder dysfunction. Manual expression of bladders wa s performed 2-3 times daily, as necessary, and animals were monitored for the possibility of a urinary tract infection. After SCI, animals were housed individually. On postoperative day 7, open field locomotion was assessed using the Basso-Beattie-Bresnahan (BBB) locomotor scale (24) and animals that did not fall within a preset range (BBB: 4-8) were excluded from the study. Animals were subsequently assigned to one of four groups (n=6 rats/group): (1) SCI-8D and (2) SCI-2W represented untrained sp inal cord injured animals, sacrificed 8 days and 2 weeks after SCI respectively; (3 ) SCI-8DTM represented animals that were sacrificed on day 8 post SCI w ithin 36hrs of init iating three 20 minut e training bouts; (4) SCI-2WTM represented animals that were trai ned for 5 consecutive days and sacrificed at week 2 following SCI. In addition, the rats that did not receive SCI served as a control group (CON). 7.2.3 Treadmill Locomotor Training Animals in training paradigms animals were trained continuously for 5 days (2 trials/day, 20 minutes/trial), starting on pos t-operative day 8. Animals were given five minutes to explore the treadmill on the first training day and then encouraged to walk on the moving treadmill (11 mpm) (130) for a seri es of four, five-mi nute bouts. A minimum of five minutes rest was pr ovided between bouts. On the s econd day of training, animals

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88 completed two bouts of ten minutes each, twic e a day. Starting on day 3, animals trained continuously for 20 minutes with a minimum interval between trials of 6 hours. Body weight support was provided manually by the trainer. The level of body weight support was adjusted to make sure that rats coul d bear as much weight as possible without collapse of their hindlimbs. Typically, the rats started stepping when they experienced a small load on their hindlimbs. In addition, during the first week of training, when all rats had profound paraplegia, assistance was provided to place rat hindlimbs appropriately for plantar stepping positio n during training. 7.2.4 Tissue Harvest At the time points indicated above, the so leus of both legs were dissected and weighted. Half of the muscle was embedded in freezing medium and the whole muscle was snap frozen at resting length in isopentan e, pre-cooled in liqui d nitrogen and stored at 0C. The half that was embedded in freezing medium was used for immunohistochemistry. The other porti on of muscle was used for RT-PCR measurements. 7.2.5 RT-PCR Measurement RNA extraction: Total RNA was extracted from frozen muscle samples (pre weighed) by using the TRIzol Reagent (In vitrogen life technologies, Carlsbad, CA) according to the companys protocol. Briefl y, around 40 mg of muscle was homogenized with a blade homogenizer in 1ml of TRIzol Reagent. Extracted RNA was precipitated from the aqueous phase with isopropanol and, after being washed with 75% ethanol, was dried and suspended in a known volume of DE PC treated water. Th e RNA concentration was determined by optical density at 260 nm The muscle total RNA concentration was calculated on the basis of total RNA yield and the weight of the extracted muscle piece.

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89 The RNA samples were stored at 0C to be used subsequently in determining the specific mRNA expression via re lative RT-PCR procedures. Reverse transcription: A relative RT-PCR method using 18S as internal standard (Ambion, Austin, TX) was employed to st udy the expression of specific mRNAs of interest. The primers used for PCR were purchas ed from fisher Scientific and previously used by Haddad et al (2002). In each PCR reaction, 18S ribosomal RNA was coamplified with the target cDNA (mRNA) to serv e as an internal standard and to allow for correction of differences in starting amounts of total RNA. For the 18S amplification, we used the alternate 18S internal standard (Ambion), which yields a 324-bp product. The 18S primers were mixed with competimers at an optimized ratio that ranged from 1:8 to 1:10, depending on the abundance of the target mRNA. Inclusion of the 18S competimers was necessary to decrease the 18S signal, whic h allowed its linear amplificaiton in the same range as the coamplified target mR NA according to Ambions relative RT-PCR kit protocol. For each specific target mRNA, the RT-PCR reactions were carried out under identical conditions by using the same reagent premix for all the samples to be compared in the study. To validate the consistency of the analysis procedures, at least one representative from each group was included in each RT-PCR run. Polymerase chain reaction: One microliter of each RT reaction was used for the PCR amplification. The PCR reactions were ca rried out in the presence of 2 mM MgCL2 by using standard PCR buffer, 0,2 mM dNTP 2 M specific primer set, 1 M 18S primer/competimer mix, and 0.75 unit Taq DNA polymerase in 50 ul total volume. Amplifications were carried out in a Biometra cycle with an initial denaturing step of 3 min at 94 0C, followed by 25 cycles of 45 sec 94 0C, 30 sec at 55 0C 60 0C, 1.5 min at

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90 72 0C, and a final step of 3 min at 72 0C. PCR products were se parated on a 2% agarose gel by electrophoresis and staine d with ethidium bromide. The signal quantification was conducted by scanning densitometry. In each approach, each specific mRNA signal was normalized to its corresponding 18S. 7.2.6 Immunohistolochemistry Tranverse cryosta t sections (10 m) were prepared from the central portion of each muscle and mounted on gelatin-coated gla ss slides. Rabbit anti-laminin (Neomarker, Labvision, Fremont, CA) was used to outline the muscle fibers. Sections were incubated with rabbit anti-laminin and anti-Embryonic myosin (1:10) (40C over night), followed by incubation with rhodamine-conjugated anti-ra bbit IgG and Fitc-conjugated anti-mouse IgG (Nordic Immunological Laboratories). St ained sections were mounted in mounting medium for fluorescence (Vector Laborato ries, Burlingame, CA) and kept at 40C to diminish fading. Stained cro ss sections were photographed (10X magnification) by using a Leica fluorescence microscope with a digi tal camera. The proportions of positive fiber type were determined from a sample of 150-250 fiber across the entire section of each muscle. In addition, the proportion of centr al nuclei in the soleus fibers was determined by H & E staining, as an indicator of muscle fiber regeneration and satellite cell activation. 7.2.7 Data Analysis All statistical analysis was perfor med using SPSS for Windows (Version 10.0). Results were expressed as mean standard error of mean (SEM). Research hypotheses were tested at an alpha level of 0.05. On e-way analysis of variance (ANOVA) was performed to test gene expression between gr oups. Post hoc tests were performed using the Bonferroni-Dunn procedure for multiple pair-wise comparisons.

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91 7.3 Results 7.3.1 mRNA Expression of IGF-I and It s Receptor and Binding Proteins Eight days following SCI, IGF-I mRNA levels in the soleus muscle relative to 18S were not different in injured animals compared to control animals, but IGF-I mRNA levels were increased 2.5 fold at day 14 in untrained, injured animals (Figure 7-1). mRNAs encoding the IGF-IR and IGF-I BP5 were elevated 1.2 to 1.75 fold at either 8 days or 2 weeks following SCI (Figure 7-3 and 7-5). In contrast, the IGF-I BP4 mRNA level did not change significantly throughout the experimental period in all untrained animals, although there was a trend toward s a decrease at 2 weeks (Figure 7-4). Compared with the control and untrained soleus muscles, the expression of the mRNAs for IGF-I and the loading-sensitive IGF-I isoform, MGF, were significantly increased (range of 3.8-4.7 fold) after three tr ials of treadmill trai ning (Figure 7-1 and 72). One week of training fu rther increased MGF and IGF -I expression (range of 4.5-6.2 fold). The magnitude of these changes was si gnificantly higher than that seen with only three trials of training. Both three bout s and 1-week treadmill training resulted in significant increases in IGF-BP4 and IGFR mRNA expression (Figure 7-3 and 7-4). However, there were no significant differences in the levels of mRNA for IGF-R between the training and no training groups. In a ddition, mRNA expression of IGF-BP5 was decreased significantly in the trained animal s compared with the untrained animals at both time points (Figure 7-5).

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92 Figure 7-1. IGF-I mRNA expression relative to 18s in soleus muscle. Significant differences compared with both control and SCI no training group. Significant differences comp ared with control group. Figure 7-2. MGF mRNA expression relative to 18s in soleus muscle. Significant differences compared with both control and SCI no training group.

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93 Figure 7-3. IGF-R mRNA expre ssion relative to 18s in soleus muscle. Significant differences compared with control group. Figure 7-4. IGF-BP4 mR NA expression relative to 18s in soleus muscle. Significant differences compared with both control and SCI no training group. Figure 7-5. IGF-BP5 mR NA expression relative to 18s in soleus muscle. # Significant differences compared with SCI no training group.

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94 7.3.2 Embryonic Myosin Immunohistochemical analysis of the soleus muscle revealed the presence of small fibers expressing embryonic myosin in bot h SCI groups (Figure 7-6). However, the proportion of small fibers expressing em bryonic myosin was 6.4 2.0% and 1.6 0.9% in the trained SCI and untrained SCI animal s, respectively (p=0.001). We did not observe any expression of embryonic myosin in the control adult soleus muscle. Figure 7-6. Cross-section of soleus muscle stained with monoclonal antibody against embryonic myosin isoform. (A) Contro l soleus; (B) SCI-2 W soleus; (C) SCI2WTM soleus. 7.3.3 Central Nuclei Hemotoxilin and eosin staining was utili zed to analyze the proportion of central nuclei in the soleus fibers as an indicator of muscle fiber regeneration (Figure 7-7). Our

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95 data showed the presence of a small number of fibers cont aining central nuclei (~1% of the fibers) in the soleus muscle following 1week of treadmill training. However, central nuclei were not visible in the c ontrol and SCI untrained soleus. Figure 7-7. Cross-section (hemotoxilin & eosin stained) of soleus muscle showing the presence of central nuclei (arrow ma rks a central nucleus) after 1 week treadmill training (C), which were absent in control (A) and SCI untrained soleus (B). 7.4 Discussion Following SCI, the soleus muscle experiences significant muscle. In chapter 6 we reported a 20.5 % and 17.0 % decrease in the CSA of the type I and IIa fibers, respectively. However, one week of treadmill training significantly ameliorated the loss in fiber size. To better u nderstand the mechanisms involved in mediating muscle plasticity in the paralyzed muscle following contusion SCI, we measured IGF-I and its

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96 associated receptor and binding protein mRNA expression in the soleus, an important postural muscle. IGF-I acts as an autocrine and paracrine gr owth factor that has skeletal muscle anabolic properties. Therefore, we hypothe sized that the IGF-I system might be downregulated in response to SCI. However, our results suggest that the IGF-I system components were either unchanged (8 days) or upregulated (2 weeks) in the paralyzed muscles following contusion SCI. Other studie s have also reported contradictory findings regarding IGF-I expression in atrophic muscle in various disuse models (13, 55, 177). For example, Awede (13) reported that unload ing by hindlimb suspension resulted in atrophy of soleus muscle (20%) a ssociated with a 30% decreas e of IGF-I mRNA. However, Haddad et al (103) reported that IGF-I mRNA in soleus mu scle increased by 40% at 15 days after SCI, despite the muscle experien cing severe atrophy. In addition, Criswell et al. also showed that overexpres sion of IGF-I in skeletal mu scle does not prevent atrophy following limb unloading (55). Unlike the ambiguous results of studies examining the ro le of IGF-I in muscle atrophy, numerous studies have consistently shown that IGF -I plays a critical role in mediating muscle mass after a variety of ex ercises and training programs (1, 19, 51). A number of studies have show n that increased load on musc le can significantly increase expression of IGF-I and the loading sensit ive IGF-I isoform, MGF (1, 4, 19, 20). In addition, a number of studies demonstrat ed that IGF-I mRNA and peptide levels increased during compensatory hypertrophy in both animal models and humans (1, 5, 13, 29). In the present study, we observed that treadmill training significantly increased soleus IGF-I and MGF expression in the sole us muscle. In specific, mRNA expression

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97 for IGF-I and MGF was signifi cantly increased (range of 3.8 4.7 folds) after only three bouts of training. One week of training result ed in a further increase of MGF and IGF-I expression. The most important feature of the treadmill is the rhythm ic loading of limbs and force feedback from the hindlimb muscle s. Thus, our study pr ovides further evidence to suggest that limb loading may play an important role in skeletal muscle IGF-I expression. The cellular effects of IGF-I may be me diated by its associated receptor and binding proteins. After binding with IGF-I, IG F-IR activates two signaling pathways: the Ras-Raf-MEK-ERK pathway (53) and the PI3K -Akt pathway (152), which are important in regulating muscle mass (159). These processes can be mediated by IGF-I binding proteins. Among six known IGF binding protein, IGFBP-4 and IGFBP-5 may play a role in skeletal muscle adaptations (14). Both IG F-BP4 and BP5 are suggest ed to inhibit IGF-I function, whereas IGFBP-5 also has been show n to promote IGF-I action (50, 66). In the present study, the expressions of IGFBP-4 and IGF-R were in creased significantly after either 3 bouts or 5 days of treadmill training. In contrast, mRNA expression of IGFBP-5 was decreased significantly following locomoto r training. These results were similar to observations previously reported using resist ance exercise where Haddad (101) reported an increase of IGFBP-4 and no changes of IGFBP-5. Unfortuna tely, to date, the function of IGF binding proteins in muscle hype rtrophy is still not clear (50, 66) IGF-I has been shown to promote satellite cell proliferation and differentiation. As a result of satellite differen tiation, new myoblasts are formed, which subsequently fuse to form small cells known as myotubes. In this study, we observed th e presence of smallsized muscle fibers expressing embryonic isoforms following treadmill training to a

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98 larger extent than without training followi ng SCI. The expression of embryonic myosin isoforms in the adult muscle suggests that new muscle fibers are forming (12). In addition, the presence of central nuclei after tr eadmill training in the soleus also suggests ongoing muscle regeneration. In summary, 1 week of treadmill training significantly augmented IGF-I and MGF mRNA expression. Treadmill training also modulated mRNA expression of IGFR and IGFBPs. We speculate that the IGF-I signaling pathway pl ays an important role in mediating muscle plasticity following cont usion SCI. Future st udies utilizing viralmediated gene delivery of IGF-I may have the potential to induce greater muscular plasticity following SCI.

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99 CHAPTER 8 CONCLUSION In this dissertation, we used a set of in vivo, in situ, and in vitro measures to examine changes in muscle characteristics following locomotor training in a rat model of incomplete SCI. The objective of chapter 4 was to impl ement magnetic resonance imaging to characterize the changes in rat lower hindlimb muscle morphology following contusion SCI and to determine the therapeutic potenti al of two modes of locomotor training. The non-invasive nature of MR imaging allowe d us to quantify skeletal muscle size longitudinally. Following SCI, we observed significant atrophy in all rat hindlimb muscles. The greatest amount of atrophy (11.1 -26.3%) was measured at 2-week postinjury, and spontaneous recovery in muscle size was observed in th e untrained SCI rats by 4 weeks post SCI. Both cycling and treadm ill training halted the atrophic process and accelerated the rate of recovery. The therapeu tic influence of both training interventions was observed within 1 week of training, and a significant positive correlation was found between locomotor functional scores and total hindlimb muscle CSA. Based on the findings of chapter 4, the next two chapters (5 and 6) systematically studied the adaptations in soleus muscle at a very critical time point, one week of training. Functional and immunohistochemical m easurements were used to characterize soleus muscle function (peak force, fatigab ility, contractile properties, fiber types), muscle size, as well as overall locomotor function (BBB) after SCI in rats. Locomotor training resulted in a significant improvement in BBB scores (32%), muscle fiber size

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100 and function. Compared to untrained injured an imals, injured animals that trained for one week exhibited 38% greater peak soleus tetani c forces, a 9% decrease in muscle fatigue, and 23% larger muscle fiber CSA. In a ddition, there was a strong correlation between BBB scores of injured animals and peak soleus muscle force (r=0.704). Insulin-like growth factor I (IGF-I) is a potent myogenic and neurotrophic factor and has been shown to play a critical role in muscle regeneration, muscle hypertrophy, inhibition of neuronal cell death and motor ne uron regeneration. Finally, in chapter 7, we determined the impact of 1-week treadmill training on mRNA expression of IGF-I and its related receptor and binding proteins in rat skeletal muscle following contusion SCI. Our findings demonstrated that tr aining significantly increased th e expression of mRNAs for IGF-I, MGF (the loading-sensitive IGF -I isoform), IGFR, and IGFBP4. Treadmill training also significantly decreased IGFBP5 mRNA expression. In addition, immunohistochemical analysis showed an incr eased presence of small fibers expressing embryonic myosin following treadmill training, an indication that new muscle fibers are being formed. Taken together, the findings from the present work demonstrate that early therapeutic intervention induces significant muscular plasticity in the rat hindlimb muscles following contusion midthoracic SCI. We hope that this study will provide essential feedback for the development of early rehabilitation interventions in SCI individuals.

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106 60. de Leon RD, Reinkensmeyer DJ, Ti moszyk WK, London NJ, Roy RR, and Edgerton VR Use of robotics in assessing the adaptive capacity of the rat lumbar spinal cord. Prog Brain Res 137: 141-149, 2002. 61. Demirel HA, Powers SK, Naito H, Hughes M, and Coombes JS Exerciseinduced alterations in skeletal muscle myosin heavy chain phenotype: doseresponse relationship. J Appl Physiol 86: 1002-1008, 1999. 62. Dietz V, Colombo G, Jensen L, and Baumgartner L Locomotor capacity of spinal cord in paraplegic patients. Ann Neurol 37: 574-582, 1995. 63. Dietz V, and Harkema SJ Locomotor activity in spin al cord-injured persons. J Appl Physiol 96: 1954-1960, 2004. 64. Dietz V, Wirz M, Colombo G, and Curt A Locomotor capacity and recovery of spinal cord function in paraplegic patie nts: a clinical a nd electrophysiological evaluation. Electroencephalogr Clin Neurophysiol 109: 140-153, 1998. 65. Dietz V, Wirz M, and Jensen L Locomotion in patients with spinal cord injuries. Phys Ther 77: 508-516, 1997. 66. Duan C Specifying the cellular responses to IGF signals: role s of IGF-binding proteins. J Endocrinol 175: 41-54, 2002. 67. Ducker TB, Lucas JT, and Wallace CA Recovery from spinal cord injury. Clin Neurosurg 30: 495-513, 1983. 68. Dunn SE, Burns JL, and Michel RN Calcineurin is required for skeletal muscle hypertrophy. J Biol Chem 274: 21908-21912, 1999. 69. Dupont-Versteegden EE, Houle JD, Dennis RA, Zhang J, Knox M, Wagoner G, and Peterson CA Exercise-induced gene expression in soleus muscle is dependent on time after spinal cord injury in rats. Muscle Nerve 29: 73-81, 2004. 70. Dupont-Versteegden EE, Houle JD Gurley CM, and Peterson CA Early changes in muscle fiber size and gene e xpression in response to spinal cord transection and exercise. Am J Physiol 275: C1124-1133, 1998. 71. Dupont-Versteegden EE, Murphy RJ, Ho ule JD, Gurley CM, and Peterson CA Mechanisms leading to restoration of muscle size with exercise and transplantation after spinal cord injury. Am J Physiol Cell Physiol 279: C16771684, 2000. 72. Edgerton VR, Leon RD, Harkema SJ, Hodgson JA, London N, Reinkensmeyer DJ, Roy RR, Talmadge RJ, Tillakaratne NJ, Timoszyk W, and Tobin A Retraining the injured spinal cord. J Physiol 533: 15-22, 2001.

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120 234. Wernig A, Muller S, Nanassy A, and Cagol E Laufband therapy based on 'rules of spinal locomotion' is effective in spinal cord injured persons. Eur J Neurosci 7: 823-829, 1995. 235. Wernig A, Nanassy A, and Muller S Laufband (treadmill) therapy in incomplete paraplegia and tetraplegia. J Neurotrauma 16: 719-726, 1999. 236. Wernig A, Nanassy A, and Muller S Maintenance of locomotor abilities following Laufband (treadmill) therapy in paraand tetraplegic persons: follow-up studies. Spinal Cord 36: 744-749, 1998. 237. West SP, Roy RR, and Edgerton VR Fiber type and fiber size of cat ankle, knee, and hip extensors and flexors following low t horacic spinal cord transection at an early age. Exp Neurol 91: 174-182, 1986. 238. Willis PE, Chadan SG, Baracos V, and Parkhouse WS Restoration of insulinlike growth factor I action in skeletal muscle of old mice. Am J Physiol 275: E525530, 1998. 239. Winiarski AM, Roy RR, Alford EK Chiang PC, and Edgerton VR Mechanical properties of rat skeletal muscle after hind limb suspension. Exp Neurol 96: 650660, 1987. 240. Wirz M, Colombo G, and Dietz V Long term effects of locomotor training in spinal humans. J Neurol Neurosurg Psychiatry 71: 93-96, 2001. 241. Wirz M, Zemon DH, Rupp R, Scheel A, Colombo G, Dietz V, and Hornby TG Effectiveness of automated locomotor trai ning in patients with chronic incomplete spinal cord injury: a multicenter trial. Arch Phys Med Rehabil 86: 672-680, 2005. 242. Wrathall JR, Pettegrew RK, and Harvey F Spinal cord contusion in the rat: production of graded, reproducible, injury groups. Exp Neurol 88: 108-122, 1985. 243. Wu H, Naya FJ, McKinsey TA, Mercer B, Shelton JM, Chin ER, Simard AR, Michel RN, Bassel-Duby R, Olson EN, and Williams RS MEF2 responds to multiple calcium-regulated signals in the c ontrol of skeletal muscle fiber type. Embo J 19: 1963-1973, 2000. 244. Yang S, Alnaqeeb M, Simpson H, and Goldspink G Cloning and characterization of an IGF1 isoform expressed in skel etal muscle subjected to stretch. J Muscle Res Cell Motil 17: 487-495, 1996. 245. Yarkony GM, Roth E, Lovell L, Heinemann A, Katz RT, and Wu Y Rehabilitation outcomes in complete C5 quadriplegia. Am J Phys Med Rehabil 67: 73-76, 1988. 246. Young W Spinal cord contusion models. Prog Brain Res 137: 231-255, 2002.

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122 BIOGRAPHICAL SKETCH Min Liu was born in Tianjin, China, in May 1970. He received his M.D. Degree from Tianjin Medical Univers ity in 1993. He worked as an orthopedic and hand surgeon for 7 years before entering the rehabilitati on science doctoral program at the University of Florida.


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ADAPTATIONS IN SKELETAL MUSCLE FOLLOWING SPINAL CORD INJURY
AND LOCOMOTOR TRAINING
















By

MIN LIU














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


2006

































Copyright 2006

by

Min Liu

































This document is dedicated to my parents, wife, sister and daughter.















ACKNOWLEDGMENTS

The completion of this project would not have been possible without the support

and guidance of many people. At the moment when I am finishing my journey of

graduate study at UF, I am extremely grateful to all of those involved in my education,

research, and life.

I would first like to express my sincere thanks to my advisor, Dr. Krista

Vandenbome, who welcomed me as a researcher into her laboratory and permitted me to

pursue the area of research most compelling to me. Her excellent guidance and constant

encouragement were invaluable throughout my entire doctoral studies. I am very grateful

to Dr. Glenn Walter for his remarkable wisdom and invaluable ideas. His passions on

science and sharp comments will always be my model in the future. I thank Dr. Floyd

Thompson for his broad knowledge. I am blessed to have learned so much about science

from him. I thank Dr. Prodip Bose for his immeasurable support and assistance during

my doctoral program. I also would like to express my appreciation to Dr. Orit Shechtman

and Dr. David Fuller for serving my dissertation committee and for their incredible

scientific advice.

It has been a pleasure and honor to work in Dr. Vandenborne's lab. My deepest

thanks go to the members in the lab. I am grateful for the assistance of Dr. Jennifer

Stevens. This project would not have been completed without her support. I owe great

thanks to Ye Li. Her assistance for developing new assay was instrumental in the

completion of this project. Special thanks go to Chris, Tiffany, Neeti, Arun, Prithvi,









Gabe, Fan, and Shiv. I am grateful to all for their help and friendship. Due to the presence

of all these wonderful people, work is more than enjoyable.

Finally and most importantly, I would like to express my deepest gratitude to my

family members. It is with much love that I acknowledge and deeply appreciate the

sacrifices and difficulties that my loving wife, Qing Yang, patiently endured. I also thank

my lovely daughter, Melodie, who made my graduate study colorful. I am indebted to my

father and sister for their relentless support, care and encouragement.
















TABLE OF CONTENTS


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

LIST OF TABLES .............. ................................ ................... .... .... x

LIST OF FIGURES ......... ............................... ........ ............ xi

A B S T R A C T .......................................... ..................................................x iii

CHAPTER

1 INTRODUCTION ............... .................................. ................... 1

2 REVIEW OF RELATED W ORK ........................................ .......................... 3

2.1 Spinal Cord Injury Anim al M odels ............................................. ............... 3
2.1.1 Spinal Cord Transection M odel......................................... ............... 3
2.1.2 Spinal Cord Isolation M odel ............................................. ............... 4
2.1.3 Spinal C ord H em section M odel ........................................ .....................5
2.1.4 Spinal Cord Contusion M odel ........................................... ............... 6
2.1.4.1 Early contusion m odels ........................................... ............... 6
2.1.4.2 Rat spinal contusion models............................ ................... 7
2.1.4.3 The New York university impactor........................................8
2 .1.5 Sum m ary .................................. .. ..................................... 9
2.2 Muscular Adaptations Following Spinal Cord Injury .......................................10
2.2.1 Muscle Atrophic Response Following SCI..............................................10
2.2.2 Force Mechanical Properties Following SCI ............................................10
2.2.3 Myosin Heavy Chain Expression Following SCI ....................................12
2.3 Treadmill Locomotor Training Following Spinal Cord Injury...........................16
2.3.1 The Recovery of Walking Ability Following SCI and Locomotor
Training .............................................. ........ ........ .. ......... ...............17
2.3.1.1 Locomotor recovery following SCI in animals............................ 17
2.3.1.2 The recovery of walking ability in individuals after SCI ...............18
2.3.2 The Impact of Locomotor (Treadmill or Cycling) Training on Skeletal
M uscle Following SCI ............................... ..... .. ................. 20
2.4 IGF-1 Signaling and M uscle Plasticity...................................... ...... ............... 22
2.4.1 IGF-I and Its Related Receptor and Binding Proteins.............................22
2.4.2 Protein Synthesis Induced by IGF-I/PI3K/Akt Pathway............................25
2.4.3 IGF-I/PI3K/Akt Pathway and Protein Degradation ................................26









2.4.4 Role of IGF-I in Satellite Cell Proliferation. ........ ....... ..... .............27

3 OUTLINE OF EXPERIMENTS ........................................ .......................... 29

3 .1 E x p e rim e n t 1 ................................................................................................... 2 9
3.1.1 Specific A im ................................................ .. ...... ................. 29
3.1.2 H ypotheses ...................................................................... ........................29
3 .2 E x p erim en t 2 ................................................................................................... 3 0
3 .2 .1 Sp ecifi c A im ............................................................... .................... .... 30
3 .2 .2 H y p o th e se s ........................................................................................... 3 0
3 .3 E x p e rim e n t 3 ................................................................................................... 3 1
3.3.1 Specific Aim ................... ........... .... ............... 31
3 .3 .2 H y p o th e se s ........................................................................................... 3 1
3 .4 E x p erim en t 4 ................................................................................................... 3 2
3.4.1 Specific Aim ................... ........... .... ............... 32
3 .4 .2 H y p o th e se s ........................................................................................... 3 2

4 A LONGITUDINAL STUDY OF SKELETAL MUSCLE FOLLOWING
SPINAL CORD INJURY AND LOCOMOTOR TRAINING ............... ...............33

4 .1 A b stra ct .......................................... ... ......................................... ..................... 3 3
4.2 Introduction....................................34
4 .3 M materials and M ethods ................................................................................... 36
4.3.1 E xperim mental A nim als ..................................................... ...... .... ... ..36
4.3.2 Spinal Cord Contusion Injury ................................................. 36
4.3.3 Treadmill and Cycling Locomotor Training ...........................................37
4.3.4 M magnetic Resonance Im aging .............................................. ......38
4.3.5 Open Field Locom otor Function ................................. ..........39
4.3.6 Statistical Procedures.......... ................................ .... .......................40
4 .4 R results ...................... .... ........ ... .. ..... .......................... ............. 40
4.4.1 Muscle Size After Spinal Cord Contusion Injury....................................40
4.4.2 Effect of Locomotor Training on Muscle Size............................ ......... 42
4.4.3 Relationship Between Hindlimb Muscle Size and Locomotor Function...44
4 .5 D iscu ssio n ........................... ..............................................................4 6

5 CHANGES IN SOLEUS MUSCLE FUNCTION AND FIBER MORPHOLOGY
WITH ONE WEEK OF LOCOMOTOR TRAINING IN SPINAL CORD
CON TU SION IN JU RED RA TS ....................................................................52

5 .1 A b stra c t ................... ...................................................................5 2
5.2 Introduction.................... .............. 53
5.3 M materials and M ethods ................................................................................... 55
5.3.1 E xperim mental A nim als......... ................................................................ 55
5.3.2 Open Field Locom otor Function ................................. ..........56
5.3.3 Spinal Cord Contusion Injury............................................56
5.3.4 Treadmill Locomotor Training .................. .........................................57
5.3.5 In situ Soleus Force M easurem ents ...........................................................58









5.3.6 Tissue H arvest .............................. .................. .. ............ ............. 59
5.3.7 Im m unohistolochem istry ................................................. ................. 60
5 .3 .8 D ata A n aly sis........... ...... .................................................. .. .... ..... .. 60
5.4 Results................................... ... ......... 61
5.4.1 BBB Open Field Locomotor Scores.......................................................61
5.4.2 Soleus Contractile Properties ........................................ ............... 62
5.4.3 Soleus Muscle Fiber Cross Sectional Area ............................................66
5 .5 D iscu ssio n ................................................... ................ 6 7

6 CHANGES IN SOLEUS FIBER TYPE COMPOSITION WITH ONE WEEK OF
TREADMILL LOCOMOTOR TRAINING IN SPINAL CORD CONTUSION
IN JU R E D R A T S .............................................................................. ................ .. 7 3

6 .1 A b stract ........................................................ ............................ 73
6 .2 Introdu action ............................ ...................................................... ............... 74
6 .3 M materials and M ethods .............................................................. .....................75
6.3.1 Experim ental Anim als .............. ... ................................ ............... 75
6.3.2 Spinal Cord Contusion Injury.................................. ...................... 75
6.3.3 Treadmill Locomotor Training................ ..... ............ .............76
6.3.4 Im m unohistolochem istry ................................................. ................. 77
6 .3 .5 D ata A n aly sis........... ...... .................................................. .. .... ..... .. 7 8
6 .4 R e su lts ............................................................................................................. 7 8
6.4.1 Soleus fiber type com position ........................................ .....................78
6.4.2 Soleus fiber cross-sectional area ..................................... ............... 80
6 .5 D iscu ssion ................................................... .................. 8 1

7 EFFECTS OF TREADMILL TRAINING ON IGF-I EXPRESSION IN RAT
SOLEUS MUSCLE FOLLOWING SPINAL CORD INJURY..............................85

7.1 Introduction........................................................................ ....... ...... 85
7 .2 M materials and M ethods .............................................................. .....................86
7.2 .1 E xperim ental A nim als ..................................................................... ...... 86
7.2.2 Spinal Cord Contusion Injury.................................. ...................... 86
7.2.3 Treadmill Locomotor Training.......................... ......................87
7.2.4 Tissue H arvest ................................. .......... .. ...............88
7.2.5 R T-PCR M easurem ent ........................................ .......... ............... 88
7.2.6 Im m unohistolochem istry ................................................. ................. 90
7 .2 .7 D ata A n aly sis........... ...... .................................................. .. .... ..... .. 90
7.3 R esults.................................................... ........ ........ .................91
7.3.1 mRNA Expression of IGF-I and Its Receptor and Binding Proteins .........91
7 .3 .2 E m bry onic M y osin ......................................................... .....................94
7.3.3 C central N uclei............ ......................................................... .... .... .... 94
7.4 D discussion ...................................................................... .. ....... ...... 95

8 C O N C L U SIO N ......... ...................................................................... ......... .. ..... .. 99









L IST O F R E FE R E N C E S ......................................................................... ................... 10 1

BIOGRAPHICAL SKETCH ............................................................. ..................122
















LIST OF TABLES


Table page

4-1. Maximal cross-sectional area of individual muscles..............................................51

6-1. M onoclonal antibody specificity ........................................ ......................... 78
















LIST OF FIGURES


Figure page

4-1. A representative trans-axial MR image of the rat lower hindlimb...........................39

4-2. Relative changes in the CSAmax of the tibialis anterior, triceps surae, extensor
digitorum, and flexor digitorum muscles following SCI.. .................................41

4-3. Relative change in total CSAmax in the no training group, cycling training group
and treadm ill training group......................................................... ............... 42

4-4. Relative change in the CSAmax of the triceps surae in the no training group,
cycling training group and treadmill training group.. ...........................................44

4-5. Relative change in the CSAmax of the tibialis anterior in the no training group,
cycling training group and treadmill training group.. ...........................................45

4-6. Relationship of rat locomotor function and total lower hindlimb CSA ...................45

5-1. Average BBB locomotor scores for the SCI + TM and SCI no TM group at 1
and 2 w weeks post SC I. ......................... ...... ................ ... .... .... .......... .....61

5-2. Soleus m uscle peak tetanic force.......................................................... ..... ......... 62

5-3. Soleus m uscle specific force.......................................................... ............... 63

5-4. Peak soleus tetanic force vs BBB score............................................. ........... 64

5-5. Soleu s m u scle fatigue e ...................................................................... ...................64

5-6. Soleus muscle force-time integral during a fatiguing protocol .............................65

5-7. Soleus m uscle peak tw itch force ........................................ ......................... 65

5-8. Soleus m uscle tim e to peak ............................................. ............................. 66

5-9. Soleus m uscle 1/2 relaxation tim e ..................................................... ... .......... 66

5-10. Average soleus muscle fiber CSA ................................. ...............67

6-1. Serial cross sections of a control soleus stained with monoclonal antibodies
directly against specific M HC isoform s........................................ ............... 79









6-2. Serial cross sections of a SCI no TM soleus stained with monoclonal antibodies
directly against specific M HC isoform s ....................................... ............... 79

6-3. Serial cross sections of a SCI + TM soleus stained with monoclonal antibodies
directly against specific M HC isoform s........................................ ............... 80

6-4. MHC based fiber type percentage composition. ................... ............................. 81

6-5. Soleus muscle fiber type specific CSA................... ............................................. 81

7-1. IGF-I mRNA expression relative to 18s in soleus muscle. ......................................92

7-2. MGF mRNA expression relative to 18s in soleus muscle................... ............92

7-3. IGF-R mRNA expression relative to 18s in soleus muscle................... ................93

7-4. IGF-BP4 mRNA expression relative to 18s in soleus muscle...............................93

7-5. IGF-BP5 mRNA expression relative to 18s in soleus muscle..............................93

7-6. Cross-section of soleus muscle stained with monoclonal antibody against
embryonic myosin isoform......................................... 94

7-7. Cross-section (hemotoxilin & eosin stained) of soleus muscle............................95















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

ADAPTATIONS IN SKELETAL MUSCLE FOLLOWING SPINAL CORD INJURY
AND LOCOMOTOR TRAINING

By

Min Liu

May 2006

Chair: Krista Vandenborne
Major Department: Rehabilitation Science

Spinal cord injury (SCI) is one of the most devastating human afflictions, which

leaves its victims paralyzed. Skeletal muscles distal to the injury site experience

significant muscle atrophy and loss of function, leading to impaired walking and motor

function. Recently, novel intervention therapies, focusing on repetitive locomotor

training, have shown great promise in promoting spinal plasticity and recovery in motor

function following SCI. Recovery of motor function after SCI likely requires both neural

and muscular adaptations. The major goal of this study was to investigate adaptations in

skeletal muscles following SCI and locomotor training. A combination of MR imaging,

in situ functional measurements, immunohistochemical assays and RT-PCR was

performed in an animal model of incomplete SCI. Our findings demonstrate that SCI

results in significant atrophy in all rat hindlimb muscles, and that locomotor training halts

the atrophic process and accelerates the rate of recovery. Additionally, our data suggest

that early therapeutic intervention using treadmill training significantly increases animal









locomotor function and soleus muscle size and function. Compared to untrained SCI

animals, one week of treadmill training results in a 32% improvement in BBB scores, a

38% increase in peak soleus tetanic force, a 9% decrease in muscle fatigue, larger muscle

fiber CSA (23%), and a reduced shift toward faster fiber types. Finally, our findings

demonstrate that treadmill training significantly increases mRNA levels for IGF-I, MGF,

IGFR, IGFBP4 and decreases IGFBP5 mRNA expression in the soleus muscle. In

addition, immunohistochemical analysis shows the increased presence of small fibers

expressing embryonic myosin, a hallmark of muscle regeneration, following treadmill

training. Taken together, the findings from the present work demonstrate that early

therapeutic intervention promotes muscular plasticity following SCI. We anticipate that

this study will provide essential feedback for the development of early rehabilitation

interventions in SCI individuals.














CHAPTER 1
INTRODUCTION

Spinal cord injury (SCI) is one of the most devastating human afflictions, which

leaves its victims paralyzed or with impaired motor control (57, 67, 107, 132, 245). It is

estimated that there are approximately 200,000 persons with SCI living in the United

States, with roughly 11,000 new cases occurring each year, making SCI a leading cause

of disability (200). Skeletal muscles distal to the injury site experience significant muscle

atrophy and loss of function, leading to impaired walking and motor function. The neural

responses to spinal cord injury vary from patient to patient, depending on the severity of

the injury. As a result of new developments in the acute management of spinal cord

injury, the majority of spinal cord injuries sustained are clinically incomplete.

Traditionally, orthotic and assistive devices have been used as compensatory

strategies to counter muscle weakness in SCI patients in an attempt to facilitate functional

walking. Successful mobility is often dependent on learning a new behavior requiring

either a wheelchair and/or bracing with assistive devices. More recently, locomotor

training has emerged as an alternative modality for retraining walking after incomplete

SCI and has revealed encouraging breakthroughs (27, 62, 64, 65, 105, 106, 234).

Improved gait speed, improved balance, less reliance on assistive devices and orthoses,

less physical assistance from caregivers, and improved functional performance have all

been documented with locomotor training (63, 235).

The purpose of this study was to investigate the impact of locomotor training on

skeletal muscle plasticity and muscle recovery as well as growth factors known to play a






2


role in muscle regeneration in a rat contusion SCI model. In addition, we set out to

determine the effect of locomotor training on muscle function in contusion spinal cord

injured animals and to perform a preliminary investigation of the relationship between

alterations in muscle size/function and locomotor behavior.














CHAPTER 2
REVIEW OF RELATED WORK

2.1 Spinal Cord Injury Animal Models

Animal models of SCI can be used to characterize the lesion development, study

the mechanism of recovery, develop therapeutic interventions, and also to study skeletal

muscle adaptations with decreased use (7, 18, 44, 59, 72, 73, 76, 79, 91, 98, 117, 118,

137, 138, 152, 155, 161, 170, 194, 200, 222, 248). Currently, the experimental SCI

animal models include the transaction, isolation, hemisection, and contusion model. Due

to differences in limb loading, neural activation and behavioral outcomes resulting from

each of these methods, it is important to compare the similarities and differences across

these models.

2.1.1 Spinal Cord Transection Model

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

ascending information between the caudal cord and the brain is mechanically eliminated

(83). In this case, SCI is created by an incision into the spinal cord. The spinal cord can

be either completely transected and left in place or a small section of the spinal cord can

be removed (83). Following transaction injury, there is an initial flaccid paraplegia stage

in which animals drag their limbs (135, 187). The animals are able to move using their

forelimbs, and have no difficulty reaching food and water. At approximately 3 to 4

weeks, the paralyzed hind limbs of the animals change from flaccid to spastic. After

spasticity develops, the limbs are almost always held in extension and no recovery of

voluntary activity is observed (135, 187). EMG recordings monitored over 24-hour









periods show a 75% decrease in the total integrated EMG and a 66% decrease in the total

duration of muscle activity in the SOL muscle, 5 to 6 months after transaction when

compared to control (7). Thus, in the spinal transaction model hind limb muscles

experience a significant reduction in both electrical activation and loading.

The complete transaction model has been used extensively to evaluate the

effectiveness of interventions with regard to both axonal regeneration and functional

recovery (83). The advantage of this model is a relative stabilization of pathological

changes and subsequent neurological outcomes (210). Therefore, the effectiveness of

particular strategies can be readily assessed (201). However, the transected spinal cord

model also has some disadvantages. First, due to the natural tension present in spinal

cords, the two ends of a cut cord will separate. Such a gap is rarely present in human SCI.

In addition, in order to cut the spinal cord, the dura has to be opened, allowing invasion

by external cells. Therefore, the spinal cord fails to demonstrate spontaneous spinal

regeneration (210). Finally, due in part to the advanced emergency care, the number of

spinal cord injuries classified as incomplete has risen dramatically (http://Ref-

www.spinalcord.uab.edu/). Thus, the transaction model, while valuable for certain

applications, may not be the best model to study the potential of rehabilitation

interventions to promote neuromuscular plasticity.

2.1.2 Spinal Cord Isolation Model

Spinal cord isolation has been referred to as the classic "silent preparation", which

was first described in dogs by Tower (228). In this preparation, the lumbar region of the

spinal cord is functionally isolated via complete spinal cord transactions at two sites. In

addition, all the dorsal roots are cut bilaterally between the two transaction sites (190).

Thus, this model eliminates supraspinal, infraspinal, and peripheral afferent input to









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

muscle fiber connections intact. The motoneurons within the isolated segment of the spinal

cord do not receive sensory input from the dorsal roots, or neural signals originating from

either above or below the two transaction sites. Based on 48 hours of continuous

intramuscular EMG recordings from a representative ankle extensor and a representative

ankle flexor, the muscles innervated by these motoneuron pools are essentially

electrically silent, even during passive manipulation (190). Acute recordings, during

tactile stimulation of the legs or feet, also showed virtually no EMG activity in a variety

of hindlimb muscles (75, 76, 209). Moreover, based on observations during cage activity,

it can be assumed that minimal forces are generated in these paralyzed muscles (187).

Therefore, spinal cord isolation represents one model that has been successfully used to

study the effects of a complete elimination of neuromuscular activity on muscle

properties (221).

2.1.3 Spinal Cord Hemisection Model

In partial transaction models, an attempt is made to cut tracts of the spinal cord

selectively. Depending on the severity of the lesion, the resulting neurologic deficit can

be relatively mild, thus making the postoperative animal care fairly easy, particularly

with regard to bladder function. Partial injury models also may allow for comparison of

the regenerative response in a particular tract with its uninjured partner on the

contralateral side (92).

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

the descending respiratory pathways and causing respiratory muscle paresis or paralysis.

Thus, this model has long been used to understand the mechanisms related to plasticity

and recovery of the respiratory pathways after spinal cord injury. Unfortunately, partial









injury models also suffer from difficulties determining whether observed functional

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

from other systems that are spared. For this reason, this model is not commonly used to

study hind limb muscle adaptations.

2.1.4 Spinal Cord Contusion Model

Most human spinal cord injuries result from fracture and dislocation of the spinal

cord column (88). Although penetrating wounds of the spinal cord can result from a knife

or gunshot, most human spinal cord injuries are caused by transient compression or

contusion of the spinal cord (39, 119, 125, 223). Even in the setting of complete

paraplegia after blunt injury, the cord rarely is completely transected, but rather leaves

some residual, normal-appearing cord parenchyma peripherally at the injury zone (39).

The first observation of human SCI neuropathology begins with an early phase of

spreading hemorrhagic necrosis and edema. Then it reaches an intermediate phase of

tissue repair and reorganization. Finally it ends up with a chronic phase characterized by

the formation of cystic cavities (108). These injury patterns are well simulated by spinal

cord contusion injury (37, 99, 100). Therefore, scientists have long used animal spinal

cord contusion models to study the pathophysiology of spinal cord injury and

regeneration (23, 35, 37, 44, 87, 99-101, 121, 131, 147, 166, 167, 182, 190, 235, 253). In

our investigation we utilize the contusion injury model and in the section below a brief

overview of the history of this model is provided.

2.1.4.1 Early contusion models

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

weight onto dog cords exposed by laminectomy (9). In 1914 (199), he reported that

midline myelotomy reduced progressive tissue damage in the contused spinal cord.









Unfortunately, Allen died in World War I and his work was discontinued for nearly 50

years.

In 1968, Albin and colleagues (8) revived the contusion model when they used a

primate spinal cord contusion model to assess the efficacy of hypothermic therapy

following SCI. After that, several investigators started using the canine spinal cord

contusion model again. Parker and colleagues (168, 169) assessed the effects of

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

time, Koozekanani and colleagues (128) examined the causes of variability in this model,

while Collmans and others (52) measured edema, blood flow and histopathological

changes in the contused dog spinal cord. In 1971, Osterholm and Mathews proposed that

catecholamine accumulation explains the progressive central hemorrhagic necrosis in the

contused cat spinal cord (165). Although subsequent studies did not confirm the predicted

spinal cord catecholamine changes, this was the first excitotoxic theory of neural injury

(109).

2.1.4.2 Rat spinal contusion models

In 1985, the Wrathall group (85, 163, 242) described morphological and behavioral

changes in a rat weight-drop contusion model. The weight-drop device was similar to that

used for the feline spinal cord contusion model, i.e., a weight dropped dorsally onto

thoracic spinal cord exposed by laminectomy. In addition, Wrathall and colleagues

developed a combined behavioral score to assess motor, sensory, and reflex recovery in

spinal injured rats, correlating these scores with quantitative axonal counts, neuronal and

glial loss, and evoked potentials (175). In 1987, Somerson and stokes described a

feedback controlled electromechanical device that indented the spinal cord at a defined

force, duration, and extent (206). This device has two levels of indentations that cause









mild or moderate injury, producing consistent 3D morphological and locomotor changes.

The device was subsequently used to assess the neuroprotective effects of MP and other

drugs (84).

2.1.4.3 The New York university impactor

The most commonly used rodent spinal cord contusion model is the New York

University (NYU) impactor, developed at the NYU Neurosurgery Laboratory and first

described by Gruner (98) in 1992. This model uses a weight-drop device that differs from

previous devices in several respects. First, the impactor dropped a steel rod directly onto

the spinal cord exposed by a laminectomy, achieving more consistent contusions. Second,

the device used digital optical potentiometers to measure the trajectory of the falling rod

with a precision of 20 um and 20 us, providing accurate measurements of the

delivered trauma. Third, the impactor measured movement of the spinal column at the

impact site, subtracting this movement from the cord movement. Fourth, the device was

designed to deliver three different levels of injury to rat spinal cord by dropping a 10-g

rod 12.5, 25.0, or 50.0 mm onto the spinal cord, respectively producing mild, moderate,

and severe injuries (98).

In 1993, NIH funded a Multicenter Animal Spinal Cord Injury trial (MASCIS), in

which a group of eight spinal cord injury laboratories validated and standardized the

Impact model (246). The group demonstrated that the impactor produced consistent

spinal cord injuries, reflected in a variety of measurements. This device allows for the

precise measurement of a number of biomechanical parameters including the impact

velocity of the rod, the distance of cord compression, the cord compression rate, and the

dynamic force applied to the cord (23). In addition, the MASCIS group standardized the

ages of the rats (77 1 day), anesthesia, and injury timing (60 5 min after anesthesia).









Using these guidelines, this device apparently can produce extremely consistent injuries

in terms of the resulting neuropathology. Finally, MASCIS validated the Basso-Beattie-

Bresnahan (BBB) locomotor score, a 21-point ordinal behavioral scale that linearly

predicts spinal cord histological changes (246).

The BBB locomotor rating scale was described by Basso, Beattie, and Bresnahan in

1995 (24) as a measure of motor performance in rats. With this 21-point scale, animals

achieving scores in the lower third (1 to 8) are capable of hindlimb joint movements

without weight support. Those rating in the middle part of the scale (9 to 13) demonstrate

varying degrees of hindlimb weight support and forelimb-hindlimb coordination, and

those achieving scores in the upper one third (14 to 21) show improvements in paw and

tail position, toe clearance, and trunk stability during a fully supported and coordinated

gait. The BBB scale has been shown to be a relatively reliable measure of locomotor

function and a sensitive reflection of the degree of tissue injury after spinal cord

contusion (23). Its fairly widespread use has been valuable for allowing the

communication and standardized comparison of results from different institutions (43,

142, 145).

2.1.5 Summary

Animal models have proved to be invaluable for the development of experimental

therapies, and undoubtedly will continue to play an essential role in the field of spinal

cord injury research. Models in which the spinal cord is sharply transected, either

completely or partially, are useful for studying the anatomic regeneration of axons,

whereas the contusion models better simulate the biomechanics and neuropathology of

human spinal cord injury. In order to study the effect of locomotor training on muscle









plasticity and recovery we selected to use a moderate mid-thoracic rat contusion injury

model.

2.2 Muscular Adaptations Following Spinal Cord Injury

2.2.1 Muscle Atrophy Following SCI

Following SCI, due to the reduced muscle activity and limb unloading, skeletal

muscles show a significant reduction in their size and mass (45, 46, 59, 65, 70, 72-74,

118, 137, 141, 150, 155, 174, 178, 194, 200, 201, 210, 212, 218, 248). In addition,

muscle atrophy is more pronounced in paralyzed muscles that normally bear weight,

especially those that cross single joints (148, 186, 237). These muscles often contain a

large proportion of slow fatigue-resistant muscle fibers and are largely responsible for

maintaining posture and bearing weight (112, 186). For example, the soleus muscle, a

postural muscle that extends the ankle, undergoes significant muscle atrophy following

SCI. In contrast, the tibialis anterior muscle, which flexes the ankle and does not

normally contract against a high load, atrophies considerably less in a number of species,

including humans (148). The medial gastrocnemius muscle, which crosses both the knee

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

as a synergist to the soleus muscle during plantar flexion (186).

2.2.2 Force Mechanical Properties Following SCI

Neural activity is very important to determine the mechanical properties of a

skeletal muscle. In 1960, Buller et al. (38) first reported that cross-reinnervation of the cat

slow soleus muscle with the nerve of the fast flexor digitorum longus resulted in

incomplete conversion of the isometric twitch properties from slow to fast. Similarly,

cross-reinnervation of the fast flexor digitorum longus muscle with the nerve of the slow

soleus muscle led to incomplete conversion of the isometric twitch properties from fast to









slow (38). These findings demonstrate that the pattern or amount of motoneuron

activation strongly influences the muscle properties.

Following SCI, animal hindlimb muscles show pronounced decrease in maximal

force-generating capacity and the acquisition of faster mechanical properties (185, 191-

193, 215, 217, 220, 221). In these studies, muscle force mechanical properties were

determined using an in situ set up. Briefly, the animal leg was securely positioned in an

apparatus such that the muscle was in a near-horizontal plane. The muscle distal tendon

was cut and then attached to a force transducer. The muscles were stimulated via bipolar

silver electrodes placed on the transected tibial nerve. They found that the maximum

isometric twitch and tetanic tension of the cat soleus muscle were reduced by 38 and

39%, respectively, -10 mo after spinal cord transaction (185, 191). In these same cats,

the time to peak tension and half-relaxation times were 41 and 50% shorter, when

compared to control cats (185). The changes in force generating capacity and contractile

speed were accompanied by a rightward shift in the force-frequency relationship and a

small, but significant, decrease in fatigue resistance (193). The adaptations in the

mechanical properties of the cat medial gastrocnemius, a fast ankle extensor, were less

pronounced than that in the soleus, in both young and adult animals (185, 191).

Presently, rat is the most prevalent animal model used to assess the effects of SCI

and to determine the potential beneficial influence of rehabilitative procedures in the

recovery of function after SCI (60, 69-71, 115). In general, the adaptations in the

mechanical properties of the rat soleus after a complete spinal transaction are relatively

similar to those reported for cat muscles, although the magnitude of the effects appears

somewhat greater in cats than rats (194, 214). Talmadge and his colleagues (214) showed









that in rats maximal tetanic force is reduced by -44% at 3 months post spinal cord

transaction. In addition, spinal cord transaction resulted in faster twitch properties as

evidenced by a shorter time to peak tension (-45%) and half-relaxation time (-55%). In

addition, a significant reduction in fatigue resistance of the soleus was observed (214).

The adaptations in the mechanical properties of muscle following SCI are similar to

those observed in other models of reduced neuromuscular activity. For example, chronic

muscle unloading, via either hindlimb suspension or actual space flight, results in

significant reductions in tetanic force, and twitch contraction times of the rat soleus (41,

42, 219). Most unloading and spaceflight studies have also reported a decrease in half-

relaxation time and an increase in Vmax (41, 42, 239). The similarity in the adaptations in

SCI and chronic unloading models further indicates the role of loading and

neuromuscular activity in mediating muscle adaptations following spinal cord injury.

2.2.3 Myosin Heavy Chain Expression Following SCI

Myosin is one of the molecules that regulates contractile speed in mammalian

skeletal muscle and is highly correlated with the myofibrillar ATPase histochemical

identification of specific muscle fiber types (216, 217). It is a hexamer composed of two

heavy and four light chains (197). To date, four different myosin heavy chain (MHC)

isoforms have been identified in varying proportions in the hindlimb muscles of rats.

These have been identified as a slow isoform called MHC-I and three fast isoforms called

MHC-IIa, MHC-IIx, and MHC-IIb (224). A number of studies have closely linked the

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

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

containing a given isoform in the order of (fastest to slowest) IIb > IIx > IIa > I (36).









The complement of MHC isoforms expressed by mammalian hindlimb muscles can

be regulated by conditions that alter the levels of neuromuscular activation (defined as

electrical activation and load-bearing) of a muscle (97, 170, 187, 193, 217). Interventions

that result in increased neuromuscular activation, such as chronic electrical stimulation,

functional overload and endurance exercise, result in MHC shifts towards the "slower"

isoforms. In contrast, factors that reduce neuromuscular activation, including space flight,

hindlimb suspension, and spinal cord injury, result in MHC shifts towards the "faster"

isoforms (222).

With a spinal cord injury there is no direct damage to the muscle, and innervation

of muscles is not physically disrupted; therefore, the interruption of transfer of electrical

activity through the motoneurons likely stimulates the changes that are observed (136,

171). Spinal motoneurons caudal to a lesion site lose neural input from descending brain

stem pathways, which influences their tonic and phasic firing patterns and consequently

affects muscle contractile and metabolic activity. Studies involving cross-reinnervation of

muscles have further defined the influence of motoneuron input on the structural and

functional characteristics of a muscle (188).

Myosin type adaptations following spinal cord transaction. Almost all muscles

studied show an increase in the percentage of fast fibers and a decrease in the percentage

of slow fibers following spinal cord transaction (15, 114, 121, 137, 147, 166, 213, 217,

222). In cat soleus muscle, as many as 50% of the fibers react with only a fast MHC, and

a small percentage of the fibers react with both a fast and a slow MHC antibody

following transaction (121, 122). In contrast, control adult soleus muscle does not appear

to express the fast MHC (121, 122, 215). The fast fibers post-SCI tended to be found









disproportionately at the boundaries of the fascicles, suggesting that myogenic spatial

factors had influenced the ATPase conversion of the fibers. Roy et al (186, 192) also

showed that cat fast muscle tibialiss anterior) also shows an increase in the fast fiber

proportion and MHC-IIx expression after 6 months of spinal cord transaction, but the

increases are less prominent than in the soleus.

Rodents show a higher degree of MHC isoform transformation after spinal

transaction than cats. The proportion of MHC-I in the rat soleus is reduced from -90% in

controls to -25% only 3 months following a complete mid-thoracic spinal cord

transaction (222). The MHC-IIx, which is normally not found in the rat soleus, increased

to nearly 50% and that of MHC-IIa to -30% 6 months after spinal transaction (217, 222).

Myosin type adaptations following spinal cord isolation. In cats, spinal isolation

also has been shown to result in an increase in the percentage of fibers labeled by

antibodies specific for fast MHC in the cats soleus and tibialis anterior (93). Based on

mATPase staining at an alkaline preincubation, Graham (93) found 64% fast fibers in

regions sampled in the soleus of control cats and 100% in similar regions in the spinal-

isolated cats. In addition, essentially all fibers in the MG of the spinal isolated cats

reacted exclusively with a fast MHC antibody (93). These fiber type data are consistent

with the observation that the myosin ATPase activities in spinal isolated cats are 85% and

30% higher in the SOL and MG, respectively, 6 months after spinal isolation (123). In

rats, spinal isolation also resulted in a slow-to-fast shift in the myosin isozyme pattern

(97). Similar to spinal transaction, the MHC isoform that is primarily up-regulated at the

expense of MHC-I is the MHC-IIx isoform (97).









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

isolation has been shown to be greater than that observed for spinal transaction alone. For

example, 6 months following spinal transaction the cat soleus contains only 67% MHC-I

fibers (215) compared to 48% after spinal isolation (93). Thus, the magnitude of the

adaptations observed following spinal isolation is greater than that observed after spinal

transaction. This suggests that the residual amount of electrical activation in the cat

soleus after spinal transaction plays a role in maintaining the levels of MHC-I expression.

Myosin type adaptation following spinal cord contusion injury. Hutchinson

(116) found that there was no change in the relative percent of MHC expression at 1

week post spinal cord contusion injury. However, at the 3 week time point, there was an

upregulation of the transitional IIx heavy chain (116). Although the increased expression

of IIx MHC after SCI was not statistically significant, it appears to have been sufficient to

induce effects of biological significance (speeding up of 12 RT). That such a modest

increase in IIx MHC profiles could produce significant physiological differences

indicates the tight regulatory control MHC phenotype imparts on contractile speed.

Skeletal muscle MHC expression in individuals with SCI. A few studies have

directly assessed the effects of SCI on MHC isoform expression. In 1999, Castro (45)

reported that there is very little adaptation in MHC isofrom expression in the vastus

lateralis muscle during the first several months after complete SCI in patients. This study

investigated vastus lateralis biopsy samples in 12 patients as soon as they were clinically

stable (45). No significant slow-to-fast changes in MHC isoform composition of the

vastus lateralis were found at any time point (6, 11, 24 weeks post SCI), and the mean

proportions of each of the MHC isoforms were comparable to non-disabled control









subjects. However, there was an apparent conversion among type II fibers, with a

significant MHC-IIa to MHC-IIx shift, in the vastus lateralis at 24 weeks post SCI. Thus,

at 24-week after injury there was an elevation in the proportion of MHC-IIx, the fastest of

the human limb MHC isoforms. These data are consistent with a previous study showing

that in long term SCI subjects (3-20 years post SCI), the vastus lateralis muscle contained

a higher percentage of MHC-IIx or in combination with MHC-IIa (11).

Histochemical data support the hypothesis that slow to fast MHC isoform

transformations occur in human muscle after SCI, even though to less of an extent than

observed in animal models. For instance, the proportion of type lib fibers was elevated

and type I fibers decreased in the tibialis anterior and VL of SCI subjects (95, 148). The

decrease in the proportion of histochemically identified type I fibers appeared to be

related to the duration of time following the SCI injury. In fact, Round (184) found that

there was no decrease in the proportion of type I fibers in 2 individuals who had suffered

a SCI less than 15 months prior to biopsy of the VL. In contrast, in those individuals that

had suffered a SCI 3 years or more prior to biopsy, the proportion of type I fibers

decreased relative to non-injured control values (184). These data also highlight that the

change in fiber type composition in human skeletal muscle occurs slower, a phenomina

that has also been observed in other models of unloading (184).

2.3 Treadmill Locomotor Training Following Spinal Cord Injury

Motor recovery following spinal cord injury can be enhanced or accelerated by

repetitive locomotor training (26, 27, 64, 86, 174, 233-235, 240, 241). The most

frequently used locomotor training is treadmill training. Treadmill training is performed

using a body weight support and manual assistance for stepping. The underlying premise

of locomotor training relates to the hypothesis that rhythmic loading of the limbs and









force feedback from the hindlimb muscles induces task appropriate activity-dependent

plasticity. The following several paragraphs review the effects of locomotor training on

functional recovery in both SCI animals and subjects.

2.3.1 The Recovery of Walking Ability Following SCI and Locomotor Training

2.3.1.1 Locomotor recovery following SCI in animals

Experimental studies have shown that locomotor training of SCI animals can

effectively improve the ability to step on a treadmill in cats following complete SCI (18,

54, 58, 59, 113, 140). Over the last ten years or so, the Edgerton group has published

several papers to address this issue. For example, de leon (58) characterized the effects of

training by comparing groups of untrained with trained spinal cats and reported that step-

trained spinal cord cats can reach considerably higher speeds and make more consecutive

steps. Treadmill training also improved interlimb coupling, overall limb excursion and

joint excursion significantly (59). In addition, Edgerton's group demonstrated a certain

task-specificity in the improvement of locomotor function. Therefore, spinal cats trained

to stand were not able to walk. More importantly, they claimed that the spinal cord can

'learn' or 'store' memories of simple motor responses that are acquired through

conditioning (59). For instance, the acquired motor function can last up to six weeks

followed by a graduate decay of locomotor ability (59). This reduction can be reinstated

within one week of training.

However, studies using rats with incomplete SCI are scarcer and have provided

contradictory results even if it appears clinically and pathophysiologically relevant.

Fouad et al. (81) found no beneficial effect on free locomotor activity after a very partial lesion

of the cord, whereas Thota et al. (227) reported improved functional recovery after 7 weeks

treadmill training in rats with partial spinal cord lesion. However, Fouad (81) proposed a









possible explanation for the absence of observed benefits in their study. They noted that

animals in their study had a high level of motor function and were very mobile after SCI,

limiting the potential beneficial effects of the treadmill training. Recently, a group in

Belgium (158) studied the effect of treadmill training on motor recovery in adult rats after

severe contusion injury induced by balloon inflation. Multon et al. (158) reported that early

treadmill training improved locomotor recovery and showed that the beneficial effect was

observable after only 2 week, with a maximal effect at 4 weeks. As a result, severely

contused injured rats could support their body weight after 12 weeks of treadmill training,

while non-trained rats could not (158).

2.3.1.2 The recovery of walking ability in individuals after SCI.

Based on animal research, the interactive approach using treadmill and body weight

support system for locomotor training has been extended to human individuals and

several studies have yielded positive reports (18, 26, 27, 64, 80, 89, 110, 233-235). In

1987, Barbeau and colleagues (18) first reported suspending a human over a treadmill to

assess the feasibility for walking retraining. During locomotor training, SCI individuals

were suspended over a treadmill in a harness and an overhead support system. In

addition, Behrman (27) emphasized the following training principles: 1) generating

stepping speeds approximating normal walking speeds (0.75-1.25 m/s); 2) providing the

maximum sustainable load on the stance limb; 3) maintaining an upright and extended

trunk and head; 4) approximating normal hip, knee, and ankle kinematics for walking; 5)

synchronizing timing of extension of the hip in stance and unloading of limb with

simultaneous loading of the contralateral limb; 6) avoiding weight bearing on the arms

and facilitating reciprocal arm swing; 7) facilitating symmetrical interlimb coordination;

and 8) minimizing sensory stimulation that would conflict with sensory information









associated with locomotion. Each of these principles is designed to maximize sensory

stimulation that matches the kinetic and kinematic properties associated with phases of

stepping. Therefore, this system provides an environment in which one can facilitate

balance control and manually assist trunk and leg movement during weight-bearing

stepping.

In 1992, Wemig and Muller reported that patients with varying degrees of paralysis

increased their overground speed following treadmill training with BWS (233). In

addition, subjects with unilateral limb paralysis were able to walk a short distance

without using knee-stabilizing braces and could negotiate stairs with the help of cane and

handrails. In 1993, Barbeau and colleagues (17) reported an increase of overground

walking speed and endurance in nine subjects with incomplete SCI after walking on the

treadmill with BWS. Moreover, subjects walked with a more normal gait pattern and

lower limb muscle EMG profiles (17).

Subsequently, Wernig (234) did a comprehensive study of the efficacy of

locomotor training in 44 chronic and 45 acute clinically incomplete SCI subjects. In

addition, those subjects were compared with 64 patients (24 chronic and 40 acute) treated

conventionally. Following locomotor training, 25 subjects learned to walk independently

and 7 patients could walk with assistance. In addition, those patients who could already

walk before therapy improved their walking speed and endurance. By comparison, there

were much fewer people improving their walking ability in the conventionally treated

group. In a follow-up study, it was reported that all subjects retained and in some cases

further improved their ability to walk over ground (236). Since then, this intervention has









been widely applied in the incomplete SCI subjects (18, 26, 27, 64, 80, 89, 110, 233-

235).

Unfortunately, to date, there has not been a similar training effect on overground

walking in humans with clinically complete SCI. However, treadmill training can

improve several aspects of walking on a treadmill. For example, Dietz et al. (36, 62) reported

that, after several weeks of treadmill training, the levels of weight bearing significantly

increased during training. In addition, when stepping on a treadmill with BWS, rhythmic

leg muscle activation patterns can be elicited in clinically complete subjects who are

otherwise unable to voluntarily produce muscle activity in their legs (144). Another study

has demonstrated that the levels of leg extensor muscle activity recorded in clinically

complete SCI subjects significantly improved over the course of several weeks of step

training (240). Thus clinically complete SCI subjects can improve their stepping ability

by locomotor training, but the level of improvement has not reached a level that allows

them to walk without assistance.

2.3.2 The Impact of Locomotor (Treadmill or Cycling) Training on Skeletal Muscle
Following SCI

Recovery of locomotor function after spinal cord injury likely requires neural

plasticity as well as the maintenance or restoration of skeletal muscle. Some of the

adaptations observed in the muscles following SCI are effectively ameliorated by

inducing full weight-bearing in spinalized animals while they are stepping on a treadmill

(112, 173, 189). In spinal cats trained to step, the atrophic response of the muscles with a

high proportion of slow fibers, i.e., the SOL, is significantly reduced (186). The largest

effect has been shown in the soleus muscle, the extensors at the ankle and knee which is

expected to be highly recruited during treadmill exercise (112, 173, 189).









Compared with treadmill training, however, cycling exercise is less frequently used

and studied. The cycling training consists of a circular pedaling motion, which flexes one

limb while extending the other. The animal is suspended in the harness, and the hindlimb

feet are strapped onto the pedals of the motorized cycle, which is controlled by a speed

controller (115, 203). Houle (115) studied the hind limbs of spinalized rats by daily

cycling exercise for relatively long duration (60 min) at low intensity (45 rotations per

minutes, 30 minutes per trials, two trials per day, 5 days per week); that is, there was no

apparent load on the muscle. He found that 3 months cycling exercise diminished the

extent of atrophy that was observed without exercise (115).

In addition, treadmill training has been demonstrated to prevent conversion of SOL

myofibers from slow oxidative to fast glycolytic properties in spinalized cats. For

example, Roy et al. (192) trained the adult spinal cord transected cats on a treadmill for around

5 months and found that step trained cats showed 100% type I MHC in soleus. In

contrast, cycling did not show a similar effect. Houle et al. (115) reported that cycling exercise

did not prevent or reverse the observed changes in muscle fiber type following SCI. The

expression of primarily fast MHC isoforms persisted in soleus muscle, and in fact, there

was an exaggerated decrease in the expression of type I MHC following 3 months of

cycling exercise.

Treadmill training has also been shown to have a positive effect on muscle

function. Lovely and his colleagues (141) found that 5 months treadmill training

significantly improve hindlimb muscle tetanic force and specific tension. The magnitude

of the forces produced at the soleus tendon while stepping on the treadmill during the last

2 weeks of experiment were close to normal levels (141). In addition, Roy et al. (192) studied









the mechanical and biochemical response of a slow extensor (SOL) and fast extensor

muscle (MG) in the adult cat hindlimb following a complete low-thoracic spinal cord

transaction, with and without a daily short bout of stepping training on treadmill. He

found that step training had no effect on the adaptations in the contractile properties of

the MG associated with chronic spinal transaction. However, step training had a positive

effect in maintaining force in the soleus muscle.

2.4 IGF-1 Signaling and Muscle Plasticity

IGF-I has been shown to play an important role in muscle regeneration following

injury (4, 5, 16, 127, 154, 195, 204), muscle hypertrophy (51, 90, 160). Systemic release

of IGF-I may contribute to an increase in protein content and a reduction in protein

degradation in skeletal muscle (247). Incubation of muscle cells with IGF-I (1 MicroM)

for 3-7 day stimulates both cell hyperplasia and myofiber hypertrophy (230). In addition,

over-expression of IGF-I using a muscle specific promoter has been associated with

myofiber hypertrophy in transgenic mice (51), and local infusion of IGF-I has been

shown to contribute to skeletal muscle hypertrophy (1), as well as block the aging-related

loss of muscle mass in mice (19). Moreover, numerous in vivo activity models, such as

increased loading, stretch and eccentric contraction are known to result in increases of

IGF-I peptide and IGF-I mRNA expression in skeletal muscle (4, 5, 16, 127, 195, 204).

Resistance training also has been associated with increased IGF-I mRNA expression in

both animal models (3) and individuals with complete spinal cord injuries (29).

2.4.1 IGF-I and Its Related Receptor and Binding Proteins

IGF-I: mature IGF-I is a 70-amino acid single-chained polypeptide with many

three-dimensional structural similarities to the proinsulin (see Review (202)). IGF-I is

synthesized in the liver as a consequence of growth hormone. Systemic IGF-I produced









in the liver promotes cell division and is generally responsible for normal growth and

development (157). More recently, it has been recognized that IGF-I is also produced

locally by skeletal muscle in a growth hormone independent manner (244). There is

significant evidence that locally produced, autocrine and/or paracrine IGF-I, is important

for muscle regeneration and hypertrophy (4, 5, 16, 127, 195, 204). Specifically, Jennische

(120) showed IGF-I immunoreactvity in the cytoplasm of myoblasts and myotubes and in

satellite cells during muscle regeneration. LeFaucheur (134) further showed that

antibodies that neutralized IGF-I reduced the number and size of regenerating myofibers

after muscle damage. Experimental manipulation of muscle of IGF-1 levels, in the

absence of changes in loading state, also has been shown to induce muscle hypertrophy.

For example, transgenic mice in which IGF-I is over-expressed using a muscle specific

promoter undergo hypertrophy (51, 160). In addition, direct infusion (1) of IGF-I in

muscle results in hypertrophy, whereas inhibition of IGF-I function can prevent this

response (32). Over-expression of IGF-I in muscle has also been shown to prevent some

of the age-related decline in muscle mass (19).

Muscle specific isoforms and mechano-growth factor (MGF): IGF-I has different

isoforms by alternative splicing. One of them is only detectable following injury and/or

mechanical activity. Yang (244) cloned the cDNA of a splice variant of IGF-I that is

produced by active muscle and that seems to be the factor that controls local repair,

maintenance, and remodeling. This protein has been called mechano growth factor

(MGF). Because of a reading-frame shift, MGF has a different 3' sequence and a

different mode of action compared with systemic or liver IGF-I. Although MGF has been

called a growth factor, it may be regulated as a local repair factor (244). In addition,









MGF has been shown to be significantly upregulated in response to stretch and increased

loading (151, 167). Yang (244) showed that MGF was markedly upregulated in rabbit

extensor digitorum longus muscle, which had been subjected to acute stretch by

immobilizing the hindlimb in the extended position. This work was further supported in a

study in which mRNA expression of the muscle-specific isoforms of IGF-I was up-

regulated in rabbit muscle after electrical stimulation at 10 Hz for 4 days (151). It appears

that the expression of both systemic and autocrine IGF-I in muscle provides an

interesting link between the mechanical signal and tissue remodeling and repair.

IGF-I receptor (IGF-IR): the cellular effects of IGF-I are mediated by the activation

of a specific receptor, which has two binding alpha subunits and two transmembrane beta

subunits (101). It is known that the biological activity of growth factor depends on the

concentration of the receptor and the affinity of its interaction (238). It has been shown

that there is a change in IGF-IR mRNA expression during muscle inactivity (102, 103)

and resistance exercise (101). Willis (238) also reported an increase of IGF-IR density in

aged skeletal muscle, suggesting that animals retain plasticity for IGF-IR.

IGF binding protein (IGFBP): the interaction between IGFs and their binding

proteins represents prereceptor regulation. So far, six IGFBP were identified. Their

functions are: 1) stabilize and transport IGFs from the circulation to peripheral tissues, 2)

maintain a reservoir of IGFs in the circulation, 3) potentiate or inhibit IGF function, and

4) mediate IGF-independent biological effects. Of the six systemic-type binding proteins,

IGFBP-3 is primarily responsible for maintaining IGF-1 levels in the circulation in

conjunction with another protein called acid labile subunit (82, 207). In contrast, IGFBP-

4 and IGFBP-5 are located in skeletal muscle (207). There is some evidence that









alterations in IGFBP expression occur during unloading or overloading and that IGFBPs

play a role in skeletal muscle adaptation (14, 101-103).

After binding with the receptor, the IGFR subsequently recruits the insulin receptor

substrate, which results in the activation of two signaling pathways: the Ras-Raf-MEK-

ERK pathway and the PI3K-Akt pathway (53). The Ras-Raf-MEK-ERK pathway is

crucial for cell proliferation and cell survival in mitosis-competent cells (152). However,

in adult skeletal muscle, the function of the Ras-Raf-MEK-ERK pathway is less clear. By

contrast, activation of PI3K is thought to play an important role in inducing skeletal

muscle hypertrophy (159).

2.4.2 Protein Synthesis Induced by IGF-I/PI3K/Akt Pathway

The binding of IGF-I to its receptor induces a conformational change in the IGF-I

receptor tyrosine kinase, resulting in the activation of several intracellular kinases,

including phosphatidylinositol-3-kinase (PI3K) (180). PI3K is a lipid kinase, which

phosphorylates phosphatidylinositol-4-5-bisphosphate to phosphatidylinositol -3-4-5-

trisphosphate (PtdIns(3,4,5)P3). PtdIns(3,4,5)P3 provides a binding site for

serine/threonine kinase Akt. After translocation to the membrane, Akt is phosphorylated

and activated by phosphoinositide-dependent protein kinase (PDK).

Akt plays a very important role in mediating muscle hypertrophy. Recently, it has

been demonstrated that activation of Akt is sufficient to induce hypertrophy in vivo

(131). Lai (131) showed that acute activation of Akt induces dramatic increases (> 2

folds) in the size of skeletal muscle in a transgenic mouse in which a constitutively active

form of Akt can be inducibly expressed in adult skeletal muscle. In addition, skeletal

muscle atrophy is coupled with a decreased ability to activate the Akt pathway in burned









rats (211) and expression of active form of Akt in skeletal muscle cells is sufficient to

cause hypertrophy in normal mouse gastrocnemius muscle (212).

Akt can phosphorylate mammalian target of rapamycin (mTOR) directly and

indirectly by inhibition of Tscl-Tsc2 complex (117). In turn, activation of mTOR results

in an increase in protein translation by two mechanisms: first, mTOR activates p70S6k

(153, 164), a positive regulator of protein translation; second, mTOR inhibits the

activation of PHAS-1, a negative regulator of the protein initiation factor eIF-4E. In

addition, glycogen synthesis kinase (GSK) is another substrate of Akt that has been

shown to play an important role in mediating hypertrophy. GSK is inhibited following

phosphorylation of Akt and inhibition of GSK is sufficient to stimulate myogenic

differentiation (56). In addition, expression of a kinase-inactive form of GSK induces

dramatic hypertrophy in skeletal myotubes (180) and blocks protein translation initiated

by the eIF2B protein (104).

2.4.3 IGF-I/PI3K/Akt Pathway and Protein Degradation

Even though it is difficult to identify the muscle specific mediators of atrophy, a

search for markers of the atrophy suggests that two genes are up-regulated in multiple

models of skeletal muscle atrophy. They are called MuRF1 (32) and MAFbx (91). Both

genes encode ubiquitin ligases (E3), proteins that mediate ubiquitination of specific

substrates. MuRF has recently been shown to be able to induce the ubiquitination of the

cardiac form of TroponinI and possibly the myofibrillar protein titin at the M line (126).

MAFbx is suggested to bind to substrates including MyoD and calcineurin (91).

Recently, studies have shown that the upregulation of MAFbx and MuRF is

antagonized by treatment of IGF-I (196). The mechanism is focused on the function of

FOXO. The phosphorylation of Akt can inhibit the FOXO (133), whose activation is









required for the upregulation of MuRF and MAFbx (196). Thus, the activation of Akt

can block the upregulation of MuRF and MAFbx by inhibition of FOXO.

2.4.4 Role of IGF-I in Satellite Cell Proliferation.

Satellite cells are quiescent muscle precursor cell in adult skeletal muscle. They are

located under the basal lamina of the muscle fiber, but separate from the muscle fiber

itself. Satellite cells are the main, if not only, cell type that contributes to muscle

regeneration (30, 31).

During muscle hypertrophy, there appears to be a myogenic response where

satellite cell-derived myoblasts are thought to fuse with existing myofibers (49, 205). The

importance of this response stems from the observations that mature mammalian skeletal

muscle fibers appear to maintain a relatively finite relationship between the size of the

myofiber and the number of myonuclei present in a given myofiber (10, 111, 150, 219).

However, mammalian myofibers become permanently differentiated shortly after birth

and cannot undergo mitotic division or directly increase their myonuclear number (47).

The requirement for additional nuclei to support hypertrophy appears to be met via the

proliferation, differentiation, and finally the fusion of muscle satellite cells or their

progeny with the enlarging myofibers, providing the new myonuclei needed to support

the hypertrophy process (10, 172, 181, 182). Among the well-characterized growth

factors, IGF-I is the only one that has been consistently reported to facilitate each of these

processes.

Increasing evidence indicates that IGF-I stimulates both myoblast proliferation and

differentiation (78). IGF-I acts via the mitogen-activated protein (MAP) kinase pathway,

activating the expression of the cell cycle progression markers, such as cycling D, cdk4,

c-fos, and c-jun (78, 124). After withdrawal of the Akt pathway, IGF-I subsequently









modulates expression of terminal muscle differentiation markers, such as p21, MyoD,

myocyte enhancer factor 2 (MEF2) and myogenin (161).

However, although it is not clear what role the PI3K pathway plays in

differentiation, recent evidence demonstrates a key role for the PI3K pathway in primary

satellite cell proliferation (46). Chakravarthy et al. (46) demonstrated that IGF-I-

stimulated proliferation of primary satellite cells isolated from transgenic mice

overexpressing IGF-1 is associated with the activation of the PI3K/Akt signalling

pathway and the downregulation of the cell-cycle inhibitor p27Kipl (46). In addition,

ectopic expression of p27Kip 1 has been shown to block the IGF-I-induced increase in

satellite cell proliferation (46). Furthermore, Machida et al. (143) recently reported that

IGF-I represses p27Kip 1 transcriptional activity through phosphorylation of Akt. Thus,

p27Kipl has been proposed to be a key regulatory factor, particularly in its ability to

regulate satellite cell cycle progression.














CHAPTER 3
OUTLINE OF EXPERIMENTS

The purpose of this study was to investigate locomotor training impacts on

muscular adaptations following incomplete SCI. An outline of the experiments performed

is provided in the section below:

3.1 Experiment 1

3.1.1 Specific Aim

To quantify changes in rat hindlimb muscle cross-sectional area following

moderate T8 spinal cord contusion injury and 3 months of locomotor training (treadmill

vs. cycling).

Animals (n=8/group) were assigned to either a treadmill training group, cycle

training group or a SCI (no training) group. Moderate spinal cord contusion injuries were

produced using a standard NYU (New York University) impactor. Animals assigned to

either training group were trained continuously for 3 months (5 days/week, 2 trials/day,

20 minutes/trial), starting on post-operative day 8. Non-invasive 3D magnetic resonance

(MR) images were collected from the lower hindlimb muscle at pre-injury as well as at 1,

2, 4, 8, and 12 weeks post injury. Based on the MR images, the in vivo maximal muscle

cross-sectional area of the tibialis anterior, triceps surae, extensor digitorums and flexor

digitorums were determined.

3.1.2 Hypotheses

a) Following moderate T8 spinal cord contusion injury, rat hindlimb muscles show

an acute decrease in maximal CSA, followed by spontaneous recovery.









b) Following moderate T8 spinal cord contusion injury, hindlimb extensor muscles

show a greater decrease in maximal CSA than hindlimb flexor muscles.

c) Both cycling and treadmill locomotor training attenuate the decrease of muscle

CSA following T8 spinal cord contusion injury and facilitate the recovery of muscle

CSA.

3.2 Experiment 2

3.2.1 Specific Aim

a) To determine the impact of moderate T8 spinal cord contusion injury on the rat

soleus morphology (fiber CSA) and in situ contractile properties. b) To determine the

effect of 1-week treadmill locomotor training on the rat soleus morphology (fiber CSA)

and in situ contractile properties following T8 spinal cord contusion injury.

Adult female rats (n=8 rats; 16 muscles/group) were assigned to either a SCI-

treadmill training group, a SCI-no training group, or a control group. Animals assigned to

the training group were trained continuously for Iweek (5 days/week, 2 trials/day, 20

minutes/trial), starting on post-operative day 8. Morphological and contractile properties

of the predominantly slow twitch soleus muscle were assessed at 2 weeks post-injury.

Specifically, we measured the soleus fiber CSA, in situ isometric force, twitch properties

and fatigability.

3.2.2 Hypotheses

a) Two weeks after moderate T8 spinal cord contusion injury, rat soleus muscle

experiences a significant decrease in muscle fiber CSA and in situ isometric force

production, compared to the normal control rat soleus. In addition, the rat soleus muscle

is more fatigable compared to the normal control soleus.









b) One-week treadmill locomotor training attenuates the decrease in rat soleus fiber

CSA and loss in in situ isometric force production observed following moderate T8

spinal cord contusion injury. In addition, the rat soleus muscle is less fatigable following

1-week treadmill training, compared to the soleus muscle in the SCI-no training group.

3.3 Experiment 3

3.3.1 Specific Aim

a) To determine the impact of moderate T8 spinal cord contusion injury on the rat

soleus fiber type composition. b) To determine the effect of 1-week treadmill locomotor

training on rat soleus fiber type composition following moderate T8 spinal cord contusion

injury.

Adult female rats (n=12 rats; 24 muscles/group) were assigned to either a SCI-

treadmill training group, a SCI-no training group, or a control group. Animals assigned to

the training group were trained continuously for Iweek (5 days/week, 2 trials/day, 20

minutes/trial), starting on post-operative day 8. Fiber type composition of the

predominantly slow twitch soleus muscle was assessed at 2 weeks post-injury.

Specifically, the soleus fiber type composition was determined using

immunofluorescence techniques with monoclonal antibodies (BA-D5, SC-71, BF-F3, and

BF-35).

3.3.2 Hypotheses

a) Two weeks after moderate T8 spinal cord contusion injury, the rat soleus muscle

will show a fiber type shift from MHC-I towards MHC-II compared to the normal control

soleus.

b) One-week treadmill locomotor training will attenuate the rat soleus fiber type

shift observed following T8 spinal cord contusion injury.









3.4 Experiment 4

3.4.1 Specific Aim

a) To determine the impact of moderate T8 spinal cord contusion injury on mRNA

expression of IGF-I and its related receptor and binding proteins in rat skeletal muscle.

b) To determine the impact of 1-week treadmill locomotor training on mRNA

expression of IGF-I and its related receptor and binding proteins in rat skeletal muscle

following T8 spinal cord contusion injury.

Adult female rats (n=6 animals, 12 muscles/group/time point) were assigned to

either a SCI-treadmill training group, a SCI-no training group, or a control group.

Treadmill training started on post-operative day 8. mRNA expression levels of IGF-I

were assessed at both 36 hours (4-6 hrs after last (3rd) bout of training) and 1 week post-

treadmill training. Specifically, a semi-quantitative RT-PCR was used to quantify mRNA

expression of IGF-I, MGF, IGF-R, IGFBP4, and IGFBP5.

3.4.2 Hypotheses

a) Two weeks after moderate T8 spinal cord contusion injury, rat soleus IGF-I and

MGF mRNA expression levels will not be significantly different than that of control

animals. However, mRNA expression of IGF-R, IGFBP4, and IGFBP5 will be

significantly altered after spinal cord contusion injury.

b) One week of locomotor training increases rat soleus IGF-I and MGF mRNA

expression levels in the spinal cord contused injured rat, compared to non-trained SCI

animals. In addition, 1-week treadmill locomotor training attenuates the alterations

observed in soleus mRNA expression of IGF-R, IGFBP4, and IGFBP5 following

moderated thoracic contusion injury.














CHAPTER 4
A LONGITUDINAL STUDY OF SKELETAL MUSCLE FOLLOWING SPINAL
CORD INJURY AND LOCOMOTOR TRAINING

4.1 Abstract

Spinal cord injury (SCI) results in loss of muscle mass and motor function.

Recently, novel intervention therapies, focusing on repetitive locomotor training, have

shown great promise in promoting spinal plasticity and recovery in motor function

following SCI. Recovery of motor function after spinal cord injury likely requires both

neural and muscular adaptations. The objective of this study was to implement magnetic

resonance imaging to characterize the longitudinal changes in rat lower hindlimb muscle

morphology following contusion SCI and to determine the therapeutic potential of two

modes of locomotor training. After moderate midthoracic contusion SCI, Sprague

Dawley rats were assigned to either treadmill training, cycle training or an untrained

group. Lower hindlimb muscle size was examined at pre, 1-, 2-, 4-, 8-, and 12-weeks post

injury. Following SCI, we observed significant atrophy in all rat hindlimb muscles. The

greatest amount of atrophy (11.1 -26.3%) was measured at 2-week post-injury and

spontaneous recovery in muscle size was observed by 4 weeks post SCI. Both cycling

and treadmill training halted the atrophic process and accelerated the rate of recovery.

The therapeutic influence of both training interventions was observed within 1 week of

training. Finally, a significant positive correlation was found between locomotor

functional scores and hindlimb muscle size following SCI.









4.2 Introduction

Spinal cord injury (SCI) is one of the most devastating human afflictions, leaving

its victims paralyzed or with impaired motor control (107, 132). Animal models have

long been used to characterize spinal cord lesions, study the mechanisms of recovery, and

develop effective therapeutic interventions. Among SCI animal models, contusion injury

is the most clinically relevant since it reproduces the histpathological features observed in

approximately 40% of human trauma cases (39). Mid-thoracic spinal cord contusion

injury of moderate severity disrupts connectivity in the lumbar spinal cord, but allows

some communication between the supraspinal centers and caudal regions of the spinal

cord(24).

Novel intervention therapies, focusing on repetitive locomotor training, have shown

promise in promoting spinal plasticity and recovery in motor function after spinal cord

injury in animal models (18, 58, 73, 74, 140). Clinical research has also shown

encouraging results for treadmill locomotor training in incomplete paraplegic patients(27,

62, 106). The underlying premise of locomotor treadmill training relates to the hypothesis

that rhythmic loading of the limbs and force feedback from the hindlimb muscles induces

task appropriate activity-dependent plasticity (63, 74, 105). Depending upon the

requirements of each subject, treadmill training benefits from the use of partial body

weight support and a team of experienced gait facilitators (27). A less equipment and

personnel intensive alternative training intervention is cycling locomotor training. Cycle

locomotor training is accomplished by the simple circular movement of the hind limbs on

a bicycle-type device, driven by a motorized belt (203). Since there are significant

practical differences in the equipment and personnel requirements for the performance of









treadmill versus cycle locomotor training, there are important questions to be addressed

regarding the relative efficacy of each type of training modality.

Recovery of locomotor function after spinal cord injury likely requires neural

plasticity as well as the maintenance or restoration of skeletal muscle. Patients with

chronic complete spinal cord injury display extensive muscle atrophy and associated

secondary health related complications (44, 45). In addition, a large number of studies

have shown a rapid loss in muscle mass following spinal cord transaction and spinal

isolation in animals (70, 137, 149, 178, 186). Both treadmill locomotor training and

cycling training have demonstrated a positive influence on muscle size after spinal cord

transaction. Spinal cord transected cats showed a significant reduction in the atrophic

response of muscles with a high proportion of slow twitch muscle fibers after about 6

months of treadmill training. The largest effect of treadmill training was observed in the

soleus, a muscle containing approximately 90% slow fibers (112). A similar benefit has

been reported in spinalized rats following cycling training. Interestingly, the impact of

either cycling or treadmill locomotor training on skeletal muscle following contusion SCI

has not been explored, even though the remaining communication between the

supraspinal input and peripheral skeletal muscles may create a better target for activity

dependent plasticity. In addition, only a limited amount of studies have investigated the

atrophic response in skeletal muscle following contusion injury (116).

The purpose of this study was to utilize magnetic resonance (MR) imaging to

investigate the longitudinal changes in muscle morphology in the rat lower hindlimb

muscles following midthoracic (T8) contusion SCI. A second aim of this study was to









determine the potential of treadmill versus cycling locomotor training to ameliorate

muscle atrophy and to induce muscle plasticity following spinal cord contusion injury.

4.3 Materials and Methods

4.3.1 Experimental Animals

Twenty-four Sprague Dawley rats (12 week, 228-260g; Charles River, NJ) were

studied before SCI, and at early (1, 2 weeks), intermediate (4 weeks), and late (8, 12

weeks) time points following contusion SCI. The rats were housed in a temperature-

controlled room at 21 OC with a 12:12 hours light:dark cycle and were provided rodent

chow and water ad libitum. All procedures were performed in accordance with the U.S.

Government Principle for the Utilization and Care of Vertebrate Animals and were

approved by the Institutional Animal Care & Use Committee at the University of Florida.

4.3.2 Spinal Cord Contusion Injury

Spinal cord contusion injuries were 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 (details in (179,

226)). 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 were monitored for the possibility of urinary tract

infection. Animals were housed in pairs with the exception of the first few hours

following surgery. At post-operative day 7, open field locomotion was assessed using the









Basso-Beattie-Bresnahan (BBB) locomotor scale (24) and animals that did not fall within

a preset range (0-7) were excluded from the study. Animals were subsequently divided

(randomly) into three groups (n=8/group). Two groups were assigned to either treadmill

or cycling locomotor training, and the third group did not receive training.

4.3.3 Treadmill and Cycling Locomotor Training

In both training paradigms animals were trained continuously for 3 months (5

days/week, 2 trials/day, 20 minutes/trial), starting on post-operative day 8. Animals

assigned to the treadmill training group were given five minutes to explore the treadmill

on the first training day and then encouraged to walk on the moving treadmill (11 mpm)

(130) for a series of four, five-minute bouts. A minimum of five minutes rest was

provided between bouts. On the second day of training, animals completed two bouts of

ten minutes each, twice a day. Starting on day 3, animals trained continuously for 20

minutes with a minimum interval between trials of 2 hours. Training consisted of

quadrapedal treadmill stepping. Body weight support was provided manually by the

trainer. The level of body weight support was adjusted to make sure that rats could bear

their weight and there was no collapse of their hindlimbs. Typically, the rats started

stepping when they experienced some small load on their hindlimbs. In addition, during

the first week of training, when all rats had profound paraplegia, assistance was provided

to place the rat hind paws in plantar stepping position during training. In general,

following 3-4 weeks of training, rats were independent in stepping with occasional

assistance.

The design of the cycle trainer used in these studies was adapted from the one

developed by Houle and Skinner (115). The rat bicycle is composed of a direct drive gear

box, adjustable foot pedals, and a support harness, as described previously (34). The









animal was suspended in the harness, and the hindlimb feet were strapped onto the pedals

of the motorized cycle, which is controlled by a speed controller and an on/off switch.

The exercise consisted of a circular pedaling motion, which flexes one limb while

extending the other. Care was taken not to overstretch either one of the limbs. The

pedaling rate was set at 31 rpm as it approximates the self-selected cadence at which

animals walk on the treadmill. During the first week of training, the rat tail was attached

to an aluminum support beam with surgical tape to maintain the trunk stability during

exercise. Gradually the load was increased by positioning the body harness towards the

chest, so that the hind portion of the body falls over the pedal. A similar exercise method

has been used in spinalized rabbits (231). The timing and duration of bicycle training was

equivalent to the treadmill training protocol.

4.3.4 Magnetic Resonance Imaging

All imaging procedures were performed in a horizontal, 4.7 Tesla magnet with

Paravision 2.1.1 software (Bruker Medical, Ettlingen, Germany). The spectrometer was

equipped with the Bruker S116 actively shielded gradient coil and a custom-built, 5-cm

long, 3.3-cm inner diameter birdcage extremity coil. Rats were initially anesthetized with

4% gaseous isoflourane in oxygen (1 L/min flow rate) and maintained with 1-2%

isoflourane in oxygen for the duration of the MR experiment. Animals were placed on a

cradle in the prone position with the right leg secured in the center of the coil covering

the region from mid-thigh to ankle. Pulse rate, blood oxygen saturation, respiration rate,

and body temperature were monitored continuously.

3D proton MR images were obtained at pre-injury as well as at 1, 2, 4, 8, and 12

weeks post injury, using a fast gradient echo imaging sequence. The data were acquired

with an encoding matrix of 516 x 256 x 64, field of view of 2.5 x 2.5 x 4 cm, pulse









repetition time of 100 ms, and an echo time of 6.4 ms. The total region imaged extended

from the mid-thigh to the calcaneus. Chemically selective fat suppression was used to

enhance the definition between muscle groups. The CSA of the tibialis anterior, triceps

surae, extensor digitorums and flexor digitorums was determined for each slice and the

maximal CSA (CSAmax) was recorded (Fig 1). Image analysis was performed using a

custom-designed interactive computer program, EXTRACTOR (77). The total CSAmax

was defined as the sum of the individual CSAmaxS.


















Figure 4-1. Representative trans-axial MR image of the rat lower hindlimb. A) Data
were acquired with a slice thickness of 1 mm and field of view of 2.5 x 2.5
cm. B) Outline of tibialis anterior, triceps surae, extensor digitorum, and
flexor digitorum muscles.

4.3.5 Open Field Locomotor Function

In order to determine the relationship between changes in muscle size and

locomotor function, a standardized test for open field locomotion known as the Basso,

Beattie, Bresnahan (BBB) Locomotor Rating Scale was implemented (24). Rat locomotor

function was evaluated at pre-injury as well as at 1, 4, 8, and 12 weeks post injury. The

animal was placed in a test apparatus, observed for 4-minute, and scored in real time by









2-blinded observers (24). All open field locomotor testing was video-taped for further

analysis and review.

4.3.6 Statistical Procedures

All statistical analysis was performed using SPSS for Windows (Version 10.0).

Results were expressed as mean standard error of mean (S.E.M). Research hypotheses

were tested at an alpha level of 0.05. One-way analysis of variance (ANOVA) for

repeated measurements was performed to test CSAmax changes following contusion SCI.

In addition, one-way ANOVA was used to compare individual muscles at each

experimental time point. Training effects on the muscle CSAmax of the hindlimb muscles

were assessed using two-way repeated measures ANOVA (group x time). Post hoc tests

were performed using Bonferroni-Dunn procedure for multiple pairwise comparisons. In

addition, Regression analysis was performed to assess the correlation between BBB

locomotor rating scale and hindlimb total CSAmax in all rats studied.

4.4 Results

4.4.1 Muscle Size After Spinal Cord Contusion Injury.

MR images acquired before and after spinal cord contusion injury showed

significant atrophy in all hindlimb muscles studied (Table 4-1). The amount of atrophy

was both muscle- and time-dependent. The rate of atrophy was greatest during the first

week after SCI across all muscles. At Iw-SCI, the CSAmax of the extensor digitorums,

flexor digitorums, tibialis anterior, and triceps surae was reduced to 90.6 1.9%, 86.8 +

2.4%, 88.3 1.9%, and 83.4 2.2% of pre-injury values, respectively (Figure 4-2). The

most extensive amount of atrophy was observed at 2w-SCI, and varied across muscles.

The triceps surae demonstrated the greatest amount of atrophy (73.7 4.1% of pre-injury










values), the extensor digitorums the least (88.9 2.6%), followed by the tibialis anterior

(79.5 + 3.2%) and flexor digitorums (84.6 + 7.5%) muscles (Figure 4-2).


MED
120- FD
EI TA
EM TS
110

100- *

90-

80-

70

60

50
1 2 4 8 12
Weeks post SCI


Figure 4-2. Relative changes in the CSAmax of the tibialis anterior (TA), triceps surae
(TS), extensor digitorum (ED), and flexor digitorum (FD) muscles at 1, 2, 4,
8, andl2 weeks following SCI. Data are expressed as a percentage of the
CSAmax measured at pre-injury. Statistically significant differences
between ED and TS (P < 0.05). Statistically significant differences between
FD and TS (P < 0.05).

Starting at the fourth week post SCI, a steady increase in the muscle CSAmax was

observed. The rate of increase in muscle CSAmax ranged from 0.48 to 6.26 mm2/week. By

12 weeks post-SCI, the CSAmax of all muscles studied had recovered to values that were

no longer statistically different from pre-injury control values. At 12w-SCI, the CSAmax

was 102.2 1.6% in the extensor digitorums, 102.1 4.4% in the flexor digitorums, 98.3

+ 3.4% in the tibialis anterior and 95.4 2.1% in the triceps surae. As a result, the total

CSAmax for all muscles combined was 98.3 1.6% of pre injury values.











105-

100 *

95\

I 90

85-
-o- No training
-0- Treadmill training
80 : -A- Cycling training

75
0 2 4 6 8 10 12
Weeks post SCI


Figure 4-3. Relative change in total CSAmax in the no training group, cycling training
group and treadmill training group. Data are expressed as a percentage of the
CSAmax measured at pre-injury. Training started at 1 week post injury
(dashed line). Statistically significant differences between no training group
and treadmill training group (P < 0.05). Statistically significant differences
between no training group and cycling training group (P < 0.05).

4.4.2 Effect of Locomotor Training on Muscle Size.

Both cycling and treadmill training decreased the rate and magnitude of atrophy in

the lower hindlimb muscles following spinal cord contusion injury and accelerated the

rate of recovery in muscle CSAmax. The influence of locomotor bicycle and treadmill

training on CSAmax was observed as early as 1 week after the onset of training (Figure 4-

3). The control untrained triceps surae muscles showed a significant decrease (-10.6 +

4.0 mm2/week) in muscle CSAmax from 1 week to 2 weeks post-SCI. In contrast, the

triceps surae muscle in both training groups revealed significant muscle hypertrophy,

+3.6 + 1.4 mm2/week in the bicycle training and +4.7 0.9 mm2/week in the treadmill

training group. As a result, at 2 weeks post-SCI the CSAmax of the triceps surae was 73.4









+ 4.1% of pre-injury values in the non-training SCI group and 88.8 1.4% and 91.2

0.9% in the cycling and treadmill training groups, respectively (Figure 4-4). As shown in

Figure 4-4, a similar acute response to training was observed in the tibialis anterior

muscle as well as the extensor digitorum and flexor digitorum muscles (data not shown).

Consequently, at 2 weeks post-SCI the total CSAmax of the lower hindlimb muscles was

on average 10.5% and 12.4% higher in the cycling trained and treadmill trained animals,

respectively, compared to the non-trained group (Figure 4-3).

The therapeutic effect of locomotor training was observed not only at the early time

point but also throughout the remaining training period (Figure 4-3). The total CSAmax in

both cycling and treadmill locomotor training animals was significantly greater than that

of non-trained SCI animals. By 12w-SCI, both cycle and locomotor training groups

demonstrated a full recovery in the total CSAmax, with values of 101.3 0.8% and 101.8

+ 1.3%, respectively compared to pre-injury control values. In addition, a direct

comparison between the cycling and treadmill trained animals showed no significant

difference between the two locomotor training interventions for all muscles studied,

except the tibialis anterior muscle. The CSAmax of the tibialis anterior muscle was fully

recovered at 4-week post SCI in the treadmill training group, whereas in the cycling

training group pre injury values were only reached at 8-week post SCI (Figure 4-4).











110-
105- *
100-1 *

95g *\ "
S90-
0 85

S-o- No training
75 / -0- Treadmill training
0 -A- Cycling training
70 -
65-
0 2 4 6 8 10 12
Weeks post SCI


Figure 4-4. Relative change in the CSAmax of the Triceps surae (TS) in the no training
group, cycling training group and treadmill training group. Data are expressed
as a percentage of the CSAmax measured at pre-injury. Training started at 1
week post injury (dashed line). Statistically significant differences between
no training group and treadmill training group (P < 0.05). Statistically
significant differences between no training group and cycling training group
(P < 0.05).

4.4.3 Relationship Between Hindlimb Muscle Size and Locomotor Function.

Our animals participated in a parallel study evaluating the influence of locomotor

training on several functional measures after spinal cord injury (34). In the present study,

we examined the relationship between the hindlimb muscle CSA and locomotor function

following SCI. Our data demonstrated a significant positive correlation between scores

on the BBB locomotor rating scale and hindlimb total CSAmax (r =0.71, P < 0.001).

Figure 4-5 provides the results of the regression analysis.




























Weeks post SCI


Figure 4-5. Relative change in the CSAmax of the Tibialis anterior (TA) in the no
training group, cycling training group and treadmill training group. Data are
expressed as a percentage of the CSAmax measured at pre-injury. Training
started at 1 week post injury (dashed line). Statistically significant
differences between no training group and treadmill training group (P < 0.05).
Statistically significant differences between no training group and cycling
training group (P < 0.05).


0 0



I m o o
oc
E
U

O A A No training
O 1D 0 Treadmill training
0 & Cycling training

140 150 160 170 180 190 200 210 220
Total CSA


Figure 4-6. Relationship of rat locomotor function (BBB scale) and total lower hindlimb
CSAmax (r =0.71, P < 0.001). Data of all the rats at each time point were
pooled together. Note that the line is drawn for visual purposes only and not to
indicate a linear function.









4.5 Discussion

Following midthoracic spinal cord contusion injury, we observed significant

atrophy in the rat hindlimb muscles. The degree of atrophy appeared to be muscle-

specific, with the anti-gravity extensor muscles showing greater atrophy than the flexor

muscles. In the non-trained animals, the greatest amount of atrophy (11.1 -26.3%) was

measured at two weeks post-injury and spontaneous recovery in muscle size was

observed by 4 weeks post SCI. Both cycling and treadmill training halted the atrophic

process and significantly accelerated the rate of recovery. The therapeutic influence of

both treadmill and bicycle training was observed within 1 week of training. Finally, a

direct comparison between the cycling and treadmill trained animals showed no

significant difference between the two locomotor training interventions for all muscles

studied, except the tibialis anterior muscle.

Previous studies have examined the atrophic response of skeletal muscle after

complete and incomplete SCI in humans (44, 45, 96) as well as in a variety of animal

models (70, 136, 149, 178, 186). A large number of studies have shown a rapid loss of

muscle mass in spinal cord transected and spinal isolated animals, with more extensive

atrophy in slow twitch muscles compared to fast twitch muscles. However, only a limited

amount of studies have investigated the effect of contusion SCI on skeletal muscle

morphology. Specifically, Hutchinson et al (116) found a significant decrease in muscle

wet weight (20-25%) in all lower hindlimb muscles, except the EDL, compared to age-

matched controls at Iweek following moderate contusion injury. They also reported that

muscle atrophy occurred in flexor as well as extensor muscle groups and that the severity

was similar in fast and slow muscles. In contrast, a pattern of differential decrease was

noted following contusion injury in the present study. At 2 weeks post spinal cord









contusion injury, when muscle CSA measures revealed the greatest degree of atrophy,

MR quantitative assessments showed the following atrophic hierarchy: triceps surae >

tibialis anterior > flexor digitorums > extensor digitorums. This differential muscle

response may be attributed to the higher neuromuscular activity in extensor muscles

relative to flexor muscles (173). The greater degree of atrophy in the extensor vs. flexor

muscles may also be influenced by the ankle joint position post-SCI. The paralyzed

hindlimbs of spinalized or contusion-injured rats are maintained in an extended position

and dragged by the forelimbs during cage walking (23, 187, 213). Larger muscle loss has

also been observed when muscles are immobilized in a shortened position, compared to a

neutral or stretched position (13).

Muscle atrophy following midthoracic contusion injury of moderate severity was

transient and restoration of muscle morphology mirrored the spontaneous recovery in

locomotor function. In the non-trained SCI-animals increases in CSAmax (or hypertrophy)

were first noted at the 4 week post-SCI time point. The rate of spontaneous hypertrophy

ranged from 0.1 to 6.3 mm2/week, with the largest gains in muscle size at the

intermediate time point (4 weeks). As a result, by 12 weeks post-SCI, the total CSAmax

for all muscles combined was no longer statistically different from pre-injury control

values. In comparison, Hutchinson and colleagues (116) reported that muscle atrophy,

assessed by wet weights, was attenuated starting at 3-week post spinal cord contusion

injury. By 10 weeks post-injury muscle wet weights were no longer different from

control values, except for the medial and lateral gastrocnemius. In contrast, spinal

transaction animal models, in which communication between the supraspinal centers and









caudal region of the spinal cord is eliminated, have shown little spontaneous recovery in

muscle size (97).

The restoration in muscle size in the moderate contusion injury model appears to

mirror the spontaneous improvement in locomotor function. Basso et al. (23) showed that

after a moderate thoracic spinal cord contusion injury, animals demonstrate hindlimb

paralysis until 7 days post injury, which is followed by a progressive recovery in

locomotor function over the next 5 weeks. In the present study, we observed a significant

positive correlation between scores on the BBB locomotor rating scale and hindlimb total

CSAmax. The consistency between the muscle morphometry and behavioral data supports

the contention that neuromuscular activity is an important determinant of skeletal muscle

size and vice versa.

One of the primary goals of this study was to determine the effect of both treadmill

and cycle training on the posterior and anterior hindlimb muscles of moderate contusion

spinal cord injured animals. The impact of treadmill training on skeletal muscle has been

studied in a variety of animal models. Roy et al. (186) demonstrated that in spinal cats

trained to step, the atrophic response of the muscles with a high proportion of slow fibers

is significantly reduced. The largest effect of training was observed in the soleus muscle,

followed by the extensors at the ankle and knee; muscles which are expected to be highly

recruited during treadmill exercise (112). In contrast, treadmill stepping appeared to have

little effect on a large number of muscles with a high proportion of fast twitch fibers,

such as the tibialis anterior (186). In this study we found that treadmill training

effectively facilitated muscle hypertrophy in both the tibialis anterior and triceps surae

muscles of contusion injured animals. Throughout the 12 weeks of training, the tibialis









anterior and triceps muscles were significantly larger than the corresponding muscles in

the untrained animals. The largest difference between the treadmill trained and untrained

animals was noted at the early and intermediate time points post-SCI.

Depending upon the access and availability of treadmill locomotor training, cycle

training may provide a practical alternative training strategy, contingent upon the

continued demonstration of safety, feasibility, and efficacy of this approach. In the

present study, we performed a direct of comparison between cycle and treadmill

locomotor training and showed that both locomotor interventions induce significant

muscle plasticity in all hindlimb muscles, as early as 1 week after initiation of training.

While the treadmill training protocol used in this study was designed to maximize

loading on the hindlimb muscles, cycling exercise uses a different strategy and is

accomplished by simple circular movements of the hindlimbs on a bicycle-type device,

driven by a motorized belt (34, 203). EMG data acquired in previous studies show that

during cycling locomotor training the left and right hind limb muscles are stretched in an

alternating pattern, which results in alternating bursts of muscle activity (115). Thus,

cycling training also initiates sensory input to the spinal cord and subsequently influences

the firing pattern of the motoneurons that innervate the hind-limb muscles. Houle (115)

studied the hind limbs of spinalized rats following cycling training (45 rotations per

minutes, 30 minutes per trials, two trials per day, 5 days per week) and found that cycling

locomotor training ameliorates muscles atrophy in spinalized rats. Similarly, in this study

we found that in contusion-injured rats cycling training effectively halts the atrophic

process and accelerates muscle recovery. While untrained triceps surae muscles

demonstrated a significant loss in muscle size from 1 week to 2 weeks post-SCI (-10.6 +









4.0 mm2/week), 1 week of bicycle training induced significant muscle hypertrophy (+3.6

+ 1.4 mm2/week). However, a direct comparison between cycling and treadmill training

showed that while cycling training was equally effective in promoting muscle

hypertrophy in the triceps surae muscles throughout the 12 weeks of training, treadmill

training induced a larger hypertrophic response in the tibialis anterior muscle compared

to cycling at the intermediate and late time points. One explanation for this discrepancy

may be that during cycling revolutions the ankle was kept in a dorsiflexed (shortened)

position on the pedal, minimizing the stretch reflex in the tibialis anterior muscle.

A large number of investigators are studying the mechanisms responsible for neural

repair, neuroplasticity and muscle hypertrophy (69). Based on our data and the well-

established role of loading for muscle growth, we speculate that the training itself is not

of sufficient duration to effectively induce muscle hypertrophy. Consequently we propose

that repetitive muscle activation, even if induced by passive motion (e.g. during motor-

driven bicycling), promotes the restoration of the neuromuscular interface and the

recovery of "functional" motor units. The activation of these functional motor units under

loaded conditions, such as is observed during cage reambulation, may provide the

appropriate stimulus to induce muscle fiber regeneration and hypertrophy. This in turn

may result in improvements in motor function, increased ambulatory activity and use-

dependent neuroplasticity and muscle hypertrophy.

In conclusion, this study demonstrates that rats after spinal cord contusion injury

suffer significant atrophy in the lower hindlimb muscles, with the greatest amount of

atrophy noted 2 weeks following injury. Starting at 4 weeks post-SCI, rat hindlimb

muscles show spontaneous recovery resulting in near normal values at 12 weeks post-









SCI. This study also demonstrates that both treadmill and cycle training diminish the

extent of atrophy and facilitate muscle plasticity after contusion injury. The therapeutic

influence of training on skeletal muscle was observed within the first week of training.

Since muscle atrophy is associated with a myriad of secondary health problems, the

potential of maintaining muscle mass with repetitive locomotor training is exciting and

warrants further research. In addition, the recovery of locomotor function after spinal

cord injury likely requires both neural and muscle plasticity, as well as recovery of the

neuromuscular interface.


Table 4-1. Maximal cross-sectional area (mm2) of individual muscle. Values are means +
SME. Significantly different with pre injury value (p< 0.05)


pre injury 1 week 2 week 4 week 8 week 12 week

Extensor digitorums 14.50.1 13.20.3* 12.90.4* 13.90.4 14.50.3 14.80.3

Flexor digitorums 32.10.7 27.80.7* 26.91.1* 28.61.1 31.10.1 32.30.9

Tibialis anterior 41.80.9 36.91.4* 33.31.7* 35.91.8* 38.81.4 40.31.5

Triceps surae 106.43.3 88.73.5* 78.14.4* 91.52.6* 98.53.3* 101.33.5














CHAPTER 5
CHANGES IN SOLEUS MUSCLE FUNCTION AND FIBER MORPHOLOGY WITH
ONE WEEK OF LOCOMOTOR TRAINING IN SPINAL CORD CONTUSION
INJURED RATS

5.1 Abstract

Recently, a new approach in the treatment of individuals with SCI involves

rehabilitation to maximize residual function using locomotor training. The purpose of this

study was to examine the influence of midthoracic contusion SCI on skeletal muscle

function and to evaluate the therapeutic influence of early treadmill locomotor training on

soleus muscle function (peak force, fatigability, contractile properties), size (fiber area),

as well as overall locomotor function (BBB) after SCI in rats. Thirty five adult Sprague

Dawley rats (female, 16-20 weeks, weighing 250-290g) were studied. Histological

measurements of muscle fiber size were made on all animals. Twenty four animals were

designated for muscle contractile measurements (8 controls and 16 receiving a moderate

T8 spinal cord contusion injury). Eight of the SCI rats received treadmill locomotor

training (TM) starting 1 week after SCI for 5 consecutive days, 20 minutes/trial, 2

trials/day. The additional eight injured rats received no exercise intervention (no TM).

Locomotor training resulted in a significant improvement in BBB scores (32%), muscle

fiber size and function. Compared to untrained injured animals, injured animals that

trained for one week exhibited 38% greater peak soleus tetanic forces, a 9% decrease in

muscle fatigue, and 23% larger muscle fiber CSA. In addition, there was a strong

correlation between BBB scores of injured animals and peak soleus muscle force

(r=0.704). Collectively, these results indicate that early therapeutic intervention using









treadmill locomotor training can significantly improve functional locomotor recovery

following SCI. The magnitude of these changes is remarkable considering the relatively

short training interval, and clearly illustrates the potential that early exercise intervention

has for countering some of the early functional deficits resulting from SCI.

5.2 Introduction

Spinal cord injury (SCI) is a devastating human condition that results in paralysis

or impairments in motor control that lead to significant disability (62, 73, 229). Currently,

a major therapeutic problem centers around the profound dysfunction of the primary

locomotor skeletal muscles following SCI (176). Recently, a new approach in the

treatment of individuals with SCI involves rehabilitation to maximize residual function

using locomotor training (63, 176). Such locomotor training uses principles derived from

animal and human studies showing that stepping can be generated by virtue of the

neuromuscular system's responsiveness to phasic, peripheral sensory information

associated with locomotion (18, 27, 58, 63, 106, 140). Although these locomotor training

programs may promote functional recovery, the particular contributions of this therapy

for addressing primary locomotor skeletal muscle dysfunctions are not well understood

and investigation regarding these important issues is needed.

Animal models of SCI can offer a practical approach to efficiently evaluating the

safety, feasibility, and efficacy of therapeutic procedures on skeletal muscle adaptations

during recovery (21, 74). Current animal models of SCI include transaction, compression,

and contusion (24, 183). The transaction model has been widely used to study limb disuse

because it allows for a reproducible complete SCI (23, 69, 70, 166, 218), yet the

contusion model most closely parallels the mechanism of injury of the majority of human

SCIs (incomplete injuries) (116, 183) and has been shown to reproduce the









histopathologic sequence of SCI observed in about 40% of human trauma cases (39). In

contrast to the transaction model where animals experience a complete loss of locomotor

capabilities (23, 87), animals with a moderate contusion injury regain some locomotor

function without specific training (2, 22, 23, 28, 183, 225). Although contusion injuries

have been performed in a variety of animal species, electrophysiologic, behavioral, and

imaging studies have indicated that SCI in rats can reasonably model events occurring

after human SCI (94, 146).

While much attention has been focused on neural damage and recovery after SCI

(35, 72, 74, 87, 155), less attention has focused on skeletal muscle adaptations after

injury, especially following a contusion injury. One of the few studies to closely examine

muscle adaptations after a contusion injury in rats found that, soleus and extensor

digitorum longus muscle phenotype and contractile properties were significantly affected

within the first 1-3 weeks after SCI (116). With the recovery of weight supported

hindlimb stepping over the course of 10 weeks, there was a concurrent return to baseline

levels for contractile properties and muscle morphology, which suggests that weight

bearing plays a critical role in maintaining normal muscle physiology. In addition, since

changes occur within weeks after SCI, early exercise interventions may offer the greatest

potential to counter these muscle adaptations and restore normal function. In Chapter 4

we showed that exercise training (bicycle or treadmill) after a contusion SCI results in

faster recovery of muscle size (anterior and posterior compartment muscles) and

preservation of normal muscle morphology after 3 months of training. In this

investigation, we also found that the greatest differences in muscle size between trained

and untrained rats occurred within the first weeks of training (chapter 4). The time course









of muscle adaptations after SCI in the aforementioned study are consistent with those of

Hutchinson et al. (2001). Therefore, we were interested in investigating the short term

benefits of early locomotor training in reducing muscle atrophy and improving muscle

function. It has been reported that the greater disuse adaptations occur in slow twitch

than in fast twitch muscle fibers and in postural extensorr) muscles more than flexor

muscle groups (2, 28, 225). Accordingly, we chose to study adaptations of the soleus

muscle because it is composed of slow twitch muscle fibers and functions as a postural

muscle, which makes it more susceptible to muscle atrophy.

The purpose of this study was to examine the influence of SCI on skeletal muscle

function and to evaluate the therapeutic influence of early treadmill locomotor training on

soleus muscle function (peak force, fatigability, contractile properties), size (fiber area),

as well as overall locomotor function (BBB) after SCI in rats. Our hypothesis was that

early locomotor training would attenuate some of the functional changes in muscle seen

early after injury by improving muscle force and contractile properties, decreasing

muscle fatigue, and preserving normal muscle morphology.

5.3 Materials and Methods

5.3.1 Experimental Animals

Thirty five adult Sprague Dawley rats (female, 16-20 weeks, weighing 250-290g)

were studied. Histological measurements were made on 35 animals (11 controls, 12

treadmill-SCI, and 12 no treadmill-SCI). Twenty four animals were designated for

muscle contractile measurements, with eight serving as controls and sixteen receiving a

moderate T8 spinal cord contusion injury using a standard NYU impactor (23). Eight of

the injured rats received treadmill locomotor training (TM) starting 1 week after SCI for

5 consecutive days, 20 minutes/trial, 2 trials/day. The additional eight injured rats









received no exercise intervention (no TM). All rats were housed in a temperature-

controlled room at 210C with a 12:12 hours light:dark cycle and were provided

unrestricted access to food and water. All procedures were performed in accordance with

the U.S. Government Principle for the Utilization and Care of Vertebrate Animals and

were approved by the Institutional Animal Care & Use Committee at the University of

Florida.

5.3.2 Open Field Locomotor Function

One of the most common and reproducible measures of locomotor function

following SCI contusion injury employs a standardized test for open field locomotion

known as the BBB Locomotor Rating Scale (24, 25). The BBB rates behavior ranging

from individual joint movements of the hindlimb to plantar stepping and coordinated

walking, progressing further to finer elements of locomotion, such as trunk stability, paw

position, and tail position. Rats were evaluated for locomotion prior to surgery, 1 week

after SCI, and 2 weeks after SCI. Movement was evaluated for 4 minutes by 2 examiners

using the Basso, Beattie, Bresnahan (BBB) Locomotor Rating Scale (24, 25).

5.3.3 Spinal Cord Contusion Injury

Spinal cord contusion injuries were produced using a MASCIS (Multicenter

Animal Spinal Cord Injury Study) impactor and protocol (23). Briefly, 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 Baytril (10mg/kg)

per day for 5 days, starting the day of surgery. Procedures were performed under

ketamine (100mg/kg)-xylazine (6.7mg/kg) anesthesia (179, 226). Subcutaneous lactated

Ringer's solution (5 ml) was administered after completion of the surgery. Animals were

given Buprenorphine (0.05mg/Kg IM) and Ketoprofen (5.0 mg/Kg SC) for pain and









inflammation over the first 36hrs after SCI. The animals 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 were monitored for the possibility of urinary tract

infection. After SCI, animals were housed individually. On post-operative day 7, open

field locomotion was assessed using the Basso-Beattie-Bresnahan (BBB) locomotor scale

(24, 25) and animals that did not fall within a preset range (BBB, 4-8) were excluded

from the study. Injured animals were subsequently divided into two groups (n=12/group)

and were randomly assigned to either the treadmill locomotor training or no training

groups. The third group consisted of non-injured control rats (n=12/group).

5.3.4 Treadmill Locomotor Training

Animals that received locomotor treadmill training were trained for 5 consecutive

days (hereafter defined as 1 week of training), 2 trials/day, 20 minutes/trial, starting on

post-operative day 8. Training consisted of a quadrapedal treadmill stepping. On the first

day of training, animals were given five minutes to explore the treadmill and then

encouraged to walk on the moving treadmill (11 mpm) (130) for a series of four, five-

minute bouts. A minimum of five minutes rest was provided between bouts. Body weight

support was provided manually by the trainer. The level of body weight support was

adjusted to make sure that rats could bear their weight and there was no collapse of their

hindlimbs. Typically, the rats started stepping when they experienced some small load on

their hindlimbs. In addition, during the first week of training, when all rats had profound

paraplegia, assistance was provided to place rat hindlimbs appropriately for plantar

stepping during training. On the second day of training, animals completed two 10

minute bouts, twice a day. Starting on day 3, animals trained continuously for 20 minutes









with a minimum interval between trials of 6 hours. Bodyweight support through the trunk

and the base of the tail was provided as necessary and gradually removed as locomotor

capability improved.

5.3.5 In situ Soleus Force Measurements

In situ soleus force measurements were performed in control animals and 2 weeks

after SCI (following 1 week of training). Animals were anesthetized with isoflurane (4%

for induction, 1-2% for maintenance). A small dorsal, midline incision was made along

the posterior lower hindlimb to expose the gastrocnemius-soleus complex. The

gastrocnemius and soleus muscles were dissected free from each other and surrounding

connective tissue with care not to disrupt the blood supply. A non-compliant steel wire

suture (5/0 AESCULAP Inc.) was attached to the tendon at the distal end of the soleus

muscle. A stimulating bipolar electrode cuff was placed around the tibial nerve, proximal

to its innervation of the soleus muscle. The sciatic nerve was crushed proximal to the site

of the bipolar electrode cuff and the cutaneus tibial branch was severed to ensure that all

electrical stimuli were transmitted directly to the soleus muscle. The animal was then

placed in a supine position in a specially fabricated experimental set up that allowed the

animal to be secured in a reproducible position over a circulating warm water bath to

maintain body temperature (37C). The left leg was secured in place by a pair of screw-

driven pins at the condyles of the femur, while the foot was securely clamped such that

soleus muscle was oriented in the horizontal plane. The distal end of the soleus tendon

was attached to the variable range force transducer (Biopac Systems Inc, TSD105A) via

the wire suture. The room temperature was set at 280C and monitored throughout testing.

A mineral oil drip (30C) was used to maintain a consistent muscle temperature and

prevent drying of the muscle during testing.









Testing began by stimulating the soleus muscle using supramaximal (-7V, 0.2ms

duration) unidirectional square-wave pulses via an S88 Grass stimulator (Grass-

Telefactor, RI). The transducer force output was amplified and digitized using a Biopac

MP100 System (BIOPAC Systems, Inc. Goleta CA) and Acqknowledge 3.7 computer

software. The physiological tests were performed on the soleus adjusted to the isometric

optimum length, determined by measuring maximal isometric forces generated at graded

muscle lengths. Supramaximal stimulus intensity was verified for each animal by

progressively increasing the intensity until a plateau in twitch force was achieved. A

force frequency relationship was generated for each animal using supramaximal 1500ms

stimulating trains ranging from 1-120Hz to determine the optimal frequency for maximal

tetanic force production. This optimal frequency (70-80Hz) was used for all subsequent

testing. Using the optimal stimulation frequency, maximal isometric tetanic force was

repeated 3 separate times using 1500ms trains with a 5 minute rest interval between each

stimulation. The maximum tetanic force of 3 attempts was recorded. In addition, three

twitch stimulations were evoked for maximal twitch tension and contractile property

measurements. Soleus specific tension was calculated by maximal titanic force / muscle

weight (N/g). A fatigue test was also performed and consisted of a modified Burke's

fatigue test with 300ms trains delivered every second for 2 minutes at the predetermined

optimal frequency. Force time integral was calculated as the area under the force-time

curve during muscle contraction as a measure of muscle isometric work.

5.3.6 Tissue Harvest

At the conclusion of the contractile measurements, the soleus muscles were

removed from both hindlimbs of the animal. The muscles were subsequently rapidly









frozen in isopentane precooled in liquid nitrogen (storage at -80C) for the following

histological measurements.

5.3.7 Immunohistolochemistry

Cryostat sections (10 [pm) in a transverse plane were prepared from the central

portion of each muscle taken from the both legs and mounted serially on gelatin-coated

glass slides. Immunocytochemical reactions were performed on each cryostat section

with anti-laminin to outline the muscle fibers for cross sectional area quantification. The

soleus fiber CSAs were analyzed using The NIH image program (version 1.62). The

pixels setting used for conversion of pixels to micrometer were 1.50 pixels- 1 um2 for a

10 X objective. The average fiber CSA of all the fibers in each fiber type was determined.

5.3.8 Data Analysis

One way ANOVAs with Bonferroni-Dunn post hoc testing were used to compare

results across groups (controls, trained and untrained animals) for peak soleus tetanic and

twitch forces, peak fatigue, average fiber CSA, time to peak tension, /2 relaxation time. A

p value of <0.05 was considered significant. The force decrease during fatigue was

calculated as (initial-final force)/initial force. Independent t-tests were used to compare

differences in BBB scores between groups before training (1 week post SCI) to ensure

that there were no differences between groups prior to training. Independent t-tests were

also used to compare differences in BBB scores after 1 week of training/no training (2

weeks post SCI). A Pearson's correlation was used to examine the relationship between

the soleus peak force and BBB scores for the left leg across both injured groups 2 weeks

after SCI.










5.4 Results

5.4.1 BBB Open Field Locomotor Scores

Intact control animals consistently demonstrated normal locomotor behavior on the

BBB scale (21 point scale). One week post SCI, injured animals displayed no significant

differences in BBB scores between groups designated for TM training (5.44 1.61) vs no

training (5.79 1.98) (p>0.05) (Figure 5-1). At two weeks after SCI, and after one week

of training for the TM group, BBB scoring was repeated and scores were significantly

different between untrained (6.31 2.8) and trained (10.63 1.67) animals (p<0.05)

(Figure 5-1). The significant difference between these scores indicated that one week of

training had significantly improved open field locomotion scores in the trained compared

with the untrained animals.


= SCI no TM
12, C SCI +TM
11-
10
9
8 *
7
6-
5 5
m 4
3
2


1 wk post SCI 2 wk post SCI



Figure 5-1. Average BBB locomotor scores for the SCI + TM and SCI no TM group at 1
and 2 weeks post SCI (n=8/group). *Significantly different between SCI no
TM and SCI + TM at 2wks post SCI (p<0.05). SCI + TM received 5 days of
TM locomotor training beginning 1 wk post SCI (mean+SEM).










5.4.2 Soleus Contractile Properties

At two weeks following SCI, the mean peak soleus muscle force measured in the

untrained injured animals was 117 29mN. However, the mean peak soleus muscle force

measured in the injured TM group was 178 19mN. This difference was significantly

greater (p<0.05) than that recorded in the untrained injured group, but not significantly

different from the mean peak force of 201 14mN recorded in the intact control group

(Figure 5-2). Specific tension was significantly lower in untrained injured animals

compared control rats (p<0.05). Although a trend existed, no statistically significant

differences were measured between muscle specific tension in the treadmill trained SCI

rats and untrained SCI rats (p>0.05) (Figure 5-3).


IICON
250- ~ SCI no TM
S SCI + TM
200
z
E
2 150
0
LL
C 100
so

0.





Figure 5-2. Soleus muscle peak tetanic force for CON, SCI no TM, and SCI + TM groups
at 2 weeks post SCI (n=8/group). *Significantly lower force for SCI no TM
compared to SCI + TM and CON, p<0.05.











I- CON
1.4, SCI no TM
S SCI + TM
1.2

1.0
z
0.8
0
u- 0.6
E
S0.4

0.2

0.0




Figure 5-3. Soleus muscle specific force for CON, SCI no TM, and SCI + TM groups at 2
weeks post SCI (n=8/group). *Significantly lower specific force for SCI no
TM compared to CON, p<0.05.

The possibility of a relationship between open field locomotor score and force

production in skeletal muscle was tested by plotting the peak soleus muscle forces for all

animals vs. their respective BBB scores (Figure 5-4). Least squares linear regression of

these points revealed a correlation coefficient of r=0.704, suggesting a predictable

relationship between BBB scores and peak soleus muscle force following SCI.

In addition, injured rats that received training demonstrated comparable fatigue to

control animals (27 0.04% and 26 0.09% respectively) while SCI animals without

training were more fatigable (36 0.1%) (p<0.05) (Figure 5-5). Accordingly, the

isometric work (force time integral) generated during fatigue contractions was

significantly lower in the untrained injured rats than control animals and treadmill trained

rats (Figure 5-6). However, no significant differences were found in peak twitch force, as

well as time to peak tension and half relaxation time for controls, trained SCI, and

untrained SCI (p<0.05) (Figure 5-7, 5-8, 5-9).

























60 80 100 121401 160 180 200 220
Soleus Peak Tetanic Force (mN)


Figure 5-4. Peak soleus tetanic force vs BBB score for the left legs of SCI + TM and SCI
no TM combined (n=16). Pearson correlation coefficient=0.704 (p<0.05).
Note: controls are not included in this figure.


ICON
SSCI no TM
RSCI +TM


Figure 5-5. Soleus muscle fatigue (Initial-Final Force/Initial Force) (n=8/group).
*Significantly greater fatigue in SCI no TM compared to CON and SCI + TM
groups, p<0.05.










CON






0 2 40 0 100 120







Contractions


Figure 5-6. Soleus muscle force-time integral during a fatiguing protocol (n=8/group).
Significantly lower force-time integral in SCI no TM compared to CON and
SCI + TM groups, p<0.05.


E--ICON
50-o SCI noTM
I SCI + TM
Contractions


Figure 5-6. Soleus muscle force-time integral during a fatiguing protocol (n=8/group).
*Significantly lower force-time integral in SCI no TM compared to CON and
SCI + TM groups, p<0.05.


[I CON
50 I SCl no TM
SSCl + TM


Figure 5-7. Soleus muscle peak twitch force for CON, SCI no TM, and SCI + TM groups
at 2 weeks post SCI (n=8/group).










I CON
SSSCI no TM
1 SCI + TM


Figure 5-8. Soleus muscle time to peak for CON, SCI no TM, and SCI + TM groups at 2
weeks post SCI (n=8/group).


IICON
E SCI no TM
SSCI + TM


Figure 5-9. Soleus muscle /2 relaxation time for CON, SCI no TM, and SCI + TM groups
at 2 weeks post SCI (n=8/group).

5.4.3 Soleus Muscle Fiber Cross Sectional Area

The mean soleus muscle fiber cross sectional area (CSA) observed in the untrained

animals at two weeks following SCI was 1999 541 m2. By comparison, the mean CSA

observed in the trained injured animals was 2654 359gm2, which was significantly









greater (p<0.05) than that of the untrained injured group, but not significantly different

from the mean peak fiber CSA of uninjured control animals (2467 497[im2) (p>0.05)

(Figure 5-10).


ICON
3500 SCI no TM
SCI +TM
3000

0 2500











Figure 5-10. Average soleus muscle fiber CSA for CON, SCI no TM, and SCI + TM
groups at 2 weeks post SCI (n=8/group). *Significantly smaller average
muscle fiber CSA in SCI no TM compared to CON and SCI + TM groups,
p<0.05.

5.5 Discussion

Collectively, these results indicate that early therapeutic intervention using

treadmill locomotor training significantly increases locomotor recovery and soleus

muscle size and strength following SCI. Compared to untrained SCI animals, one week

of training resulted in a 32% improvement in BBB scores, a 38% greater improvement in

peak soleus tetanic force, and larger muscle fiber CSA (23%). The magnitude of some of

these changes is remarkable considering the relatively short training interval, and clearly

illustrates the potential that early exercise intervention may have on countering some of

the early functional deficits resulting from SCI.









Changes in limb loading and neural activation produced by repetitive motor

training early after SCI provide critical activity in remaining spinal cord circuits that

facilitate neuroplasticity and muscular plasticity for better functional recovery. The

specific mechanisms by which locomotor training facilitates plasticity are poorly

understood, although it has been proposed that plasticity is guided in an activity-

dependent manner via biochemical changes at the cellular level of the nervous system

including altered synaptic connections, altered sensitivities of neurotransmitter receptors,

and altered production of neurotransmitters and growth factors (69, 74, 156). One theory

that is receiving increasing attention is the role that exercise may play in increasing the

release of myotrophic and neurotrophic factors in the spinal cord as well as skeletal

muscle (69). Another possible explanation to account for improvements with locomotor

training after SCI is that training may facilitate reorganization of undamaged neural

pathways (58). It has been suggested that moving paralyzed limbs activates muscle, joint,

and skin afferents which provides critical guidance to existing circuits to form new

patterns of motor output (87, 106)

The results of the present study indicate that muscular plasticity occurs via a

combination of changes in muscle morphology and physiological contractile properties.

Very limited information exists regarding muscular plasticity early after a contusion SCI

(116, 198), or early adaptive changes in muscle after locomotor training following a

contusion SCI. Hutchinson et al. (2001) explored muscle adaptations in rats over 10

weeks of recovery from a moderate contusion SCI without locomotor training. They

found that 3 weeks after injury, there were significant changes in muscle morphology

(wet weight) and phenotype (MHC composition) that corresponded to changes in









contractile properties. Even as early as 1 week after SCI, muscle wet weights were

different compared to controls even if no significant differences in contractile properties

were noted. Our present study examined soleus muscle properties at an intermediate time

point to that of Huchinson et al. (2 weeks post SCI) and found that there were significant

differences in soleus tetanic muscle force between injured and control animals. These

findings are consistent with those of our untrained SCI rats at 2 weeks after injury, and

going one step further, we have found that even as little as one week of locomotor

training prevents the phenotypic changes towards faster muscle seen within weeks after

injury.

Using magnetic resonance imaging to longitudinally quantify changes in muscle

size after SCI, we have recently found that the rate of hindlimb muscle atrophy without

locomotor training was greatest during the first week after a moderate contusion SCI in

rats, although the greatest absolute amount of atrophy in untrained animals occurred 2

weeks after SCI (chapter 4). Of the hindlimb muscles studied, the triceps surae soleuss

and gastrocnemius muscles combined) demonstrated the greatest amount of atrophy

(26.3%), which is consistent with other studies of muscle disuse that have described

greater atrophy of the antigravity extensor muscles than the flexor muscles across a

variety of animal species. In addition, the aforementioned study (chapter 4) found that

one week of locomotor training reduced atrophy of the triceps surae by over 15%

compared to untrained, injured animals. The present study provides additional evidence

for the merits of early intervention in the form of not only whole muscle cross sectional

area measurements, but via measurements of muscle contractile function combined with

muscle fiber size measurements.









Additional studies that have investigated the impact of early locomotor training

after a contusion SCI have not included muscle-specific measures of adaptations, yet they

provide additional support for functional improvements with early exercise intervention

(79, 158). Engesser-Cesar et al. (2005) found that mice with a contusion SCI that

participated in one week of flat-surface wheel running starting one week after injury had

significantly better BBB scores than injured mice without training. Multon et al. (2003)

began a 12 week locomotor training study with weekly BBB measurements after

inducing a spinal cord compression injury in rats with a subdurally inflated microballoon.

Training began within a week of injury and resulted in differences in BBB scores by 2

weeks post injury, consistent with the locomotor behavior results of the present study.

In the present study, we found a good relationship between BBB scores and

soleus tetanic force production, even though the soleus only represents one of many

lower extremity muscles that are involved in hindlimb function. Nevertheless, these

results suggest that while the soleus may be more susceptible to disuse with injury (2, 28,

225), its force production appears to be fairly representative of collective hindlimb

functional deficits as measured by the BBB Locomotor Rating Scale. The BBB scale has

been shown to be a valid and predictive measure of locomotor recovery and allows for

comparisons across different injuries to evaluate treatment efficacy after SCI (24, 25). In

fact, our BBB scores 1 week after injury were similar to those reported by Hutchinson et.

al. (2001) and therefore allow for more direct comparisons across these 2 investigations

of muscle adaptations after a moderate contusion SCI.

As little as five days of locomotor treadmill training appeared to decrease soleus

muscle fiber atrophy, with no differences in fiber CSA between control and trained SCI









animals. On the other hand, SCI animals without training responded similarly to other

models of limb disuse (70, 116), such that fiber CSA without training was significantly

different from control and trained animals after only two weeks after SCI. In addition,

the transformation towards faster, more fatigable fiber types in the untrained, SCI group

was evident from our electrically elicited fatigue test with the soleus muscle of untrained

animals demonstrating -27% more fatigue than trained, SCI animals or control animals.

At the same time, twitch contraction properties (peak force, time to peak tension, 1/2

relaxation time) were not different across groups, despite evidence for initial fiber type

transformations in injured animals. Quite possibly, measurements of twitch contractile

properties were not sensitive enough to detect small changes in fiber type MHC

composition with a single electrically elicited contraction early after injury. In contrast to

single isolated twitches, fatigue testing consisted of a series of consecutive contractions

delivered over 2 minutes, the cumulative effects of which might allow for better detection

of early differences in phenotypic transformations of fiber types such as increased

fatigability.

It is not clear from the present study whether locomotor training early after injury

prevents changes in contractile function and maintains normal muscle morphology or

actually reverses any changes that have occurred within the first week after injury. But it

is clear that early exercise intervention with force hindlimb loading counters the disuse

atrophy present in the limbs of injured animals, which are flaccid and unloaded. A study

by Burnham et al. (1997) supports our findings that early changes in MHC after SCI are

present in the absence of training and suggests that interventions aimed at preventing or

minimizing the transformation would need to be implemented within weeks after SCI to









prevent changes (40). Timing appears to be critical because waiting longer after SCI

injury to initiate an exercise training program has been shown to have less benefit on the

soleus muscle of rats injured by transaction (71). Although comparisons across different

SCI methods are not always generalizable, this study suggests that waiting as long as 4

weeks after injury may not offer the protective benefits that early exercise interventions

may offer.

Although there are limitations in using animal models to understand human SCI

recovery with locomotor training, animal models allow for a better understanding of the

functional properties of complex systems. The results of the present suggest that starting

some form of exercise training after SCI as early as medical stabilization of patients

allows may offer the greatest protective benefits for maximizing functional recovery.














CHAPTER 6
CHANGES IN SOLEUS FIBER TYPE COMPOSITION WITH ONE WEEK OF
TREADMILL LOCOMOTOR TRAINING IN SPINAL CORD CONTUSION INJURED
RATS

6.1 Abstract

Neuromuscular activity plays a very important role in specifying muscle

phenotypic properties. The objective of this study was to determine the impact of one

week of treadmill training on the soleus fiber type composition following spinal cord

contusion injury. Thirty five adult Sprague Dawley rats (female, 16-20 weeks, weighing

250-290g) were studied. Twenty four animals received a moderate T8 spinal cord

contusion injury. Twelve of the SCI rats received treadmill locomotor training (TM),

starting 1 week after SCI for 5 consecutive days, 20 minutes/trial, 2 trials/day. The

additional twelve injured rats received no exercise intervention (no TM). The soleus

muscle fiber type composition was determined using immunofluorescence techniques

with monoclonal antibodies (BA-D5, SC-71, BF-F3, and BF-35). Two weeks following

spinal cord contusion injury, the proportion of type I fibers decreased to 75.1 3.1% (vs

86.1% in the uninjured animal). In addition, the soleus contained 8.8 2.0% of fibers that

were co-labeled for MHC-I and MHC-IIa and 1.4 1.1% of fibers that reacted with both

IIa and IIx. Compared to the SCI soleus, 1-week of locomotor training resulted in a

significant increase in the soleus muscle type I fibers (81.3 1.7%) and a reduction in the

proportion of fibers that were stained positively with both type I and IIa (3.4 1.2%).

There were no fibers that reacted with both type IIa and IIx in the SCI-TM soleus. In

addition, training paradigms significantly increased the soleus fiber cross-sectional area









(CSA) across the different fiber types, with an average type I fiber CSA of2170im2 and

type IIa of 1402im2 in the no TM group, 2884 im2 and 1781lm2 in the TM group,

respectively. Collectively, these results indicate that early therapeutic intervention using

treadmill locomotor training can effectively reverse both the shift in fiber type

composition and decrease in fiber type specific CSA following SCI.

6.2 Introduction

Neuromuscular activity (including electrical activation and limb loading) plays a

very important role in specifying muscle phenotypic properties (see review (213)).

Following SCI, skeletal muscle shows an increase in the percentage of fast fibers and a

decrease in the percentage of slow fibers in both human subjects and animal models (15,

114, 121, 122, 137). Unfortunately, to date, very little data exists describing muscle fiber

type changes after incomplete SCI. Interestingly, the innate plasticity associated with

incomplete SCI furnishes the potential to possess greater plasticity than complete SCI

(162, 232). In a pilot study, we found that 3 months after spinal cord contusion injury the

rat soleus muscle displays a much higher proportion of fibers expressing the fast MHC

isoforms compared to control soleus muscles, with 22% of the total number of fibers

being MHC-I compared to -10% in control muscles (138). However, limited studies have

investigated the fiber type composition of skeletal muscle early after contusion spinal

cord injury. In addition, little information is available in regard to the fiber type specific

changes in fiber CSA.

Interventions that result in increased neuromuscular activation, such as electrical

stimulation (170), functional overload and endurance exercise, have been shown to

induce shifts in MHC towards the slower isoforms (6). For example, treadmill training of

spinalized cats has been demonstrated to prevent conversion of soleus myofibers from









slow oxidative to fast glycolytic (193). Roy et al. (193) trained adult spinal cord

transected cats on a treadmill for around 5 months and found that step trained cats

showed 100% type I MHC in the soleus, compared to 82% untrained cast. Despite these

promising findings, less attention has focused on how skeletal muscle responds to acute

training interventions of shorter duration.

Therefore the purpose of this study was to determine the impact of moderate T8

spinal cord contusion injury on acute changes in rat soleus fiber type composition. A

second aim of this study was to determine the effect of 1-week treadmill locomotor

training on rat soleus fiber type composition following moderate T8 spinal cord contusion

injury.

6.3 Materials and Methods

6.3.1 Experimental Animals

Thirty five adult Sprague Dawley rats (female, 16-20 weeks, weighing 250-290g)

were studied. The rats were housed in a temperature-controlled room at 21 C with a

12:12 hours light:dark cycle and were provided rodent chow and water ad libitum. All

procedures were performed in accordance with the U.S. Government Principle for the

Utilization and Care of Vertebrate Animals and were approved by the Institutional

Animal Care & Use Committee at the University of Florida.

6.3.2 Spinal Cord Contusion Injury

Spinal cord contusion injuries were produced in all rats except controls using a

NYU (New York University) impactor. Briefly, 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 Baytril (10mg/kg) per day for 5 days, starting

the day of surgery. Procedures were performed under ketamine (100mg/kg)-xylazine









(6.7mg/kg) anesthesia (details in (179, 226)). Subcutaneous lactated Ringer's solution (5

ml) was administered after completion of the surgery. Animals were given

Buprenorphine (0.05mg/Kg IM) and Ketoprofen (5.0 mg/Kg SC) for pain and

inflammation over the first 36hrs after SCI. The animals 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 were monitored for the possibility of urinary tract

infection. After SCI, animals were housed individually. On post-operative day 7, open

field locomotion was assessed using the Basso-Beattie-Bresnahan (BBB) locomotor scale

(24) and animals that did not fall within a preset cutoff (BBB<8) were excluded from the

study. Animals were subsequently randomly assigned to either SCI-treadmill training

group (SCI-TM, n=12) and SCI no training control group (SCI, n=12). In addition, the

rats that did not receive SCI were served as control group (CON, n=l 1).

6.3.3 Treadmill Locomotor Training

Animals in training paradigms animals were trained continuously for 5 Day (2

trials/day, 20 minutes/trial), starting on post-operative day 8. Animals assigned to the

treadmill training group were given five minutes to explore the treadmill on the first

training day and then encouraged to walk on the moving treadmill (11 mpm) (130) for a

series of four, five-minute bouts. A minimum of five minutes rest was provided between

bouts. On the second day of training, animals completed two bouts often minutes each,

twice a day. Starting on day 3, animals trained continuously for 20 minutes with a

minimum interval between trials of 2 hours. Body weight support was provided manually

by the trainer. The level of body weight support was adjusted to make sure that rats could

bear their weight and there was no collapse of their hindlimbs. Typically, the rats started









stepping when they experienced some small load on their hindlimbs. In addition, during

the first week of training, when all rats had profound paraplegia, assistance was provided

to place rat hindlimbs appropriately for plantar stepping position during training.

6.3.4 Immunohistolochemistry

At the end of the experiment, the soleus muscles were removed from both

hindlimbs. The muscles were subsequently rapidly frozen at resting length in isopentane,

precooled in liquid nitrogen (storage at -80C). Tranverse cryostat sections (10 tm) were

prepared from the central portion of each muscle and mounted serially on gelatin-coated

glass slides. Immunocytochemical reactions were performed on serial cryostat sections

with anti-laminin and anti-MHC antibody at various dilutions. Rabbit anti-laminin

(Neomarker, Labvision, Fremont, CA) was used to outline the muscle fibers for cross

sectional area quantification. Four anti-MHC Mabs (BA-D5, SC-71, BF-F3, and BF-35)

were selected on the basis of their reactivity toward adult MHC (Table 6-1). Sections

were incubated with rabbit anti-laminin and one of the anti-MHC antibodies (40C over

night), followed by incubation with rhodamine-conjugated anti-rabbit IgG and Fitc-

conjugated anti-mouse IgG (Nordic Immunological Laboratories). Stained sections were

mounted in mounting medium for fluorescence (Vector Laboratories, Burlingame, CA)

and kept at 40C to diminish fading. Stained cross sections were photographed (10X

magnification) by using a Leica fluorescence microscope with a digital camera. A region

of the stained serial sections from each muscle were randomly selected for MHC

composition analysis. The proportions of each fiber type were determined from a sample

of 150-250 fiber across the entire section of each muscle. In addition, the soleus fiber

CSAs were analyzed using NIH image (version 1.62). The pixels setting used for









conversion of pixels to micrometer were 1.50 pixels- 1 am2 for a 10 X objective. The

average fiber CSA of all the fibers in each fiber type was determined.

Table 6-1. Monoclonal antibody specificity


MHC isoforms
MAb
I IIa IIx IIb
BA-D5 + -
SC-71 + -
BF-F3 +
BF-35 + + +

6.3.5 Data Analysis

One way ANOVAs with Bonferroni-Dunn post hoc testing were used to compare

results across groups (controls, trained and untrained animals) for soleus muscle fiber

type composition and average CSA. A p value of <0.05 was considered significant.

6.4 Results

6.4.1 Soleus fiber type composition

As shown in Figure 6-1 & 6-4, the control soleus muscleprimarily contained fibers

reacting exclusively with type I mAb (86.1 2.2%) and a small percentage of fibers

(13.9 2.2%) reacting with type IIa mAb exclusively. Two weeks following spinal cord

contusion injury, the proportion of type I fibers decreased to 75.1 3.1%. In addition, the

soleus contained 8.8 2.0% of fibers that were co-labeled for MHC-I and MHC-IIa and

1.4 1.1% of fibers that reacted with both IIa and IIx (Figure 6-2, 6-4). Compared to the

SCI soleus, 1-week of locomotor training resulted in a significant increase in the soleus

muscle type I fibers (81.3 1.7%). There was no significant difference in fibers that were

stained only with type IIa mAb among different groups. In addition, the proportion of

fibers that were stained positively with both type I and IIa deceased to 3.4 1.2% during









the first week of treadmill training. There were no fibers that reacted with both type IIa

and IIx in the SCI-TM soleus (Figure 6-3, 6-4).



















Figure 6-1. Serial cross sections of a control soleus stained with monoclonal antibodies
directly against specific MHC isoforms. A) BA-D5 (anti-MHC-I). B) SC-
71(anti-MHC-IIa). C) BF-35(anti-all MHCs except IIx). D) BF-F3 (anti-
MHC-IIb).



















Figure 6-2. Serial cross sections of a SCI no TM soleus stained with monoclonal
antibodies directly against specific MHC isoforms. A) BA-D5 (anti-MHC-I).
B) SC-71(anti-MHC-IIa). C) BF-35(anti-all MHCs except IIx). D) BF-F3
(anti-MHC-IIb).




























Figure 6-3. Serial cross sections of a SCI + TM soleus stained with monoclonal
antibodies directly against specific MHC isoforms. A) BA-D5 (anti-MHC-I).
B) SC-71(anti-MHC-IIa). C) BF-35(anti-all MHCs except IIx). D) BF-F3
(anti-MHC-IIb).

6.4.2 Soleus fiber cross-sectional area

Compared with the control soleus muscle, spinal cord contusion injury resulted in a

significant decrease in soleus fiber CSA, across the different fiber types. At 2w-SCI, the

CSA of type I and IIa fiber was reduced by 20.5% and 17.0% respectively (Figure 6-5).

1-week treadmill training effectively prevented the atrophic response of skeletal muscle

observed following SCI. There were no significant differences in type I and IIa fiber

areas between control and treadmill trained soleus. In addition, the fiber CSA of hybrid

fibers (type I + type IIa) was significantly higher when compared with SCI soleus (Figure

6-5).










M Control
II SCI no TM
E c SCI + TM


I I Ila Ila Ila + IIx


Figure 6-4. MHC based fiber type percentage composition, as determined by
immunohistochemistry, of rat soleus muscle form control, SCI no TM and
SCI + TM group. Significant difference compared with the SCI no training
group.


M Control
II SCI no TM
S SCI + TM


S1500

U 1000.


Figure 6-5. Soleus muscle fiber type specific CSA in control, SCI no TM, and SCI + TM
groups at 2 weeks post SCI. Significantly less average muscle fiber CSA in
SCI no TM compared to control and SCI + TM groups.

6.5 Discussion

Two weeks following midthoracic spinal cord contusion injury, we observed a

significant shift in fiber type composition from slow to fast in the rat soleus muscle, as









well as a significant decrease in fiber type specific soleus fiber CSA. However, 1-week

treadmill training effectively attenuated the soleus fiber type shift as well as the fiber type

specific decrease in fiber CSA.

SCI results in measurable reductions in the amount of electrical activation in

mammalian muscles. For example, in cats, a complete SCI results in a 75% reduction in

daily integrated electromyographic activity of the soleus muscle (7). As a result, complete

spinal cord transaction has been shown to induce a large increase in the expression of fast

MHC isoforms (121, 122, 215, 217, 222). Talmadge et al. (222) showed that the

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

months following a complete mid-thoracic SCI. One of the few studies to closely

examine muscle adaptations after a contusion injury in rats showed an upregulation of the

transitional type IIx in the soleus and extensor digitorum longus muscle within the first 3

weeks after SCI (116). In addition, we previously showed in a pilot study that the soleus

MHC isoforms were shifted from MHC-I towards MHC-II 3 months after SCI, with

approx. 77.9% (vs 85.1% in the uninjured animal), 19.9% (vs 15.0% in the uninjured

animal), and 4.3% (vs 0% in the uninjured animal) of the total fibers containing MHC-I,

MHC-IIa and MHC-IIx, respectively (138). In the present study, we observed a shift in

the fiber type composition towards type II as early as 2 week post SCI, providing

additional evidence to support the role of neuromuscular activity in specifying muscle

phenotypic properties.

In this study we also found an increase in the presence of hybrid fibers following

contusion SCI. 2 weeks after contusion SCI nearly 8.2% of the soleus muscle fibers in the

untrained SCI rats were hybrid fibers. Our findings are consistent with observations









previously reported (222). For example, Talmadge (222) found that there were high

proportions of type I/IIa hybrids in the soleus muscle at 15 days after spinal cord

transaction. The mechanism behind the presence of hybrid fiber is not clear. Originally, it

was thought that muscle was in a transitional state of fiber transformation. However,

studies have found that hybrid fibers could exist for up to one year after SCI, suggesting

that hybrid fibers could be a stable phenotype under a particular condition (222). In the

present study, we found there were no pure type IIx fibers. All fibers that contained IIx

were mixed with IIa.

The unique findings of the present study were that only 1 week of daily step

training emphasizing weight support on a treadmill could effectively ameliorate, although

not reverse the shift in muscle fiber type composition observed following mid-thoracic

contusion injury. The soleus muscle of the 1-week trained SCI animals showed 81.3%

type I fibers, whereas the soleus muscle in the untrained SCI animals displayed 75% type

I fibers. Numerous studies have shown an increased proportion of type I fibers in skeletal

muscle after different exercise and training programs (6, 61, 193). For example, Demirel

et al. (61) found that treadmill training at all different durations (30, 60, or 90 min/day)

resulted in a reduction in the percentage of MHCIIb and an increase in the percentage of

MHCIIa in the plantaris muscle. Unfortunately, to date, the regulatory mechanism for

changes in muscle fiber type composition is still under debate. Recent studies suggested

that calcineurin plays a very important role in the regulation of fiber phenotypic

transformations (48, 208, 243). It has been hypothesized that calcineurin acts as a calcium

server to transform mechanical stimulis to muscle signaling pathways (208). Thus

increased muscle activation and limb loading during treadmill training may elevate









cytosolic calcium, and as such facilitate the fiber type transformation mediated by

calcineurin (see review (129)).

Collectively, the data presented here clearly demonstrate that 2 weeks of SCI result

in a fiber type shift in the rat soleus muscle towards faster fiber types. In addition, the

increased coexpression of MHC isoforms in all injured animals may point to a dynamic

process of fiber type transformation in the early weeks following contusion SCI. More

importantly, as little as five days of locomotor treadmill training appeared to attenuate the

shift in MHC composition towards faster isoforms and ameliorate fiber type specific

changes in fiber CSA. Future work should be directed towards explaining mechanisms by

which physical activity changes skeletal muscle phenotype and influences muscle

plasticity after SCI.














CHAPTER 7
EFFECTS OF TREADMILL TRAINING ON IGF-I EXPRESSION IN RAT SOLEUS
MUSCLE FOLLOWING SPINAL CORD INJURY

7.1 Introduction

One of the primary consequences of spinal cord injury (SCI) is pronounced muscle

atrophy and loss of muscle function distal to the site of injury (116, 136, 137, 178).

Recently, locomotor training programs have shown to help maintain muscle mass and

strength and thereby promote functional recovery (63, 176). In a previous study, we

showed that locomotor treadmill training ameliorates the loss in muscle size in the rat

hind limb muscles following midthoracic spinal cord contusion injury (139). Although

the effect of training was prevalent for up to 3 months, the largest therapeutic impact of

locomotor training was observed within the first week of training. In addition, we showed

that following 1 week of locomotor training muscle strength and fiber specific muscle

CSA in the postural slow twitch soleus muscle of trained spinal cord injured rats was

significantly higher than that of non-trained SCI rats (Chapter 5, 6).

Although the exact mechanisms for how locomotor training potentially confers its

benefits are not well understood, a number of signaling pathways have been proposed to

potentially regulate cellular and molecular processes involved in skeletal muscle

remodeling (33, 53, 68, 118). Insulin-like growth factor I (IGF-I) has been shown to play

a particularly important role in mediating protein synthesis, protein degradation and

satellite cell mediated repair (1). Systemic release of IGF-I may contribute to an increase

in protein content and a reduction in protein degradation in skeletal muscle (247). In









addition, overexpression of IGF-I has been associated with myofiber hypertrophy in

transgenic mice (51), and local infusion of IGF-I has been shown to contribute to skeletal

muscle hypertrophy (1), as well as block the aging-related loss of muscle mass in mice

(19). Moreover, resistance training has been associated with increased IGF-I mRNA

expression in both animal models (3) and individuals with complete spinal cord injuries

(29). Although, the effect of treadmill locomotor training on the muscle IGF-I signaling

pathway has not been studied, such information may help explain some of the positive

therapeutic benefits of locomotor training.

The purpose of this study was to determine the impact of treadmill training on

mRNA expression of IGF-I and its related receptor (R) and binding proteins (BP) in rat

soleus muscle following moderate T8 spinal cord contusion injury.

7.2 Materials and Methods

7.2.1 Experimental Animals

Thirty adult Sprague Dawley rats (female, 16-20 weeks, weighing 250-290g) were

studied. The rats were housed in a temperature-controlled room at 210C with a 12:12

hours light:dark cycle and were provided rodent chow and water ad libitum. All

procedures were performed in accordance with the U.S. Government Principle for the

Utilization and Care of Vertebrate Animals and were approved by the Institutional

Animal Care & Use Committee at the University of Florida.

7.2.2 Spinal Cord Contusion Injury

Spinal cord contusion injuries were produced in all rats except controls using a

NYU (New York University) impactor. Briefly, 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 Baytril (10mg/kg) per day for 5 days, starting