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Effects of Acute Locomotor Training with or without Baclofen Therapy on Spasticity after Contusion Spinal Cord Injury (SCI)


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1 EFFECTS OF ACUTE LOCOMOTOR TRAI NING WITH OR WITHOUT BACLOFEN THERAPY ON SPASTICITY AFTER CONT USION SPINAL CORD INJURY (SCI) By RITA I. JAIN A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2006

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2 Copyright 2006 by Rita I. Jain

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3 To the memory of my mother and grandfathe r-in-law whom I will miss throughout my life.

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4 ACKNOWLEDGMENTS I would like to thank Dr. Prodip Bose and Dr. Floyd Thomson who have been excellent mentors and supervisors during my studies at the University of Florida. Their inspiring enthusiasm and energy have been contagious, a nd their advice and constant support have been extremely helpful. I would also like to thank Dr. Ammon Peck for his guidance and for serving in my committee. I would also like to tha nk my lab personnel Ron Parmer and Yanping Cheng for their help during the entire process of my project work. I owe much of my academic and personal succ ess to my parents, my in-laws and my brother Niranjan, who, by example, provided me w ith the motivation and courage to pursue my studies. The greatest thanks to my husband, Sanjay, for his tremendous encouragement, understanding, patience, and unconditional love and to my daughter Risha, whose arrival encouraged me to complete my project at the earliest.

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5 TABLE OF CONTENTS Page ACKNOWLEDGMENTS...............................................................................................................4 LIST OF TABLES................................................................................................................. ..........7 LIST OF FIGURES................................................................................................................ .........8 LIST OF ACRONYMS AND ABBREVIATIONS......................................................................10 ABSTRACT....................................................................................................................... ............11 INTRODUCTION................................................................................................................... ......13 Spinal Cord Injury............................................................................................................. .....13 Spasticity..................................................................................................................... ............13 BACKGROUND..................................................................................................................... ......16 Spinal Cord Injury Animal Models........................................................................................16 Spinal Cord Contusion Model (Inc omplete Spinal Cord Injury)....................................17 Spinal Transection Model (Com plete Spinal Cord Injury).............................................17 Spinal Isolation Model....................................................................................................18 Anatomical (Neuromodulatory) Changes Following SCI......................................................18 A Rodent Model of Post-SCI Spasticity.................................................................................19 Mechanism of Spasticity........................................................................................................ .19 Present Antispasticity Treatment for Spinal Cord Injury.......................................................22 Locomotor Rehabilitation....................................................................................................... 24 EXPERIMENTAL DESIGN AND THE STANDARD METHODOLOGY USED IN THE PROJECT........................................................................................................................ .......29 Objective...................................................................................................................... ...........29 Animal Subject................................................................................................................. ......29 Contusion Injuries............................................................................................................. ......30 Baclofen Pump Implantation..................................................................................................31 Bicycle Training............................................................................................................... ......32 Footprints..................................................................................................................... ...........33 BBB Score...................................................................................................................... ........33 Ankle Torque................................................................................................................... .......34 Immunocytochemistry of Spinal Cord Tissue........................................................................36 RESULT......................................................................................................................... ...............38 Velocity-Dependent Ankle Torque and Associated EMGs..................................................38 Pre-injury Ankle Torque and EMG Data........................................................................38

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6 Postoperative Week 1 Ankle Torque and EMG Data.....................................................39 Postoperative Week 4 Ankle Torque and EMG Data.....................................................41 Postoperative Week 8 Ankle Torque Data and EMG Data.............................................43 Gait and Open Field Locomotion...........................................................................................46 Footprint Analysis: Gait..................................................................................................46 Open Field Locomotor Recovery....................................................................................47 Neurotransmitters.............................................................................................................. .....50 GABAb............................................................................................................................50 BDNF........................................................................................................................... ...51 GAP43..............................................................................................................................5 2 GAD67..............................................................................................................................5 2 Result Summary................................................................................................................. .....53 DISCUSSION..................................................................................................................... ...........54 Ankle Extensor Stretch Reflex...............................................................................................54 Saline Control................................................................................................................. .54 Saline Cycle................................................................................................................... ..55 Baclofen Control.............................................................................................................56 Baclofen Cycle................................................................................................................5 7 Open Field Locomotor Assessment........................................................................................58 Saline Control................................................................................................................. .58 Saline Cycle................................................................................................................... ..59 Baclofen Control.............................................................................................................59 Baclofen Cycle................................................................................................................6 0 Footprints..................................................................................................................... ...........61 Saline Control................................................................................................................. .61 Saline Cycle................................................................................................................... ..62 Baclofen Control.............................................................................................................62 Baclofen Cycle................................................................................................................6 2 Neurotransmitters.............................................................................................................. .....63 Saline Control................................................................................................................. .63 Saline Cycle................................................................................................................... ..64 Baclofen Control.............................................................................................................64 Baclofen Cycle................................................................................................................6 5 CONCLUSION..................................................................................................................... .........66 FUTURE WORK.................................................................................................................... .......69 LIST OF REFERENCES............................................................................................................. ..70 BIOGRAPHICAL SKETCH.........................................................................................................77

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7 LIST OF TABLES Table Page 4-1 Ankle torque week 1Percentage (%) change ( ) compared to pre-injury.....................41 4-2 Electromyeloencephalogram week 1 Percentage (%) change ( ) compared to preinjury......................................................................................................................... .........41 4-3 Ankle torque week 4Percentage (%) change ( ) compared to pre-injury.....................43 4-4 Electromyeloencephalogram week 4Percentage (%) change ( ) compared to preinjury......................................................................................................................... .........43 4-5 Ankle torque week 8 Percentage (%) change ( ) compared to pre-injury....................45 4-6 Electromyeloencephalogram week 8 Percentage (%) change ( ) compared to preinjury......................................................................................................................... .........45

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8 LIST OF FIGURES Figure Page 3-1 The experimental set up and procedures done in chart form.............................................30 3-2 Motorized bicycle used for training the rats......................................................................32 3-3 Ankle torque, displacement and EMGs ar e simultaneously recorded and timelocked to onset of dorsiflexion.........................................................................................34 4-1 Pre-injury ankle torque graph............................................................................................38 4-2 Pre-injury EMG graph....................................................................................................... 38 4-3 Ankle torque graph at post-op w eek 1 with significant differences..................................39 4-4 Electromyeloencephalogram graph at postop week 1 with significant differences.........39 4-5 Ankle torque graph at post-op w eek 4 with significant differences..................................41 4-6 Electromyeloencephalogram graph at postop week 4 with significant differences.........42 4-7 Ankle torque graph at post-op w eek 8 with significant differences..................................43 4-8 Electromyeloencephalogram graph at postop week 8 with significant differences.........44 4-9 Axial rotation graph of all 4 groups at different time points with significant differences.................................................................................................................... ......46 4-10 Base of support graph of all 4 groups at different time points with significant differences.................................................................................................................... ......46 4-11 Graph of BBB showing percentage change in score at post-op week8 compared to post-op week 4................................................................................................................. ..47 4-12 Open field locomotor graph showing BBB score of all 4 groups at different time points with significant differences.....................................................................................48 4-13 Immunohistochemistry of GABAb receptors in lumbar spinal cord tissues......................50 4-14 Immunohistochemistry of 5-HT (Serotonin) fibers in thoracic spinal cord tissues...........51 4-15 Immunohistochemistry of BDNF (Brain deri ved neurotropic factor) in lumbar spinal cord tissues................................................................................................................... ......51

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9 4-16 Immunohistochemistry of GAP43 fibers in central canal of thoracic spinal cord tissues........................................................................................................................ .........52 4-17 Avidine-Biotine Complex (ABC) figures of GAD67 fibers in thoracic spinal cord tissues........................................................................................................................ .........52

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10 LIST OF ACRONYMS AND ABBREVIATIONS ANOVA Analysis Of Variance BBB Basso, Beattie and Bresnahan scale BDNF Brain Derive d Neurotropic Factor Ca2+ channels Calcium channels Clchannels Chloride channels CPG Central Pattern Generator Deg/sec Degrees/second EMG Electromyeloencephalogram EPSPs Excitatory Postsynaptic Potentials GABAb Gamma Amino Butyric Acid GAP43 Growth Assosiated Protein 43 IgG Immunoglobulin G IHC Imunohistochemistry ITB Intrathecal Baclofen Kdynes Kilodynes MASCIS Multicenter Animal Spinal Cord Injury Studies Na+ channels Sodium channels PAD Primary Afferent Depolarization PIC Persistent Inward Currents SCI Spinal Cord Injury VDAT Velocity Dependent Ankle torque 5-HT 5-Hydroxytryptamine (Serotonin)

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11 Abstract of Thesis Presen ted to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science EFFECTS OF ACUTE LOCOMOTOR TRAI NING WITH OR WITHOUT BACLOFEN THERAPY ON SPASTICITY FOLLOWING CO NTUSION SPINAL CORD INJURY (SCI) By Rita I. Jain December 2006 Chair: Floyd Thompson Cochair: Prodip Bose Major Department: Medical Sciences Progress has been made in the therapeutic tr eatment of spasticity, especially utilizing intrathecal baclofen (ITB). In a ddition to medical treatment, two decades of studies in animals and humans report exciting possibilities for loco motor training to improve locomotor recovery. However, many unknowns remain regarding the timi ng of treatments, and whether they interact to facilitate or inhibit rehabilitation directed at recovery of volunt ary motor activity. Since spasticity development is progressive, we explor ed the potential for acute initiation of therapies to influence the development of spasticity. These studies were performed to evaluate the safety, feasibility, and efficacy of tw o early intervention treatments (performed alone, or in combination) on measures of spasticity and l ong term functional outcome measures following midthoracic contusion SCI. Four groups of animals received contus ion injuries to the midthoracic spinal cord using the New York University (NYU) impounder and the Multicenter Animal Spinal Cord Injury Studies (MASCIS) pr otocol for moderate injury (10 gm weight drop at a distance of 12.5mm). Two groups of animal s received ITB pumps (Azla Corp., Palo Alto, CA) at the time of injury; the other two groups recei ved pumps with saline vehicle. The tip of the intrathecal cannula was placed in the subdural space of the L1-L2 lumbar spinal cord. Beginning

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12 at day 1 following injury, two 20 minutes sessions of locomotor exercise were performed using a custom made cycle locomotor trainer on one ITB and one vehicle control group. Velocity dependent ankle torque, ankle ex tensor muscle EMGs, hindlimb axis, and open field locomotion (BBB), were recorded in all groups The data indicate that at 2 months following injury, cycle training alone produced a greater re duction in ankle torque than baclofen alone. However, ankle extensor spasticity was significantly lower, and ra te of open field recove ry was greatest in the animals that received the combin ation of ITB and cycle training, compared with animals that receiving no treatment or either of the treatments performed alone. No between group differences were observed in footprint data for acute treatments alone or combined. Terminal Immunohistochemistry of lumbar spinal cord segm ents revealed increased expression of markers for GABA (GABAb, GAD67) & monoamines (DBH (NE), and 5-HT) in combined treated animals. Treatment relate d upregulation of GABA/GABAb molecules might exert significant roles in pre-synaptic inhibition and upragulated monomamines might prevent supersensitivity of monoamine receptors (a-1 & 5-HT) and thus might block both the early onset and the late onset of spasticity. These data indicate that acute tr eatments using locomotor exercise / ITB are safe and feasible initiated as early as postcont usion day 1 and ITB and locomotor treatments differentially influenced early and late onset of the development of spasticity.

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13 CHAPTER 1 INTRODUCTION Spinal Cord Injury The spinal cord (SC) is a vital organ of the body, which is necessary for the maintenance of posture and locomotion. Trauma to the SC leads to a series of events resulting in changes in reflex excitability from spinal shock to a hyperr eflexive condition known as spasticity. Frequent causes of SC damage are trauma (car accident, g unshot, falls, etc.) or dis ease (polio, spina bifida, Friedreich's Ataxia, etc.). The SC does not have to be severed in order for a loss of functioning to occur. In fact, in most people with spinal cord in jury (SCI), the SC is in tact, but the damage to it results in loss of functioning. About half of the patients with SC cord trauma have incomplete injuries without any signs of voluntary motor or sensory perception below the level of the lesion. The victims of cord trauma are mainly young men in their early 20s to 30s (National Spinal Cord Injury Statistical Center). Efforts should be made to improve the quality of life of the young victims and to reduce the c linical burden in society. To date, no suitable therapy for the victims of SCI is available. New therapeutic strategies with the possibility of regeneration of the lesioned SC axons are needed to improve the quality of life of patients with SCI. Spinal cord injury induces secondary biochemical responses that include both neurotoxic and neuroprotective processes (Tator,1995). It is believe d that the balance between these reactions in part determines ultimate tissue damage and the degree of asso ciated neurological recovery. Although functional regeneration appears to be limite d after SCI in mammals, it is not unlikely that plasticity of surviving cells contributes to functional recovery that is often observed. Spasticity Spasticity is a form of muscular hypertonia, due to a velocity -dependent increase in tonic stretch reflexes during passive movement, whic h results from abnormal spinal processing of

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14 proprioception after SCI. Spasticity, is a major h ealth problem for patients with a SCI. It limits patients mobility and affects their independence in activities of daily living and work. Spasticity may also cause pain, loss of range of motion, co ntractures, sleep disorder s and impair ambulation in patients with an incomplete lesion. The effec tiveness of available drugs is still uncertain and they may cause adverse effects. Assessing what work s in this area is complicated by the lack of valid and reliable measurement tools (Taricco et al. 2006). The present treatment for spasticity is the use of anti-spastic drugs su ch as baclofen, which is a GABAb agonist. But the appropriate dose and the ti me frame at which it should be started to treat spasticity is not known. In humans, baclofen is given chronically i.e., 1 month after the injury, as the FDA does not approve of its use ac utely. Whether baclofen given acutely after SCI can prevent the development of spasticity is not known. It is not known that acute administration of baclofen administration is safe. In animals a nd humans following SCI, spasticity appears at a chronic stage. One of the primary goals of my thesis was to obtain relevant information regarding baclofens safety and efficacy in the acute stage, before the onset of spasticity. Rehabilitation in the form of tr eadmill training is widely used in human SCI cases to improve their gait. Treadmill training in humans is laborious and requires a lot of manpower. Setups are available in only a few centers across the nation, so the training is not accessible to all the patients. The purpose of this study was also to te st the hypothesis that a customized motorized bicycle could be used instead of the treadmill to tr eat spasticity in the acute stage and to maintain the feasibility and safety of the patient wit hout any deterioration in his condition. This study compared combination therapy of drug (baclof en) and locomotor training (bicycle) with individual therapy, started acutely after midthoracic contusion SCI in animal model, and to test if

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15 combination therapy, started acutely, is more usef ul in preventing the deve lopment of spasticity and in improving gait. The wide range of outcome measures of spasticity and gait a nd comparison of two treatment modalities (locomotor training and drug therapy) proposed in this study might provide translational data which ultimately influence the quality of SCI patients. These studies propose a strategy to utilize quantitative measures of be havior, neurophysiology, IHC, and imaging (MRI) techniques to further increase ou r understanding of neurobiology of spasticity and gait following SCI and locomotor rehabilitation. Translation of these findings may provide safe, timely, and effective intervention strategies and evidence-bas ed resources for translatable therapeutic design, which can ultimately benefit the SCI patient.

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16 CHAPTER 2 BACKGROUND This chapter presents the relevant background of SCI induced spasti city and gait problems as well as present treatment modalities to deal with these clinical problems. The scientific society studying SCI uses various types of in jury models in animals to mimic the type of injury seen in humans but the most commonly used method for SCI is mid-thoracic co ntusion SCI which has documented to develop spasticit y, even though there are various ot her types of injury models which are presented below: Recent studies of SCI models have indicat ed an increasing need for a more thorough understanding of the multiple components of sp inal cord segmental plasticity for which therapeutic interventions can be rationally target ed by various intervention s, including locomotor training (Raineteau, & Schwab 2001). In that contex t, this thesis work was focused on spasticity a common aftermath of SCI that reflects ma ladaptive changes in sp inal cord circuitry affecting motoneuron excitability and output. Sp asticity is often one of the most difficult neurological consequences to manage. Therefore, studies that can lead to a more in-depth appreciation of the underpinnings of spasticity and therap ies that can attenuate its impact are of great importance. Accordingly, this project addresses locomotor trai ning rehabilitation with or without drug (baclofen) thera py to test the effect(s) on sp asticity using multidisciplinary analytical approaches. Spinal Cord Injury Animal Models Animal models of SCI can be used to study the lesion development, study the mechanism of recovery, and to develop th erapeutic intervention. The expe rimental SCI animal models include the transection, is olation and contusion.

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17 Spinal Cord Contusion Model (Incomplete Spinal Cord Injury) Basically, human spinal cord injuries are cau sed by transient compre ssion or contusion of the spinal cord even though pene trating wounds of the spinal co rd can result from a knife or gunshot. So, we used animal spinal cord contus ion models to study the pathophysiology of SCI and its rehabilitation as it mimi cs natural human SCI. Most therapies that have gone to human trial were first validated in spinal cord cont usion models. Weight drop contusion model was used by Wrathall (1985) and desc ribed morphological and behavioral changes in that model; she also developed a combined behavioral score to assess motor, sensory and locomotor changes. Patients with complete spinal cord lesions never improve up to the stage of being able to walk without the assistance of the wei ght support system and consequen tly are not able to step on a static floor (Van de Crommert et al. 1998). The inability of patients with complete transections to achieve unassisted walki ng, unlike the fully spinalized cat, s uggests that the greater improvement observed in subjects with incomplete lesions may not solely be attributable to spinal mechanisms, since generation of stepping is pr obably more dependent on supraspinal and/or proprioceptive inputs in humans th an in cat (Edgerton et al. 2001; Van de Crommert et al. 1998). Considering these clinical and f unctional data and knowing that th e ratio of incomplete versus complete spinal cord lesions is becoming in creasing known in the population of paraplegics (Tator et al. 1993), experi mental studies using animals with incomplete spinal cord injuries appear clinically and pathophysiological ly relevant (Multon et al. 2003). Spinal Transection Model (Complete Spinal Cord Injury) In this model there is complete damage to both the descending a nd ascending fibers and there is no connection between the caudal part of the spinal cord and the brain and disrupts all the neurophil at the injury site. Th is type of injury is less common in humans as the human spinal cord is surrounded by vertebras, tissues and muscle s that provide protectio n to the spinal cord.

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18 Spinal Isolation Model In this model the lumbar region of the spinal cord is functionally isolated via complete spinal cord transection at two sites; this model eliminates supraspinal, infraspinal and peripheral afferent input to motoneurons located in th e isolated cord segments while leaving the motoneuron skeletal muscle fiber connections intact. However this model is not used in experimental set up, as cases of human isola tion models are not seen frequently so the observation in animal experiments cannot be implem ented in human subjects as it can be done in contusion animal models experiment. Anatomical (Neuromodulator y) Changes Following SCI Midthoracic spinal cord contusion injury is known to disrupt connectiv ity to and from the lumbar spinal cord which hosts important segmental circuitry that: 1) initiates and sequences lower limb locomotor behavior (central patt ern generator, CPG) 2) governs reflex excitability (Wratha ll et al.1985, Thompson et al. 1992; 1998). Impact injury of the thoracic spinal cord produces early increase followed by significant decrease in the monoamines (dopamine, norepinephr ine and serotonin), measured below the site of injury. (Thompson et al. 1999).These neuromodul ators play an important role individually and interactively in regulation of sensory transmission and the excitability of interneurons, fusimotor and alpha motoneurons (Gladden et al. 1998; 2000). It is known that locomotor exercise increases the expression of norep inephrine in the central nervous system (Dishman et al. 1997). The unifying strategy of exercise based locomo tor therapy relates to the hypothesis that repetitive activity induced by training initia tes neuronal activity in proper sequence that optimizes the utilization of the diminished but residua l central nervous system.

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19 A Rodent Model of Post-SCI Spasticity The availability of a reliable animal model of post-SCI spasticity is prerequisite to this research. Ideally, the model should be amenable to applications of rigorous outcome measures and test approaches that can have direct tran slational potential. Prev ious use of a rodent midthoracic contusion injury para digm has revealed significant neurophysiological, locomotor, neuromuscular, and histological ch anges which have collectively de monstrated the feasibility of reproducing significant features of human spasticity (Bose et al. 2002; Thompson et al. 1992, 1993, and 1998). Thompson-Bose lab developed a novel velocity-dependent ankle torque assessment protocol to reveal bonafide features of spasticity in the rat (Bose et al. 2002; Thompson et al. 1996, 2001a, 2002) and showed that changes in spasticity were time-locked to neurophysiological events in relevant areas of the spinal cord. Furthe r, a reflex test protocol (i.e., rate-depression) showed that a fundamental inhib itory process which controls sensory input to hindlimb muscle stretch reflex pathways was significantly decreased following midthoracic contusion injury (Thompson et al. 1992, 1993, 1998) These changes are progressive in onset, severe in magnitude, permanent in duration, a nd are highly relevant to features observed clinically in humans. Therefore, the methods and model developed in our lab and its employment in this project collectively provide an important a nd clinically relevant opportunity to investigate issues related to the neurobiol ogy of spasticity and how experi mental therapies may modulate this hallmark feature of SCI in a very acute setting. Mechanism of Spasticity The onset of spasticity has been correlated in time and intensity to the progressive development of several lasting changes in the exc itability of monosynaptic reflexes that compose the neural pathways from the affected muscle stretch receptors to those muscle motor neurons,

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20 (Neilsen et al. 1993).Two premotoneuronal mechan isms have been postulated to account for these changes: 1. multiplication of synaptic input by collatera l sprouting of primary afferents (Krenz and Weaver 1998). 2. decreased presynaptic inhibi tion (Calancie et al.1993). Frank and Fuortes (1957) reported that stimulation of group I a fferents from flexor muscles was able to depress the Ia monosynaptic EPSPs without changing the membrane potential, the motoneuron input resistance, or the generation of antidromic acti on potentials. The depression of the Ia EPSPs was due to presynaptic inhibi tion. Eccles and Krnjevic (1959) found that stimulation of specific sensory nerves produced a long lasting depolariza tion of group I muscle and cutaneous afferents and suggested that this primary afferent depo larization (PAD) was the mechanism for presynaptic i nhibition. Rudomin and colleague s localized a circuit of interneurons that received affere nt collaterals from primary affere nt fibers and convergent inputs from the terminals from descending fibers (Rudomin 1999). They showed the circuit for presynaptic inhibition involved a minimum of two interneurons, the second or last order being GABAergic that made axo-axonic synapses on GABAa receptors on primary afferent terminals. The activation of these receptors induces an outward Clcurrent that produces long lasting PAD. During the time course of the PAD (20-300msecs), the GABAa mediated conductance changes along the primary afferent membrane decreases the effectiveness of afferent volleys to depolarize the membrane and activate the voltage gated calc ium channels that are essential for calcium triggered release of neurotransmitters from the primary afferent terminals. The decrease in transmitter release thereby decreased the amplitude of the excitatory postsynaptic potentials produced during the PAD. The knowledge that de scending systems modulated PAD, combined with the demonstration that vi bratory inhibition was reduced in spastic patients emphasized the

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21 hypothesis that a major source of spasti city was due to the reduction of GABAa mediated presynaptic inhibition following in terruption of descending modulati on of the segmental circuitry that produced PAD. Neurotransmitters like GABAb, GAD67 also play an important role in the development of spasticity which acts as inhib itory molecules preventing the development of spasticity. GABA GABA is a spinal inhibitory interneuronal ne urotransmitter. There is a high density of GABAergic immunoreactive cell bodies and termin als predominantly within Lamina I through III of the dorsal horn. Occasionally GABA cells were observed in layers IV, V, VI and X and in the ventral horn. GABA functions in pre and pos tsynaptic inhibition in the spinal cord, via axoaxonal and axosomatic or axod entritic synapses (Shapiro 1997) In vertebrates 2 major types of GABA receptors (GABAa and GABAb) are found. GABAa contains a transmembrane ion channel that conducts chloride ions and is gated by the binding of two agonist molecules. Activation of GABAa opens the chloride channel, and the infl ux of the chloride ions inhibits the neuron by causing hyperpolarization. GABAb is present at lower leve ls in the central nervous system than is GABAa and is coupled to Ca2+ or K + channels via second messenger systems. GABAb can be distinguished from GABAa by its affinity for the agonist baclofen and its lack of affinity for muscinol and bi cuculline. Activation of GABAb by baclofen decreases Ca+ conductance and transmitter release a nd thus acts an inhibitory molecule in synaptic transmission (Bowery et al. 1980, 1989; Shapiro 1997) and prevents the development of spasticity. Since GABA is localized to interneurons, reactive synaptogenesis of interneuronal GABAergic neurons can be seen in the spinal cord of spastic animals. Thus this molecule is very important for the proposed research since we use baclofen as an antispastic drug and examine its action on the presynaptic GABAb receptors.

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22 Present Antispasticity Treatme nt for Spinal Cord Injury Baclofen has been used widely as an anti-s pasticity drug. Baclofen reduces muscle tone and spasms with similar efficacy in patients with spasticity caused by complete or incomplete SCI, or from cerebral origin (Davidoff 1985; Meythaler et al. 2001). A recent finding has proven that the effect of baclofen is more potent when ap plied intrathecally than orally (Azouvi et al. 1996). GABAb receptors are distributed extensively in the spinal cord, especially presynaptically on the primary sensory afferent terminals (Y ang et al. 2001). Baclofen, as a potent GABAb receptor agonist, has been shown to decrease synaptic transmission by bi nding to the presynaptic GABAb receptors at the afferent terminal th rough a second-messenger pathway, ultimately decreasing the calcium influx and neurotransmitter release (Batueva et al 1999; Miller 1998). In addition, baclofen's binding to the presynaptic GABAb receptors can also decrease the neurotransmitter release by activa ting potassium channels (Gage 1992) causing hyperpolarization of the post synaptic membrane, thus contributing to its presynaptic inhibitory effect (Li et al. 2004). In the paper by Li (2004) the aut hor showed that in motor neurons of normal animals (or humans) with intact sp inal cord and brain stem, there are voltage-dependent persistent inward currents (PICs) that, once activated, can remain active for many seconds after stimulation, producing sustained depo larizations (plateau potentials) and firing (selfsustained firing), thus greatly increasing their excitability (Gorassini et al. 2002; Lee and Heckman 1998a, b). A PIC is a depolarizing current generated by voltage-sens itive channels; the voltage sensitive channels stay open as long as the membrane potential remains above threshold for their activation (Heckman et al. 2004). The PICs ar e composed of a low-threshold persistent calcium current, carried by Cav1.3 L-type calcium channels, and a TTX-sensitive persistent sodium current (Lee and Heckman 2001; Li and Bennett 2003). Large PICs are not present in motor neurons immediately after spinal cord injury because of the massive loss of brain-stem-derived

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23 monoamines that normally facilitate PICs (Hounsgaard et al. 1988). Exogenous application of metabotropic receptor agonists (such as 5-HT) can enhance PICs and thus recover plateaus and self-sustained firing after acute spinal transection or in vitro slice inju ry (Lee and Heckman 1998a, b). Baclofen has been shown recently to decrease the amplitude of these enhanced PICs in motor neurons of turtle spinal cord slices and to decr ease spontaneously occurring PICs in deep dorsal horn neurons of turtles and rats (Russo et al. 1998). Thus, ra ising the possibility that baclofens clinical action may be partly postsynaptic by decreasing the PICs that play an important role in the production of spasticity (Bennett et al. 2001a, b; Li et al. 2004a). It has been found that GABAb decreased PIC only in acute SCI not in chronic SCI, it increased the PIC, as chronic spinal rats have a moderately lower (30%) sensitivity to baclofen than do acute spinal rats, which might be due to do wn regulation/desensitization of GABAb receptors or decreased background GABA levels after chronic SCI (Li et al. 2004). This finding may justify our hypothesis that baclofen starte d acutely after SCI can prevent the development of spasticity by reducing the monosynaptic reflex mainly by d ecreasing presynaptic neur otransmitter release and decreasing the PICs by reducing the Ca2+ PICs due to the upregulation of the GABAb receptors. Systemic baclofen depressed the mono synaptic excitation of Clarkes column neurons by impulse in muscle and cutaneous afferent fibers (Shapiro 1997). Baclofen, in addition to GABAb, can bind to a novel bicuculline-insensitive GABA receptor site on primary afferents of the spinal cord and reduce the amount of transmitter released (Bowery et al. 1980,1984). Baclofen is given in an intr athecal pump (ITB). Orally it cannot easily cross the blood brain barrier and only 1% of the total dose reach the central nervous system and so oral doses are generally twice or thrice the required amount which is not feasible. ITB supplies the exact amount of drug to the appropriate area for the required amount of time, thus enabling the effects of baclofen to be studied afte r immediate administration and to

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24 see the development of tolerance after withdrawal. The major draw brack of baclofen therapy in patients today is that it is started much later after the injury as the Food and Drug Administration does not approve baclofen for acute treatment. Spas ticity is usually absent in acutely injured animal preparations, and it only gradually develops w ith chronic injury (>1 month) (Bennett et al. 1999). Also it has been shown th at Baclofen started even one week after injury helps in improving spasticity (Wang et al. 2002). So the ba sic aim of this project is to acutely start baclofen therapy along with bicycl e training or alone to prevent the development of spasticity and to see whether there is rebound spasticity af ter the baclofen treatment is stopped after a certain period of time, in our case after 1 month. Locomotor Rehabilitation Rehabilitation in the form of treadmill or cust omised bicycle is widely used in human SCI and in animal models. Customized bicycle develope d as an alternative modality to treadmill, to train SCI injured rats was used in our project to prove it is as effective as treadmill. But exactly what time period after injury it should be star ted to prevent the development of spasticity and improve the gait and whether traini ng after injury may have othe r side effects on the patients body are not known. So we want to test the hypothesi s that bicycle training acutely after injury along with baclofen therapy pr events the development of sp asticity. Exercise improves SCI extensively due to its adeptne ss at enhancing sensory function which is mediated by molecular systems dependent on neurotrophic actions. Vo luntary wheel training and forced treadmill exercise increased the expression of BDNF a nd other neurotropic molecules important in synaptic function and neurite outgrowth in the spinal cord and innervated skeletal muscles (Gomez-Pinilla et al. 2005). There is increasing evidence that the human spinal cord is capable of a significant amount of plastic ity and that this plasticity is to a large extent, driven by activity-dependent proce sses (Edgerton et al. 1997, 2001). This plasticity may o ccur at any of

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25 many spinal cord regions or cell types such as motoneurons, premotor pattern-generating neurons, and/or nonneuronal cell types. Studies ha ve shown that locomotor training improves the ability to perform full weight-bearing stepping on a treadmill in cats after a complete spinal cord transection (de Leon et al. 1998; Edgerton et al 1997), which has supporte d the presence in the lower spinal cord of a central pa ttern generator (CPG) able to ge nerate rhythmic motor activities in the absence of supraspinal descending inputs (Grillner et al. 1985). How the CPG can shape its functional properties in response to the training is not clear, but it is thought that reinforcement by sensory afferents of existing sensorimotor pathways, rather than generation of new connections, might be responsible for the be neficial effects (de Leon et al. 1998, 1999, 2001). There could also be anatomically altered s ynaptic connections, increased active zones of synapses, altered sensitivities of neurotra nsmitter receptors, or altered production of neurotransmitters. Sensory input provided during locomotor traini ng are critical for driving the plasticity that mediates locomotor recovery and pharmacological treatments can be used to excite the spinal neurons that gene rate stepping (Edgerton 2001). So bicycle training along with baclofen (GABAb agonist) can show a drastic improvement in locomotor pattern of SCI subjects as the paddling movement of the bicycle give s a constant sensory i nput by activating the receptors in the joints, muscles and tendons in th e ankle, knee and hip joint of the hind limbs. Locomotor training is beneficial in main taining and even improving neural function following insult or disease (Wernig et al. 1999). It is recognized that tr ophic factors such as neurotransmitters are critical modifiers of the st ructure and function of ne ural networks such as 5-HT. Serotonin is a neurotransmitter of descending pathways from brain to spinal cord, primarily in the ventral and lateral funiculi of th e spinal cord influencing interneurons and motor neurons via postsynaptic inhibition. The serotonergic cells of origin are in the raphe nuclei of the brain stem and the reticular formation. The serot onergic cells in the nuc leus raphe obscurus and

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26 nucleus raphe pallidus project to the intermediolateral cell column and ventral horn of the spinal cord. After complete or incomplete SCI, the descending 5-HT bulbospinal tracts in the lateral and anterior funiculi undergo a slow wallerian de generation because of the small fiber size and lack of myelin, and this invol ves the axon terminals with the reduction of uptake, with the development of spastic paraparesis. The motor scores have been significantly correlated with changes in 5-HT staining in the ventral horn bu t not in the dorsal horn (Shapiro 1997). The 5-HT system surrounds the corticospinal tract in the la teral funiculus, which ex plains the correlation between losses of 5-HT and motor deficit after SC I. So an increase in the 5-HT in the lumbar spinal cord after SCI with baclofen and locomoto r training can correlate w ith the improvement in the motor functions. Physical activity even in an intact brain and spinal cord can induce the expression of trophic factors in the hippocampus and other brain regions (Gom ez-Pinilla et al. 1997; GomezPinilla et al. 1998),while exercise leads to the expression of trophic factors such as BDNF and NT-3 specific neural networks (Gomez-Pinilla et al. 2002, Ying et al. 2003) which are important in growth and neural function of the neurons. Ex ercise has been shown to induce BDNF in the lumbar enlargements of the uninjured spinal cord (Gomez-Pinilla et al. 2001) and treadmill walking increased labeling of BDNF, its recep tor, trkB, and neurotrophin-4. Neurotropin modulation induced by neuromuscular activity can pl ay a role in facilita ting functional recovery following SCI .The injured spinal cord generally losses the ability to synchronize and interpret the coordinated ensemble of afferent informati on that produces a predictable motor outcome in an uninjured patient and so pr oduces random motor pool activation. This deficit may be due to the absence or rare occurrence of synchronized events normally associated with load-bearing stepping. In the absence of thes e coordinating events, the spinal cord loses the ability to synchronize input into functional movements of the limbs. A patient who is hypo responsive to

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27 sensory input is less likely to respond to the proprioceptive input associ ated with load-bearing stepping, so the presence of spasticity, which is exaggerate d stretch reflex due to hyper responsive sensory input, is a pos itive sign of the potential for a SCI patient to regain some locomotor ability. Clearly, understanding the physi ological mechanisms that underlie spasticity will enhance our efforts to facilitate locomotor recovery in SCI patients. Activity-dependent motor training facilitates the recovery of pos ture and locomotion after a complete SCI in mammals. Because functionally recovered spinal animals i.e., SCI animals showed no evidence of regeneration of descending pathways (Joynes et al. 1999) or showed minimal changes in hind limb skeletal muscle properties (Roy et al. 1998, 1999) to account for the recovery characteristics, the functional behavior exhibite d by these animals must have been mediated by the plasticity in existing spinal pathways. It is generally accepted in th e literature that exogenous application of BDNF and NT-3 on spinal motoneur ons improves the regeneration of the fibers in the spinal cord and thus can improve th e outcome. Neurotrophins supplied by endogenous sources may have an even greater effect on comp romised cells and motor training have shown to improve the levels of neurotropins in the inju red spinal cord. The positive effects of motor training have been documented in animals (H odgson et al. 1994; de Leon et al. 1998) and humans (Harkema et al.1997). The success of reha bilitative strategies is highly task specific, which closely simulate the functi onal situation of walking are the most effective in promoting the restoration of locomotion (Edgerton et al. 1997; de Leon et al. 1998.) Reha bilitative strategies that stimulate walking, like treadmill or bicycling, are effective in improving locomotion in SCI due to the phasic sensory input produced by repe titive foot contact with the ground or the foot pad in the case of bicycle to re sult in the induction of activity de pendent events such as increased neurotropins levels in circuitry by repetitive load ing of the hind limb (Gomez-Pinilla et al. 2005).

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28 In summary, the above literature review shows that locomotor training whether it is voluntary, wheel running or treadmill prevents the development of spasticity by modifying the neurotransmitter levels and enforc ing neuroplasticity in the surviv ing sensorimotor neurons so as to develop a rhythmic reflex pattern that leads to improvement in the gait. Also baclofen therapy started acutely can prevent the development of PICs which may be another cause of the late onset spasticity and reinduce the pres ynaptic inhibition by acting on the GABAb receptors and upregulating them.

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29 CHAPTER 3 EXPERIMENTAL DESIGN AND THE STANDARD METHODOLOGY USED IN THE PROJECT Objective The whole thesis work was conducted in a rodent animal model using established techniques and protocols. Standa rd contusion SCI animal model wa s used in this project and the injury device and the animals used are describe d below. The experimental set up for spasticity measurement (Ankle torque and EMG), for locomo tor gait assessment using footprints, and the open field locomotor (BBB) scoring are all standard techniques. The bicycle training methodology was developed in this lab and will be described in more detail. The following segments will describe those techniques and protocols used in this project (Figure 3-1). Animal Subject Twenty four twelve week old female Sp rague-Dawley rats (SPF) weighing 220-260 g (Charles River Laboratories) at the start of this study were used in this project. Rats were housed two per cage, in 12 hour light/dark cycle, and gi ven food ad libitum. The total number represents here is the actual experimental animals (24) not including the ina dvertent losses: a) animals that died during surgery (2 animals), b) those exclud ed based upon post-injury selection criteria, (9 animals) or c) animals that died unaccountab ly during the 2 month tr aining and drug therapy program (none). The total number of animals used was thirty fi ve. An attending veterinarian supervised care of these animals. All procedur es were performed under the guidelines of Animal Care and Use Committee.

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30 The experimental design The experimental design Sprague Sprague dawley dawley Rats (n=24) Rats (n=24)Preinjury testing :Spasticity and locomotor assessment SCI at T8 level with pump implantation (n=6 in each group), bicycle locomotor training Contused saline control (untrained) Contused Saline control (trained) Contused Baclofen (trained) Contused Baclofen (untrained) Bicycle Training starts at POD1 Post injury testing at week1, 4, 8 Pump withdrawal at week 4 Locomotor assessment (Footprints, BBB score) Spasticity assessment (Ankle torque and EMG) Terminal IHC of spinal cord MRI of injury Figure 3-1 The experimental set up and procedures done in chart form Contusion Injuries Contusion injuries were produced using a standard New York University (NYU) Multicenter Animal Spinal Cord Injury Studies (MASCIS) impactor (Basso et al. 1996). The injury was performed under ketamine (100 mg/ kg), xylazine (10 mg/kg, 1:3 with normal saline), and glycopyrrolate (200 l in each animal) anesthesia and previously reported in detail (Bose et al. 2002, Thompson et al. 1992,1998). A laminectomy was performed at T8 segment, exposing the underlying dura. The spinal column was st abilized with angled Allis clamps on the T7 and T9 spinous processes. An incomplete sp inal contusion was made at the T8 segment of the spinal cord using the impounder tip of the MA SCIS 10-g weight impactor de vice (2.4 mm in diameter) dropped from a height of 12.5 mm (computeriz ed operation). The whole procedure was performed under aseptic conditions. The animal s were monitored routinely and were given

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31 postoperative care on a regular basis or as required until full bladder function was re-established and no evidence of pain or other discomfort was detected. Baclofen Pump Implantation All rats were anesthetized w ith subcutaneous injection of xylazine (6.7 mg/kg) followed by intraperitoneal ketamine (100 mg/kg)/glycopyrrola te (40 mg) (Reier et al. 1992; Thompson et al. 1992, 1993). Prior to implant, osmotic pumps (model 2004, 0.25 mL/h; Azla Corp., Palo Alto, CA) were filled with baclofen (3.2 g/ L; implanted in twelve an imals) or 0.9% physiological saline (implanted in twelve animals). Osmotic pump s were incubated overnight in 0.9% saline at 37C in sterile conditions for immediate deliver y of drug or vehicle. The dose rate was 0.8 g/hr. Particular efforts were taken to avoid introducing air space with in the infusion catheter. A 4cm vertical skin incision was made spanning the thoraco-lumbar juncture. A subcutaneous pocket was dissected to accommodate the osmotic pump. Musculature covering spinal processes was retracted from T12-L1, and a T13-L1 laminectomy was performed. The osmotic pump was maneuvered into the subcutaneous pocket and secured to surrounding musculature using 6.0 silk sutures. The infusion catheter was tunneled throu gh the rostral bank of dissected musculature, and secured by polyacrylamide adhesion (Vetbon d tissue adhesive; 3M, St. Paul, MN) to exposed spinal process. The wound margins of mu scle and skin were infiltrated with a longlasting local anesthetic (lidocai ne). With the aid of a dissecti ng microscope, the duramater was cut and the silastic tubing inse rted into the subarachnoid space of the lumbar enlargement. Excess tubing was secured to surrounding musculatur e with 6.0 silk sutures. Muscle layers were closed with absorbable sutures (Dexon II; Sh erwood, Davis & Geck, Wayne, NJ) and the skin closed using stainless steel wound clips (A utoclip; Becton Dickenson, Sparks, MD). Postoperative care included a 4mL subcutaneous injection of wa rmed (37C) 0.9% physiological saline and overnight assistance with body temperature regulation using a temperature controlled

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32 heating pad. The day after surgery behavioral and neurophysiological assessment were started (Figure 3-1). After 4 weeks of ITB treatment, th e pump was removed by similar procedures, and the infusion catheter was ligated with 6.0 silk sutures and left in situ. Bicycle Training Figure 3-2 Motorized bicycle us ed for training the rats. Careful attention was taken during training, as it was started acutely (PO day 1) after the injury. The animals were trained over the cour se of 2 months. The training schedule was performed 5 days a week using two 20 minute trials /day, starting from PO day 1. On the first day of training, the rats were given five minutes of training. The bicycle exer cise regimen (Figure 32) involved immobilizing the rats in a custom ma de harness with the hind limbs suspended. The hind feet were strapped onto the pe dals using cotton tapes. The exercise consisted of a pedaling motion, which fixed one limb while extending th e other without overstr etching the limbs. The cycling speed was 31 rotations/minutes (around 11 meters/min, distance wi se). During the first week of training, the rat tail was attached to the aluminum support boom by surgical tape to Protocol: 40 min/day, 5 d/wk 8 wks Initial weeks more weight support

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33 maintain the trunk stability during exercise. However, follo wing second week of training, gradually the load on the pedals was increased by positioning the body harness towards the chest, so that the hind portion of the body was more ex tended over the pedal. The basic mechanism and principle of bicycle training is that of locomo tor activity-initiated sens ory input derived from weight bearing, as well as from the receptors of the skin, bone s and muscles of the ankle, knee and hip joints of the hind limbs (Figure 3-2). Footprints To document hind limb gait abnorm alities, footprints were ac quired while rats walked at 11 m/min, along a 20 x 40 cm surface of a treadmill (Columbus Instruments, OH, USA). Prior to each trial run, hind limb footpads were coated with nontoxic blotter ink and the treadmill lined with recording paper. Footprints were acquire d from each animal with six consecutive steps considered optimal for data analysis. Hind limb axis was measured using the angle of intersection between left and ri ght hind foot axis during coordi nated stepping. Foot axis was determined by a line passing through the third di stal phalanges, metatarsophalangeal joints, and between the cuboid bone and medial cuneiform bone. The base of support was determined by measuring the horizontal distance be tween the central footpads of th e hind feet. Prior to footprint recordings, the animals walked for a few minutes to accommodate to and become familiar with the treadmill walkway. Prior to the surgery preope rative footprints were taken to compare the changes in the walking pattern after in jury and after trai ning (Figure 3-2). BBB Score An open field-testing proce dure (Basso, Beattie, and Br esnahan (BBB) 21-point scale) (Basso et al. 1995) was applied after the contusion injury at different time points to assess the locomotor deficit and its improvement following exercise and or baclofen therapy. These observations were made at POD 1, wks 4 and 8 in a blind fashion (Figure 3-2). The rats were

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34 placed in a clean molded-plastic circular enclosure to walk freely to perform this procedure. It rates behavior from individual joint movement s of the hind limb, to plantar stepping, to coordinate walking and finally the more subtle behaviors of locomotion, such as paw position, trunk stability and tail position. Observations are recorded for 4 min in an open field and then converted to a numerical score from the scale. BBB Score Inclusion Criteria: to decrease injury variability of animals with in the study, the animals were scored using the BBB, at PO da y 1 after injury. Those animals that scored > 7 were considered too mildly injure d and were excluded from the study. Statistical analyses of the data (velocity-dep endent ankle torque, f ootprints and open field locomotor recovery) included between groups ANOVA and post hoc tests to achieve group differences. Ankle Torque Figure 3-3 Ankle torque, displacement and EMGs are simultaneously recorded and timelocked to onset of dorsiflexion. Pre-operative as well as postope rative recordings utilizing the ankle torques protocol were taken. Details regarding instrumentation, animal set-up and recording pr ocedures have been 100 v 50 v 50 msec LVDT Force RMS EMG 12 deg 25 g Displacement Force EMG

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35 previously reported (Bose et al. 2002; Thompson et al. 1996) but will be described below briefly. Rats were immobilized in a custom designed tr unk restraint, without trauma or apparent agitation. All recordings were performed in awake animals. The proximal portion of the hind limbs to the mid-shank, were secured in a foam fitted cast that immobilized the limb while permitting normal range of ankle rotation (60 to 160 degrees). The lengthening resistance of the triceps surae muscles was measured indirectly by quantitating ankle to rque during 12-degree dorsiflexion rotations of the ankle from 95 through 83 degrees. Contact with the foot was achieved using a foam-fitted cradle aligned with the dorsal edge of the central footpad 2.6 cm distal to the ankle joint. The angle of contac t between the displacement shaft and the moment arm was 95 degrees (Figure 3-3). The neural activity of the triceps surae muscle was measured using transcutaneous EMG electrodes. The electrode was inserted in a skin-fold over the distal soleus muscle just proximal to the aponeur otic convergence of th e medial and lateral gastrocnemii into the tendonocalcan eousus. A reference electrode wa s placed in a skin fold over the greater trochanter. A xylocaine 2% jelly (L idocaine HCl, Astra US A Inc.) was applied over the electrode insertion points to minimize pain dur ing recording. A topical an tibiotic ointment (a combination of Bacitracin, Neomycin and Polym yxin B; Fougera Altana Inc., Atlanta, GA) was also applied on these areas after taking off the el ectrodes at the end of each trail to reduce any chance of infection. Controlled dorsiflex ion was achieved through the use of an electromechanical shaker (model 405, Ling Dynami c systems, Royston Herts, U.K.). A force transducer (LVDT) (model FT03; Grass Instruments, Quinc y, MA) was placed in series between the output shaft of the li ng shaker and the central footpad (figure 3.3). Root mean square (RMS, i.e., a 0.707-DC equivalent of the full wave rectified AC signals) of EMG bursts was also recorded on an additional cha nnel of the signal acquisiti on system. EMG magnitudes are

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36 reported as mean RMS magnitude of the EM G bursts time-locked to ankle dorsiflexion. Collectively, this arrangement allows for simultaneous monitoring of triceps surae EMG, resistive force, and velocity of shaft displacement. Recorded data were processed using Data Wave Technologies signal acquisition system (model 32C; Data Wave Technologies, Denver, CO) with an analogue to digital sampling rate of 200 KHz. Dorsiflexions of 12 degrees were performed with 3-sec intervals at 49, 136, 204, 272, 350, 408, 490, and 612 deg/sec. At each test velocity, five consecutive sets of waveforms, 5 waveforms per set, that is, 25 in total, were recorded, signal averaged, and saved for subse quent analysis. An average of two time points recording was used for data analysis. Regression of ankle torque against the ankle joint rotation yielded resistance (torque) per degree of rotation for each velocity variable of the protocol with torque values expressed in Kdynes (1000 dynes 5 1 Kdyne). The protocols were performed using the fastest rotation first, then the slowest rotations Immunocytochemistry of Spinal Cord Tissue The standard Fluorescent and Avidine-Bi otine Complex (ABC) immunohistochemical techniques were utilized for visua lizing neurotransmitters such as GABAb, 5-HT, GAD67, BDNF, GAP43. These techniques allow the visualization of varicosities as well as fibers that are interpreted as axons using 40 m thick cryostat sections of the lu mbar and thoracic spinal cord. Qualitative evaluation was done using a light microscope and bight field microscope. In brief, spinal cord segments (thoracic segm ents caudal to the injuries, and lumbar spinal cord, L3-L6) were dissected and removed after perfusi on (4% paraformaldehyde in PBS) and kept in the same fresh fixative mixture for 1 hour a nd was cryoprotected for at least 2 days in 30% sucrose in 0.1 mol PB. The specimens were cu t serially (cross section) by cryostat (40 m

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37 thickness) and processed by Avidine-Bi otine Complex (ABC) and Florescent immunohistochemistry (IHC). The immunoreactivity of GAD67, GABAb GAP43 and BDNF were identified in lumbar and thoracic spinal cord. The cryosta t cut sections were incubated w ith primary antibodies generated against GAD67 (mouse mAb; 1:1,000, National Hybridom a Laboratory, St Luis, USA), GABAb (guinea pig mAb, 1:4,000; Ch emicon International), GAP43 (mouse mAb, 1:5000; Chemicon International), and BDNF (rabbit Ab, Ch emicon International) for 24-48 h at 4 C. The sections were then washed in PBS and incubated fo r 1.5 h in alexa fluor-c ongugated appropriate antimouse, anti-guinea pig or anti-rabbit IgG (1: 1000, Molecular Probes). For ABC technique, antiguinea pig (1:200; Chemicon), anti-mouse, and an ti-rabbit (1:200; mouse and rabbit Elite kits, Vector Lab) secondary antibodi es were used to bind with appropriate primary antibodies. Sections were then washed again an d mounted for microscopic analyses.

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38 CHAPTER 4 RESULT The following sections present results of veloc ity-dependent lengthening resistance due to dorsiflexion of the ankle, ope n-field locomotion ability a nd gait (footprints) analyses. Velocity-Dependent Ankle To rque and Associated EMGs Pre-injury Ankle Torque and EMG Data Baseline measures of velocity-dependent ankl e torques and extensor EMGs were obtained from all animals before injury at post-injury weeks 1, 4 and 8. Figure 4.1. Preinjury an kle torque graph Figure 4-1 Pre-injury ankle torque graph. Figure 4-2 Pre-injury EMG graph. PREINJURY -ANKLE TORQUE0 20 40 60 80 100 120 61249040835027220413649 VELOCITY IN DEGS /SECAMPLITUDE IN KDYNE SALINE CONTROL SALINE CYCLE BAC CONTROL BAC CYCLE EMGpreinjury0.000000 0.200000 0.400000 0.600000 0.800000 1.000000 1.200000 1.400000 61249040835027220413649 saline control saline cycle bac control bac cycle

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39 Postoperative Week 1 Ankle Torque and EMG Data Figure 4-3 Ankle torque graph at postop week 1 with significant differences. Figure 4-4 Electromyeloencephalogram graph at pos t-op week 1 with significant differences. When tested at one week following injury, baclofen cycle group revealed significantly decreased magnitudes of ankle torque during ro tation at each of the eight ankle rotation ANKLE TORQUE-WEEK 10 50 100 150 200 61249040835027220413649 VELOCITY IN DEGS/SECAMPLITUDE IN KDYNES SALINE CONTROL SALINE CYCLE BAC CONTROL BAC CYCLE PREINJURY #@ @# *@# # *@*@# # $ *@# # # *@$ # # # *@$ # # # # # # #$, p 0.05, compared to salcycle *, p 0.05, compared to preinjury @, p 0.05, compared to baccont #, p 0.05, compared to baccycle* # EMGweek10 0.5 1 1.5 2 2.5 61249040835027220413649 saline control saline cycle bac control bac cycle preinjury recording *, p 0.05, compared to preinjuryTorque amplitude#, p 0.05, compared to baccycle#@* #@#@# # #@* # #@* #@#Velocit y in de g s / sec@, p 0.05, compared to baccont@* # # # # # *@# *@

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40 velocities, even less than the control values r ecorded before injury (Figure 4-3). These were about 11% lower at the hi ghest velocity (612 deg/s ec) and 0.8% lower at th e lowest velocity (49 deg/sec). Similarly the mean values observed for the baclofen cont rol group did not show significant changes compared to the pre-injury group, only 7% increase at the highest test velocity (612 deg/sec), and 28% increase at the lo west test velocity (49 deg/sec) (Table 4-1). Mean values observed for th e saline control group revealed significantly increased magnitudes of ankle torque during rotation at ea ch of the eight ankle rotation velocities when compared to corresponding pre-injury values (Figure 4-3). Approximately a 52% increase was observed at the highest velocity (612 deg/sec), and a 38% increase at the lowest test velocity (49 deg/sec) (Table 4-1). These values were signif icantly different when compared to comparable measures in both baclofen cycle and ba clofen control groups (Figure 4-3). The saline cycle group also showed a significant increase in ma gnitude of ankle torque at each of the eight ankle rotation velocities tested when compared to pre-injury values (Figure 43), approximately 39% increase at the highest te st velocity (612 deg/sec) and 37% increase at the lowest test velocity ( 49 deg/sec) (Table 4-1) There were significant differences in the velo city-dependent ankle torques between saline control and saline cycle groups wh en compared to corresponding values obtained from baclofen control, baclofen cycle and pr e-injury groups; but no significa nt differences were observed between baclofen control, baclofen cycl e or pre-injury groups (Figure 4-3). The EMG-RMS magnitudes recorded at each of th e test velocities closely paralleled the velocity dependent ankle torque measurements (Figure 4-4). Significant parallel increases in ankle torque and EMG magnitude, respectively, were observed during ankle rotations at the

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41 slowest four velocities at post-op week 1 in c ontrol and saline cycle gr oup when compared to baclofen cycle group Table 4-1 Ankle torque week 1percentage (%) change ( ) compared to pre-injury. Table 4-2 Electromyeloencephalogram week 1 percentage (%) change ( ) compared to preinjury. Postoperative Week 4 Ankle Torque and EMG Data Figure 4-5 Ankle torque graph at postop week 4 with significant differences. Groups Wk1 Ankle Torque Data % 612 490 408 350 272 204 136 49 Saline Control 52% 43% 43% 41% 41% 39% 35% 38% Baclofen Cycle 11% 7% 7% 8% 7% 8% 6% 0.8% Saline Cycle 39% 31% 25% 29% 20% 14% 19% 37% Baclofen Control 7% 10% 12% 24% 18% 17% 14% 28% Groups Wk1 EMG Data % 612 490 408 350 272 204 136 49 Saline Control 62% 89% 101% 108% 168% 289% 289% 695% Baclofen Cycle 42% 52% 50% 45% 22% 39% 67% 8% Saline Cycle 35% 40% 20% 49% 73% 143% 29% 109% Baclofen Control 38% 5% 41% 34% 6% 11% 50% 103% ANKLE TORQUE-WEEK 40 50 100 150 200 250 61249040835027220413649 VELOCITY IN DEGS/SECAMPLITUDE IN KDYNE S SALINE CONTROL SALINE CYCLE BAC CONTROL BAC CYCLE PREINJURY $ #@* *@$ # #@$ #@$ $ $ $ $ $ $*, p 0.05, compared to preinjury $, p 0.05, compared to salcycle @, p 0.05, compared to baccont #, p 0.05, compared to baccycle

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42 Figure 4-6 Electromyeloencephalogram graph at post-op week 4 with significant differences. At post-injury week 4, a signifi cant decrease in the ankle tor ques was observed at all test velocities in the saline cy cle contused group compared to thes e values observed at week1 (Figure 4-5) for example, at the highest velocity (612 deg/sec), at week 1 the ankle torques were 39% greater than control, but only 4% greater at week 4; a 35% decr ease. And at the lowest test velocity (49 deg/sec), the week-4 saline cycle an imals revealed ankle-torques that were 5% greater than controls, compared with 14 % at week 1 for these measures. On the other hand, baclofen control and bacl ofen cycle showed the same pattern as week 1. The saline control group ankle torques remained significantly elevated, and were similar to week1 data, a 61% increase at highest test velocity (612 deg/se c) and a 9% increase at the lowest velocity (49 deg/sec) (Table 4-3). The ankle torque magnitude showed the same pattern in the lower velocities as that of higher velocities. The EMG magnitudes mirrored the pa ttern of the ankle torques in all the four groups at all the velocities there was no si gnificant difference in the EMG magnitude at all the lower 4 velocities when compared to higher velocities in post operative week 4 (Figure 4-6) (Table 4-4). EMG-WEEK 40 0.5 1 1.5 2 2.5 61249040835027220413649 velocity in degs/sectorque amplitude saline control saline cycle bac control bac cycle preinjury recording @* # # # * #@ @# $ $ $ #@$@$ $*, p 0.05, compared to preinjury #, p 0.05, compared to baccycle @, p 0.05, compared to baccont $, p 0.05, compared to salcycle

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43 Table 4-3 Ankle torque week 4percentage (%) change ( ) compared to pre-injury. Table 4-4 Electromyeloencephalogram week 4percentage (%) change ( ) compared to preinjury. Postoperative Week 8 Ankle Torque Data and EMG Data ANKLE TORQUE-WEEK80 50 100 150 200 250 61249040835027220413649 VELOCITY IN DEGS/SECAMPLITUDE IN KDYNES SALINE CONTROL SALINE CYCLE BAC CONTROL BAC CYCLE PREINJURY # $ $ # # * # $ * * # $ # * $ # $ # $ # # # * **, p 0.05, compared to preinjury $, p 0.05, compared to salcycle #, p 0.05, compared to baccycle Figure 4-7 Ankle torque graph at postop week 8 with significant differences. Groups Wk 4 Ankle Torque Data % 612 490 408 350 272 204 136 49 Saline Control 61% 52% 47% 43% 27% 22% 18% 9% Baclofen Cycle 5% 5% 10% 2% 7% 9% 2% 0.05% Saline Cycle 4% 0.2% 3% 3% 1% 5% 6% 5% Baclofen Control 18% 13% 9% 6% 11% 12% 13% 8% Groups Wk 4 EMG Data % 612 490 408 350 272 204 136 49 Saline Control 60% 117% 127% 138% 122% 152% 248% 296% Baclofen Cycle 27% 27% 8% 15% 49% 20% 14% 33% Saline Cycle 39% 33% 41% 28% 23% 3% 16% 43% Baclofen Control 27% 7% 33% 17% 31% 13% 21% 128%

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44 Torque amplitude*, p 0.05, compared to preinjury EMG week 80 0.5 1 1.5 2 2.5 61249040835027220413649 saline control saline cycle bac control bac cycle preinjury data Velocity in degs/sec#, p 0.05, compared to baccycle# # # * # # # # $ $ # *$, p 0.05, compared to salcycle *, p 0.05, compared to preinjury+ $ # ++, p 0.05, compared to salcontrol Figure 4-8 Electromyeloencephalogram graph at post-op week 8 with significant differences. By post-op week 8, significant velocity-dependent ankle extensor spasticity re-appeared in the baclofen untrained cont used group, a 75% increase at the highest velocity (612 deg/sec) compared to the corresponding va lue of pre-injury c ontrol group (Table 4-5). While only an 8% increase was observed at the lo west test velocity (49 deg/se c) (Figure 4-8). The velocity dependent ankle torques in saline control group appeared similar to those observed at post-op week 1 and 4, suggesting that this significant ve locity dependent increase in ankle torque was enduring. Surprisingly, at this post-injury time point, this re -emergent spasticity was not observed in baclofen cycle group; only a 10% in crease in ankle torque was observed at the highest test velocity (612 deg/ sec) and only a 1% increase was obs erved at the lowest velocity (49 deg/sec), even after remova l of the baclofen pump. The saline cycle group showed a 41% increase in magnitude in ankle to rques at the highest velocity (612 deg/sec) and a 9% increase at the lowest velocity (49 deg/sec), relative to th ese measures in the preinjury control group. At this point, EMG magnitudes were also observed to be increased significantly at the two fastest

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45 ankle rotation velocities (490 a nd 612 deg/sec) in both saline and baclofen control and saline cycle group as compared with the baclofen tr ained contused animals. Moreover, the EMG magnitudes were observed to be decreased for the saline cycle group at the next 3 test velocities (408-272 degs/sec) (Figure 4-8). No significant increases in ankle torque or EMG magnitude were observed during ankle rotations at the slowest four velocities at post contusion week 4 and post-op week 8. The EMG pattern for baclofen cycl e remained similar to the ankle torque data for the same week i.e., not much increase in magn itude as compared to pr e-injury data (Table 46). Table 4-5 Ankle torque week 8 Percentage (%) change ( ) compared to pre-injury. Table 4-6 Electromyeloencephalogram week 8 Percentage (%) change ( ) compared to preinjury. Groups Wk 8 Ankle Torque % Data 612 490 408 350 272 204 136 49 Baclofen Control 77% 60% 60% 55% 33% 12% 14% 4% Baclofen Cycle 10% 15% 11% 2% 5% 6% 7% 1% Saline Cycle 41% 24% 20% 22% 19% 5% 5% 9% Baclofen Control 75% 63% 57% 61% 34% 12% 8% 8% Wk 8 EMG Data % Groups 612 490 408 350 272 204 136 49 Saline Control 46% 65% 42% 118% 243% 196% 12% 51% Baclofen Cycle 29% 20% 24% 33% 53% 1% 19% 43% Saline Cycle 58% 74% 36% 41% 15% 66% 17% 74% Baclofen Control 46% 128% 52% 84% 228% 142% 216% 235%

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46 Gait and Open Field Locomotion Footprint Analysis: Gait Axial rotation0 10 20 30 40 50 60 70 Pre-injuryPost injury week 4Post injury week 8 saline control saline cycle bac control bac cycle ^, p 0.01, compared to saline control preinjury @, p 0.01, compared to baccontrol preinjury $, p 0.01, compared to salcycle preinjury *, p 0.01, compared to baccycle preinjury@ ^ $ @ $#, p 0.01, compared to baccycle week 4^ # # Figure 4-9 Axial rotation graph of all 4 groups at different time points with significant differences. Figure 4-10 Base of support graph of all 4 group s at different time points with significant differences. Base of support0 1 2 3 4 5 6 Pre-injuryPost injury week 4Post injury week 8 saline control saline cycle bac control bac cycle #, p 0.01, compared to bac cycle week 4 +, p 0.01, compared to sal control week 8 ^, p 0.01, compared to saline control preinjury @, p 0.01, compared to bac control preinjury $, p 0.01, compared to sal cycle preinjury *, p 0.01, compared to bac cycle preinjury # # # + + + @ ^ $ @ $ Base of support0 1 2 3 4 5 6 Pre-injuryPost injury week 4Post injury week 8 saline control saline cycle bac control bac cycle #, p 0.01, compared to bac cycle week 4 +, p 0.01, compared to sal control week 8 ^, p 0.01, compared to saline control preinjury @, p 0.01, compared to bac control preinjury $, p 0.01, compared to sal cycle preinjury *, p 0.01, compared to bac cycle preinjury # # # + + + @ ^ $ @ $

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47 Open field locomotor recovery61.48 61.33 56.62 69.90 54.95 51.19 60.38 46.43 y = 7.4524x + 38.976 R2 = 1 y = 6.6428x + 49.976 R2 = 1 y = -0.5477x + 62.024 R2 = 1 y = 1.8809x + 49.31 R2 = 1 0 10 20 30 40 50 60 70 80 PO week 4PO week8Percentage change from PO day 1 Saline control Saline cycle Baclofen control Baclofen cycle Linear (Saline control) Linear (Saline cycle) Linear (Baclofen control) Linear (Baclofen cycle) Footprint analyses were perfor med pre-injury, post-op week 4 and week 8. .Before injury, limb axis and base of support were measured to be 28.33 2.00 degs and 2.91 0.37 cms, respectively. At post-op week 1, in saline control group the values for limb axis and base of support were 44.45 degs and 3.79 cms respectively, whereas in baclofen controls group the readings were 46.08 degs and 4.49 cms respectivel y. The baclofen training group showed the least value 34.44 degs and 3.21 cms, whereas the saline cycl e group showed 45.86 degs and 4.13 cms respectively. Thus after 1 month post-op saline control and baclofen cycle showed the least change when compared to the pre-injury values (Figure 4-9 and 4-10). Compared with the preinjury control values, these measures revealed that limb axis and base of support were significantly increased in the ba clofen untrained contusion-inju red animals and saline cycle group during week 4. At post-op week 8, the trend was reversed with baclofen cycle showing increase in the base of support and angle of axia l rotation as compared to all the other 3 control groups at same time point and post-op week 4 and also compared to pre-injury data. At post-op week 8 ironically saline control showed the most improvement in all the 4 groups even without treatment (Figure 4-9 and 4-10). Open Field Locomotor Recovery Figure 4-11 Graph of BBB showing percentage change in score at post-op week8 compared to post-op week 4.

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48 OPEN FIELD LOCOMOTOR CHART0 5 10 15 20 25 PrePO Day 1 PO week 4 PO week8BBB SCOR E saline control saline cycel bac control bac cycle + p 0.05 com p ared to sal cont r l $, p 0.05 com p ared to sal c y cle p 0.05 com p ared to bac contl p o @, p 0.05 com p ared to bac control # p 0.05 com p ared to bac c y cle $ $ + + # # @ @ && p 0.05 com p ared to sal c y cle & !! p 0.05 com p ared to bac control p oweek8 Figure 4-12 Open field locomotor graph showing BBB score of all 4 groups at different time points with significant differences. Open field locomotor capacity (BBB) was scal ed in both trained and untrained animals before injury, at post-op day 1, at post-op week 4, and at postop week 8 to evaluate recovery during the early, intermediate, a nd late phases of recovery. Befo re the start of the treatment (bicycle and drug alone or combination), at pos toperative day 1 the saline cycle group animals had the highest score as comp ared to the 3 other groups. At post-op week 4, saline cycle trained contus ed animals displayed extensive movement of all three joints of the hind lim b, (mean score, 17 2) i.e., abou t 61% recovery compared to postop day 1, but as a group, these data revealed si gnificant variability in the scores among the groups (Figure 4-11). Baclofen cycle trained an imals also showed good improvement in their score (mean, 13 2) with a total recovery a pproximately a 56% increase from post-op day 1 (Figure 4-11). In contrast, baclofen contro l group (mean score 12 1) showed about 46% recovery while saline control an imals (mean score 11 1) showed about 51% recovery when compared to post-op day 1 scores respectively (Figure 4-11 and 4-15). Bicycle trained animals ^, p 0.05, compared to sal contrl ^

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49 displayed a frequent to consistent weight suppor ted plantar stepping and occasional to frequent FL-HL coordination (mean score, 15 2). This BBB score in the bicy cle trained group was significantly greater (p< 0.05, ANOVA) than values observed in either of the untrained groups (Figure 4-11). At post-op week 8, baclofen cycle animals revealed scores that were significantly increased (mean score, 16) when compared to values of the post-op week 4 and post-op day 1. This mean represented a recovery of approximately 70% when compared to post-op week 1 and 14% improvement compared to post-op week 4. Whereas, saline cycle group did not show any increase in its BBB score, instead revealed a drop (mean score, 16 1) when compared to values of post-op week 4 score. In f act, its condition deteriorated by about 1.5% compared to post-op week 4 (Figure 4-11). Saline untrained animals s howed increase in their score (mean score,13 2), about 15% improvement as compared to postop week 4. Baclofen control group showed an increase in its score (mean score, 15 0.2) i.e ., about 15% improvement as compared to post-op week 4. Both of the BBB scores in the locomo tor trained groups were significantly greater (p<0.05, ANOVA) than the scores recorded for the untrained groups. However, at this stage, animals of both trained and untrained groups showed consis tent FL-HL coordination and consistent weight supported stepping (mean sc ores, bicycle, 14.25 1.4, control, 15.25 1.7). Please note, the terminologies ne ver (0%), occasional (less than or equa l to half, <=50%), frequent (more than half but not always, 51-94%), and consistent (nearly always or always, 95100%) used above (Basso et al. 1995) The BBB score for an uninjured rat is 21 points. In summary, open field locomotor recovery sc ores scaled at post-op weeks 4 and 8 were significantly higher in bo th of the training groups compared with untrained controls. The

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50 baclofen cycle group demonstrated the highest recovery at po st-op 2 month, which was also significantly higher than the untrained group. However, at post-op week 8, both baclofen and saline training groups showed si milar recovery (ANOVA), whereas in postop week 4, the saline cycle group showed more recovery than the baclof en cycle. The differences in the recovery may, in part, be reflected by the rela tive starting point; the saline cy cle group started at a very high score compared to baclofen cycle group at post-op day 1. Neurotransmitters Immunocytochemistry results are shown in Figures 4-13 4-17. GABAb Baclofencycle Baclofencontrol Saline cycle Saline controlGABA b Figure 4-13 Immunohistoc hemistry of GABAb receptors in lumbar spinal cord tissues. In this Figure there is an increased expression of GABAb receptors in the baclofen cycle as compared to the other three groups, with sali ne cycle group showing more expression then baclofen control and saline cont rol showing the least expression

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51 5-HT (Serotonin) Baclofencycle Baclofencontrol Saline cycle Saline control5 5 HT HT Figure 4-14 Immunohistochemistry of 5-HT (Serotonin) fibers in thoracic spinal cord tissues. In this Figure there is an increased expressi on of 5-HT fibers in the baclofen cycle as compared to the other three gr oups, with baclofen control group expressing more fibers then saline cycle and saline contro l showing the least expression. BDNF Baclofencycle Baclofencontrol Saline cycle Saline controlBDNF BDNF Figure 4-15 Immunohistochemistry of BDNF (Brain derived neurotro pic factor) in lumbar spinal cord tissues. In this Figure there is an increased expre ssion of BDNF neuromolecules in Cycle Groups both baclofen and saline as compared to the baclofen control and sali ne control, which may point to the fact that exercise tends to increase the expression of BDNF.

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52 GAP43 Baclofen Baclofen cycle cycle Baclofen Baclofen control control Saline cycle Saline cycle Saline control Saline controlGap43 Gap43 Figure 4-16 Immunohistochemistry of GAP43 fibers in central canal of thoracic spinal cord tissues. GAP43 a neurotropic factor having functions si milar to BDNF showed an increased expression in baclofen cycle and somewhat more in saline cycle then compared to baclofen control and saline control. GAD67 GAD 67VH, 10X VH, 10X VH, 10X VH, 10XBaclofencycle Baclofencontrol Saline cycle Saline control Figure 4-17 Avidine-Biotine Complex (ABC) figures of GAD67 fibers in thoracic spinal cord tissues. In this Figure GAD67 showed an increased expr ession in baclofen cycle as compared to saline cycle and also increased expression in ba clofen control, while saline control showed minimal expression.

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53 Result Summary The above results support th e conclusion that baclofen cycle showed the greatest improvement with the lowest tor que amplitude in all the velociti es tested for ankle torque and EMG. The BBB score showed greatest percentage improvement in post-op week 8 compared to post-op day 1. Immunocytochemistry images showed an increased expression of the presynaptic inhibitor molecule GABAb in baclofen cycle group as compar ed to all the other groups. Also there was an increased expression of ne urotrophic factors such as BDNF, GAP43, 5-HT and GAD67, which is an indication of improvement in locomotion and increased growth of axonal fibers. Baclofen alone and locomotor treatment groups did show some improvement until postop week 4 but later were not able to maintain the same pattern in post-op week 8. The saline control group did not show any improvement in sp asticity at all time points but an improvement in BBB score which indicates that they have an intrinsic ability to initiate stepping without any treatment.

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54 CHAPTER 5 DISCUSSION The purpose of these studies was to test the hypot hesis that acute treatments using baclofen and locomotor training, individually, and in combination, would signi ficantly decrease the development of hyper reflexive spasticity follo wing SCI by comparing velocity dependent ankle torque and time locked triceps surae EMG. Seve ral behavioral tests (e.g. footprint analysis, and open field locomotor assessment) were also perfor med to test the influence of these treatments on aspects of locomotor functional outcome. In addition, to gain a bette r understanding of the fundamental neurobiology associat ed with SCI and the treatment s, the expression of certain neurotransmitters that play an important role in spasticity and locomotion was compared in the treated and non-treated groups. Compared with non-treated animals, individual treatment using locomotor training or ITB significantly reduced the development of early on set spasticity. However, ITB treatment during the first month post-injury did not decrease the development of late onset spasticity, while cycle training reduced the late onset spasticity by ap proximately 50%. The combination therapy (ITB and cycle training) profoundly reduced the developm ent of both early and la te onset spasticity. Ankle Extensor Stretch Reflex Saline Control In saline control group there was hyper refl exive pattern (spastic ity) seen at all the velocities tested and at all time periods (i.e., at post-op week 1, 4 and 8 with the EMG signals showing the same pattern as that of the ankle torque). This shows that when no treatment is given following SCI there is development of spasticity after the initial hypo refl exive period as the repetitive proprioceptive signals from the sensor y receptors are not controlled and it leads to

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55 spasm of the flexor muscle. So some form of treat ment is essential for the treatment of spasticity following SCI. Saline Cycle The saline cycle group showed hyper reflexive pattern at post-op week 1. These data suggest that only exercise suffici ently addressed the mechanism that led to the hyper reflexia at this time point. These presumably include hyperactivity of the cells in the sp inal cord after injury due to the release of the excitatory neuromolecu les and the membrane damage which results in imbalance in the extracellular and intracellula r components which is most common till week 1 post-injury. The EMG signals at this week also showed the same pattern. In contrast, at post-op week 4, the ankle tor que amplitude data of this group showed a pattern of hypo reflexia. This decr ease in excitability may be due to the exhaustion of excitatory neurotransmitters after a certain time period af ter injury. Another possi bility is that training induced activity may initiate a balancing of the neurotransmitters and preserve descending controls of segmental regulatory processes. As it has been reported in our lab studies, a pattern of hypo reflexia was observed at post-op week 2 in untrained and trained animals without drug treatment and continued up to week 6. Immediat ely after SCI motoneur ons receive unusually large EPSPs from cutaneous stimulation consiste nt with the acute loss of descending brainstem innervations of the dorsal hor n. These EPSPs do not easily caus e reflexes immediately after injury, because of the profound lo ss of motoneuron excitability that occurs after injury from other mechanisms, such as decreased dendritic PICs. Recent studies indicate that a significant decrease in these postsynaptic, dendritic excitato ry mechanisms may play an important role in the hypo reflexia at this time (Li et al. 2004), due to the loss of intrin sic persistent inward calcium and sodium currents (PICs) that norma lly prolong and amplify synaptic inputs. In addition, these investigators studi es have revealed a spontaneous re-emergence of PICs that may

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56 significantly contribute to late onset spasticity th at develops around post contusion week 6. Accordingly, at post-op week 8, we observed a hyper reflexive pattern in all the velocities tested. These observations indicate that training alone cannot maintain th e state of hypo reflexia without any complimentary drug treatment. A similar pattern was seen in the EMG signal at all the different time periods tested and at all the different velocities tested. Baclofen Control At post-op week 1, the baclofen control group did not show any hyper reflexive pattern in all the velocities tested. These data suggest that baclofen alone can stabilize the excitatory environment that influences motoneuron excitab ility immediately after injury, such as cell membrane disruption and an imbalance of inhibi tory and excitatory neuromolecules. It is proposed that baclofen maintained an increased concentration of inhibitory neuromolecules by activating the GABAb receptors and their subsequent cont ributions to controlling excitation. At post-op week 4, a hypo reflexive pattern was obs erved due to the above described actions combined with a reduction in availability of unre gulated release of excitatory neurotransmitters. However, at post-op week 8, a hyper reflexive patt ern was observed in all the velocities tested. This may be due, in part, to the withdrawal eff ect of baclofen as the baclofen pump was removed at post-op week 4. The withdraw-induced hyper refl exia may be correlated with an increased stretch reflex excitability a ssociated with a significant lig and-mediated down-regulation of GABAb receptors in the ITB treated spinal cord, a previously reported result of the chronic exposure to baclofen (Kroin et al. 1993). This ch ange also relates to the abrupt loss of the previously described GABAb associated inhibitory processe s. In addition, it may be due to baclofen modulation of the PIC (Heckman et al 2004), which plays an important role in the development of spasticity; baclofen blocks the Ca2+ channels which play an important role in generating the PICs, and it may have generated PICs in the absence of baclofen at post-op

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57 week 8. The EMG signals showed the same pattern as that of velocity dependent ankle torque, and it was time locked at all th e different time periods tested. Baclofen Cycle ITB treatment along with exercise decreased ankle extensor stre tch reflex excitability as indicated by significant decreases in velocity dependent ankle torque and time-locked EMG magnitude. During the 1week period of ITB treatment with bicycle of the lu mbar spinal cord, the low-velocity ankle torque was unchanged, wherea s torque recorded during high-velocity ankle rotations were significantly decreased compared to pre-treatment values. The reduction in torque was also accompanied by significant reduction in th e short-latency EMG that was time-locked to ankle rotation. The reduction in EM G activation is consistent with the increase in the threshold for activation associated with the pr esumed mechanism of action of GABAb receptor mediated decrease in transmitter release at the primary afferent terminals (Peshori et al. 1998). A similar finding was reported previously from our lab (Wang et al. 2002) Similarly during post-op week 4 and week 8 the ankle torque and EMG activation were near the pre-treatment values at all the velocities tested. Current evidence suggests that following th e initial trauma, many secondary events including membrane damage, systemic and local vascular effects, altered energy metabolism, oxidative stress, inflammation, el ectrolyte imbalances, unregulated release of neurotransmitters, and a cascade of biochemical changes affect cellu lar survival, integrity, and excitability (Tator 1995). From the above data we can see that baclofen, which is a GABAb agonist, inhibited the release of the excitatory neurot ransmitters, which prevented the membrane damage and cellular imbalance in the intracellula r and extracellular compartmen t during the first week post-op, therefore preventing the development of spasticit y. During post-op week 4, exercise and baclofen maintained an inhibitory environment and prevente d the development of spas ticity most likely by

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58 blocking the PIC, which develop in the motoneurons after injury after a brief period of hypo reflexia due to the activation of Na+ and Ca2+ channels, and baclofen pr evented it by blocking the Ca2+ channels. At post-op week 8, even after the removal of the drug there was no development of hyper reflexia. This may be due to exercise, which mu st have maintained the inhibitory environment and stabilized the firing pattern fr om the motoneurons. It remains to be seen how baclofen works in presence of exercise and how exercise maintains the hypo refl exive state even after removal of the drug. This can be done in future studies by doing single cell motoneuron recordings using patch clamp. Thus it proves that without baclofen and loco motor exercise treatment spasticity develops immediately after injury and thus proves our hyp othesis that acute baclof en and bicycle training is the most effective in preventing the development of the spasticity without deteriorating the condition of the animal as we see from the da ta that neither alone baclofen or locomotor prevented the development of spasti city at all post-op week 1, 4 and 8. Open Field Locomotor Assessment Saline Control Animals with surgical lesions of the dorsal spinal cord at T8 that preserved ventral funiculi, demonstrated sufficient self-training that no detectable difference was observed in their locomotor recovery compared with animals that were systematically trained using a treadmill (Fouad et al. 2000). This is true as we also saw a 51% improvement in this group at post-op week 4 when compared to post-op day 1. At post-op week 8 group means BBB scores showed a slight improvement of about 4% fr om post-op week-4, which is attri buted to their intrinsic ability to self-train. Even though the ankle torque data showed spasticity starting from post-op week 1, 4 and 8 this group showed gait improvement without any treatment.

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59 Saline Cycle This group of animals started with a slightly higher score compared to the other entire group on post-op day 1. It showed an improvement of about 61% at post-op week 4, which is better than the baclofen cycle gr oup. Also, the ankle to rque showed hypo reflexive pattern at this time period, which may be due to the exhaustion of the excitatory neurotransmitters as reported earlier or stabilization of the neuronal circuits. While at post-op week 8, a decrease in BBB score was seen of about 2% from post-op week 4, and also the ankle torque showed hype r reflexive pattern going along with this finding. This may be due to the fact that bicycle tr aining maintained a pattern of uniformity and efficiency, which resulted in the improvement of the BBB score at post-op week 4.Training alone may not be efficient to ha ve a presynaptic inhibitory eff ect like that of baclofen on the ventral horn motoneurons which may continue to fire continuously leading to hyper reflexia in post-op week 8. After certain peri od of exercise the improvement in the segmental circuit sub serving the muscle and joints may reach the plat eau level from where further improvement is not possible. Thus exercise alone may be unable to maintain an inhibitory environment for preventing the development of late ons et spasticity and also the gait. Baclofen Control The BBB score for baclofen control group improved about 47% from post-op day 1 to post-op week 4, and also it improved about 15% from post-op week 4 to post-op week 8.The score for post-op week 4 corresponds to the ankle torque and EMG data; we observed no spasticity in this group at that period of time. Thus baclofen may have stabilized the cell membrane of the cells after in jury and prevented the imbalance in the intracellular and

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60 extracellular environment and t hus prevented the development of spasticity and also improved the gait pattern as the BBB score improved. But, in contrast to ankle tor que week 8 data; there was a re surge of hyperref lexia after the withdrawal of the drug at week 4. The BBB score improved about 14% from post-op week 4. This may be attributable to the preservation of fibers diffusely located in the ventral caudal and ventro-lateral funiculi of the ra t spinal cord (Basso et al. 2002 ; Brustein and Rossignol 1998), or gray matter of the T13-L2 spinal segments (Magnuson et al. 1999). Even after injury, baclofen may have stabilized them, and after withdrawal of the drug, their connectivity to the muscles and joints of the hind limbs may have improved. Co llectively, these may have contributed to the improvement of the BBB score. The ankle torque may be increased due to the withdrawal effect on segmental excitability processes as expl ained above in the ankle torque data. Baclofen Cycle In this group we saw an improvement in the BBB score from post-op day 1 to post-op week 4, a total improvement of 57%. This data co rresponds to the ankle to rque data where there is no hyper reflexia at post-op week 4, thus it pr oves that acute locomotor training and baclofen drug prevented the development of spasticity and also improved the gait of the injured animals instead of deteriorating them. In animals and humans with SCI, previous studies have shown improvements in gait parameters following loco motor training using body weight support on the treadmill and manual assistance (Behrman and Ha rkema 2000; Harkema et al. 1997; Dietz and Harkema 2004) but have not concurrently eval uated effects of bicycle locomotor training following animal with SCI. The findings of the pr esent study are consistent with the suggestions that as therapy, the locomotor training regimen us ing bicycle, promotes th e recovery of walking by optimizing the activity-dependent neuroplastic ity of the nervous system (Muir & Steeves

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61 1997; Bose et al. 2005). Neuronal circuits, stimulat ed by task appropriate activation of peripheral and central afferents via locomotor training, ma y also reorganize by strengthening existing and previously inactive descending connections and local neural circuits (Muir & Steeves 1997) (Bose et al. 2005). At post-op week 8, the BBB score improved from post-op week 4, even after removal of the baclofen drug. This proves that the bicycl e training may have prolonged the effects of baclofen or it might itself have prevented any secondary damaging effect s that may occur after withdrawal of the drug. Thus, it proves that acute drug therapy and locomotor treatment improves gait and maintains the reflex excitability to the pre-injury values. Footprints Hind limb axial rotation and base of support were assessed by footprints, collected while walking along a treadmill, and were measured to compare between cycle trained and control animals. Changes in these parameters in hum ans have been correlated with dysfunction of descending long tract and propriospinal systems (K unkel-Bagden et al. 1993). Saline Control Saline control group, which received no treatment, did show deterioration in the base of support and axial rotation at postop week 4, but at post-op week 8, the base of support showed near pre-injury values, while the axial rotation remained similar to post-op week 4 values. As these groups of animals were not bicycle trained and therefore not handled frequently they retained their pre-injury parameters. It has also been shown that control SCI animals have the intrinsic ability to self train.

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62 Saline Cycle Saline cycle group showed a pattern similar to that of baclofen control with deterioration at post-op week 4 and maintaining the same pattern at post-op week 8. Thus exercise alone may not be beneficial to improve the ga it parameters without the drug. This may lead to one conclusion that whatever changes occur in the segmental circuit that maintain the foot placement and locomotion occur w ithin the first 4 weeks af ter the injury and it remains the same and does not change later. Baclofen Control In this group we saw an increase in base of support and axial rotation at post-op week 4, but at post-op week 8, the values remained more or less similar to post-op week4. This may point out that bacl ofen alone may not be effective in improving the gait parameters and that some form of locomotor training is required as we see an improvement in baclofen cycle at post-op week 4. Baclofen Cycle At post-op week 4, the base of support and the axial rotation showed pre-injury values, but at post-op week 8 the base of support and axial rotation showed much increased value compared to the other group. The deficits observed in th e baclofen treated anim als of the present study could have occurred through changes in the activit y of these long tract or propriospinal systems: either at synaptic terminals, cell body of origin, or upon spinal interneuro ns modulating posture. It is known that baclofen modulation of the syna ptic actions of spinal ventromedial funicular fibers mediated presumably by GABAb receptors on or near axon term inals and last order spinal interneurons (Jimenez et al. 1991; Qu evedo et al. 1992). In addition, GABAb sensitive sites have been reported in vestibular and functional co mpanion nuclei that regulate the gain of the vestibulospinal reflex (Manzoni et al. 1994). Therefore, it is possible that the ITB treatment

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63 induced changes in posture and limb axis via acti ons within the spinal cord or within neuronal sites in the brainstem that regulate posture and equilibrium. However, the differences in the time course for the changes in hind limb axis and ba se of support with improvement in post-op week 4 and deterioration at post-op week 8 may be that after the withdrawal of the drug the GABAb receptors must have up regulated but there is no ligand (baclofen) present to bind them. But this finding goes against the fact th at in baclofen cycle animal s the ankle torque showed hypo reflexia at all the time points te sted. This could mean that the drug must be acting at different anatomical sites (Wang et al. 2002 ) to have different effect on a nkle torque and footprints. Saline cycle group also did not show much improvement in the footprint parameters at post-op week 8. One of the possibilities may be that after bacl ofen pump removal, the toll of exercise may have caused some unwanted stress on the hind limb paws that might have led to external deviation of the paw leading to anatomical defect and thus increase in ba se of support and axial rotation in post-op week 8. As baclofen pump was removed after post-op week 4, but training was continued up to post-op week 8, the residual e ffect of baclofen must have prevented the bad effects of exercise. As for the baclofen cy cle group, the combination of drug and locomotor therapy prevented the early changes, but once the drug was removed, exercise alone could not maintain the inhibitory environment to prevent the changes from taking place. Neurotransmitters Saline Control Neurotransmitters like 5-HT, GAD67, GABAb and neurotropins like BDNF and GAP43 showed a decreased expression when compared to all the other groups. This may be due to the fact that these groups did not receive any loco motor training and baclof en drug. Baclofen, which is a GABAb receptor agonist, is primarily used as an anti-spastic drug and also the ankle torque and EMG data did not show any decrease in sp asticity when their torque amplitude was

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64 measured. Thus these animals showed the least improvement when compared to all the other groups. Saline Cycle Neurotransmitters like 5-HT, GAD67, GABAb receptors and neurotropins like BDNF and GAP43 did show an increased expression when compared to saline control group. Locomotor training may have facilitated the increased expression of neurotr opins like BDNF and GAP43. As predicted from Cotmans paper (2002) exercise increases the expression of the neurotropins, which have an effect on the growth of the a xons and improves the neur onal plasticity. Since there is little increase in GABAb receptor expression this may be correlated to the improvement in spasticity seen during post-op week 4. Al so, increased expression of 5-HT and GAD67 may correlate with the improvement to the loco motion observed during post-op week 4 on the BBB scale. Baclofen Control In this group we also saw similar neurotransm itter expression as that of saline cycle group. As this group received baclof en drug only with no locomoto r training, the drug must have influenced the expression of GABAb receptor till post-op week 4, which also corresponds to the improvement of spasticity during that period. Al so, there must be some correlation between 5HT and GAD67 expression to that of baclofen drug as there was increased expression of the former neurotransmitters. Neurotropins like BDNF and GAP43 also showed an increased expression to that of saline control group, which co-relates to the improvement in the BBB score observed at post-op week 4. But this group was not able to maintain the improvement till post-op week 8 as the baclofen pump was removed at post-op week 4.

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65 Baclofen Cycle This group showed the maximum neurotransmitte r expression when compared to all the other groups. There was an increased expression of all the neurotransmitters and neurotropins and also hypo reflexive pattern when tested for spas ticity. This may be due to the fact that this group received both baclofen drug and locomotor tr aining. As we have disc ussed above, exercise improves the expression of ne urotropins like BDNF and GAP43, which promotes neuronal plasticity and improved neuronal growth in case of injury, thus leading to improvement in gait and spasticity. Baclofen drug increased the presynaptic inhibition by in creasing the expression of GABAb receptors, which was responsible for the hypo reflexive response seen during measurement of spasticity. Thus, all the above data show th at if baclofen drug and locomo tor training in the form of bicycle, if started acutely after spinal cord injury, may preven t the development of spasticity and help in improving the gait of the animal. By contra st, baclofen alone or cy cle alone did not show a continuous maintenance of hypo reflexive pattern, with later development of spasticity and not much improvement in the gait.

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66 CHAPTER 6 CONCLUSION In humans, one of the most devastating chr onic effects following SCIs is the development of spasticity. Spasticity leads to velocity depe ndent lengthening resistance in the muscles resulting in contractures and s econdary biochemical changes whic h eventually cause destruction of the muscles. In humans the present antispastic treatment is the continuous infusion of ITB, a GABAb agonist that acts on the pres ynaptic receptors leading to inhibition of both monosynaptic and polysynaptic reflexes. At present, there are no approved guidelines to administer this drug acutely before the symptoms of spasticity become evident. Locomotor training in the form of weight bearing stepping using treadmill has been shown to improve the gait of the SCI patients. Treadmill training requires extensiv e use of manpower to hold the patient while training and also this facility is present only in few selected locations across the nation. Therefore, the aim of this thesis was to provide preclinical data to test the efficacy of baclofen therapy with or without locomotor training (bicycle locomotor training) wh ich has already been shown to be as effective as treadmill (Bose et al. 2004). To test the above hypothesis spinal cord injured rats were used as their spinal cord circuitry resembles to that of human. Mo reover, contusion injury mimics most common SCI in humans and produces spasticity. The animals were divide d into 4 groups after contusion spinal cord injury: Saline control group saline pump, no bicycle training Saline cycle group saline pump, bicycle training Baclofen control group baclofen pump, but no bicycle training Baclofen cycle group baclof en pump + bicycle training.

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67 The animals were selected only if their ope n field locomotor (BBB) score fell below 3 when tested at post-op day 1. The animals were tr ained over the course of 2 months. The training schedule was performed 5 days a week using tw o 20 minute trials/day, st arting from post-op day 1. On the first day of training, the ra ts were given five minutes training. The baclofen and saline pump were removed at post-op week 4. The animals were tested for spasticity at post-op weeks 1, 4 and 8 using the velocity dependent ankle torque set up developed in our lab (Thompson et al. 1996; Bose et al. 2002) and also measuring the EMG signals from the muscles in the same set-up. The animals were tested for gait improvement using the BBB scale and footprints at post-op weeks 4 a nd 8. The animals were then sacrificed using 4% freshly prepared paraformaldehyde using an accepted protocol to remove the thoracic, injured and lumbar spinal cord. The spinal co rds were then used to do immunofluorescent experiments to test the presence of various neuromolecules such as GABAb, GAD67, 5-HT, BDNF and GAP43 which have documented roles in the motoneuron excitability and neuroplasticity following injury and therapy. As per the results we got from all the above experimental procedures we can come to the conclusion that acute baclofen drug therapy with bicycle training preven ted the development of spasticity measured by velocity dependent ankle torque and associated EMG data. Moreover, this combined treatment showed an increment in BBB score compared to post-op day 1 when compared to other 3 experimental groups. Th e immunofluorescent images showed qualitative increase of GABAb receptors as well as other neuromolecules such as 5-HT, GAD67, BDNF and GAP43 as compared to all the other 3 groups. Hypo re flexive pattern seen in baclofen cycle group might be related to GABAb receptors mediated presynaptic in hibition of the stretch reflexes. However, interestingly, the base of support and axial rotation did not show improvement when

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68 compared to other groups. While th e control groups such as baclofen drug alone or saline cycle alone did show some decrease in spasticity at post-op week 4, th ey could not retain the same improvement at post-op week 8. Thus to conclu de, we found that acute baclofen drug therapy along with bicycle training preven ted the development of spasticity. Moreover, the acute training and drug therapy did not deterior ate the condition of the injured animals. Therefore, these preclinical data have the potential to translate in human clinical trail.

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69 CHAPTER 7 FUTURE WORK In this research project, we have shown th at combination therapy of baclofen drug and locomotor training started acutely after spinal cord injury prevented the development of spasticity and improved the ga it, and overall locomotor capac ity. Surprisingly, the individual therapy of either drug (ITB) or locomotor training (customized bicy cle) did not show benefit in improving these condition. Baclofen and locomotor exercise possibly work synergistically and thus, the mechanism underlies these benefits need to be unfolded. Therefore, a detailed study involving GABAb receptor profile as well as properties of the motoneuron has to be investigated by intracellular motoneuron recording using in vivo patch clamp during different time periods of the treatment. As this research only c onducted qualitative analysis of the GABAb receptors by immunofluroscent technique, molecular techniques such as Western Blot or ELISA can be used to quantitatively measure the expres sion of receptors in the spinal cord tissue to test differences in expression in different groups Moreover, neurotropic factor, su ch as BDNF, is an important indicator of regeneration of injured axons which mediates neuroplasticity. Ther efore, a detailed molecular study to investigate the profile of BD NF and its receptor trkB can provide a better understanding of neuroplasticity mediated by B DNF following locomotor training. Furthermore, in vivo longitudinal MRI study using volumetric measurement of the lesion can be done to further predict the benefits of this combined th erapy. At present, I am planning to work on post fixed spinal cord tissue using T2-wieghed MRI imaging to study the effects of locomotor and drug therapies. These multi-dimensional studies may further enhance our understanding in the mechanism of the recovery/benefits we have observed following locomotor and drug therapies.

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77 BIOGRAPHICAL SKETCH Rita Jain was born in Mumbai, India. She comp leted her bachelors degree in medicine in 2001. After her graduation she worked as a reside nt medical officer in Niramaya Hospital in Pune, India. After working there for 6 months, sh e realized that her knowledge was not sufficient to bring a breakthrough in the medicine world an d to decrease the suffering of the patients. She decided to go for higher education. She came to United States in 2003, to pursue her masters degree in medical sciences at the University Of Florida. She chose to specialize in neuroscience. For the next 3 years, Thompsons lab was her hom e. There she worked under the guidance of Dr. Prodip Bose and Dr. Floyd Thompson on various research projects.

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Title: Effects of Acute Locomotor Training with or without Baclofen Therapy on Spasticity after Contusion Spinal Cord Injury (SCI)
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Copyright Date: 2008

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EFFECTS OF ACUTE LOCOMOTOR TRAINING WITH OR WITHOUT BACLOFEN
THERAPY ON SPASTICITY AFTER CONTUSION SPINAL CORD INJURY (SCI)




















By

RITA I. JAIN


A THESIS PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
MASTER OF SCIENCE

UNIVERSITY OF FLORIDA

2006

































Copyright 2006

by

Rita I. Jain

































To the memory of my mother and grandfather-in-law whom I will miss throughout my life.









ACKNOWLEDGMENTS

I would like to thank Dr. Prodip Bose and Dr. Floyd Thomson who have been excellent

mentors and supervisors during my studies at the University of Florida. Their inspiring

enthusiasm and energy have been contagious, and their advice and constant support have been

extremely helpful. I would also like to thank Dr. Ammon Peck for his guidance and for serving

in my committee. I would also like to thank my lab personnel Ron Parmer and Yanping Cheng

for their help during the entire process of my project work.

I owe much of my academic and personal success to my parents, my in-laws and my

brother Niranj an, who, by example, provided me with the motivation and courage to pursue my

studies.

The greatest thanks to my husband, Sanjay, for his tremendous encouragement,

understanding, patience, and unconditional love and to my daughter Risha, whose arrival

encouraged me to complete my project at the earliest.









TABLE OF CONTENTS

Page

A C K N O W L E D G M E N T S ..............................................................................................................4

LIST OF TABLES .................. .....................................................7

L IS T O F F IG U R E S ......................................................................................................... .....8

LIST OF ACRONYMS AND ABBREVIATIONS ........................................... ............... 10

A B S T R A C T ................................................................................................................................... 1 1

INTRODUCTION .................................. .. ..... ..... ................... 13

Spin al C ord Inju ry ................................................................13
S p a stic ity ................... ...................1...................3........

B A C K G R O U N D ..................................................................................................................... 16

Spinal Cord Injury A nim al M models ............................ ........................... .......... ...16
Spinal Cord Contusion Model (Incomplete Spinal Cord Injury) .................................17
Spinal Transection Model (Complete Spinal Cord Injury) ....................................... 17
Spinal Isolation M odel ........................... .. .......................... ...................18
Anatomical (Neuromodulatory) Changes Following SCI .....................................................18
A R odent M odel of Post-SCI Spasticity ................................................................ ..19
M mechanism of Spasticity .......................................................................... ... ... ..............19
Present Antispasticity Treatment for Spinal Cord Injury ................................................. 22
L ocom otor R eh ab ilitation ............................................................................ .....................24

EXPERIMENTAL DESIGN AND THE STANDARD METHODOLOGY USED IN THE
P R O JE C T ..........................................................................2 9

O bj e ctiv e ................... ...................2...................9..........
A n im a l S u b je c t ........................................................................................................2 9
Contusion Injuries............ .... ......................................... ....30
Baclofen Pum p Im plantation ......... ............ .................. .......................... ............... 31
B icy c le T ra in in g ............................................................................................................... 3 2
F o otp rin ts ......... .......................... ....................... .................................3 3
B B B S c o re ..........................................................................3 3
A nkle Torque ..............................................................................34
Immunocytochemistry of Spinal Cord Tissue ...........................................36

R E S U L T .............................................................................. 3 8

Velocity-Dependent Ankle Torque and Associated EMG's ...........................................38
Pre-injury Ankle Torque and EM G D ata ....................................................... 38









Postoperative Week 1 Ankle Torque and EMG Data ..................................... ........ 39
Postoperative Week 4 Ankle Torque and EMG Data ............................................. 41
Postoperative Week 8 Ankle Torque Data and EMG Data ..........................................43
G ait and O pen Field Locom option ............................................... ......................................46
Footprint A analysis: G ait ......................................................................... ...................46
O pen Field Locom otor R recovery .............................................................. ...............47
N eurotransm hitters .............................................................................50
G AB A b .........................................................................50
B D N F ..........................................................................5 1
G A P 43 ..................................................................................................... ......... . ....... 5 2
G AD 67 ...................................................................................................... 52
R result Sum m ary .................................................53

D ISCU SSION ................... ...................5...................4..........

A nkle Extensor Stretch R eflex ......................................................................................... 54
S a lin e C o n tro l .......................................................................................................5 4
S a lin e C y c le .....................................................................................................................5 5
B aclofen Control ....................................................... 56
B aclofen Cycle ............................................................57
O pen Field Locom otor A ssessm ent ..........................................................................................58
Saline Control ......................................................................................................... 58
S a lin e C y c le .....................................................................................................................5 9
B aclofen Control ....................................................... 59
B aclofen Cycle ................................................................60
Footprints ................... ...................6...................1..........
S a lin e C o n tro l .......................................................................................................6 1
S a lin e C y c le .....................................................................................................................6 2
B aclofen Control ....................................................... 62
B aclofen Cycle ................................................................62
N eurotransm hitters .............................................................................63
S a lin e C o n tro l ....................................................................................................6 3
S a lin e C y c le .....................................................................................................................6 4
B aclofen Control ....................................................... 64
B aclofen Cycle ................................................................65

CON CLU SION ................... ...................6...................6..........

FU TU RE W O RK ..................................................................................................................... 69

LIST OF REFEREN CE S ....................................................................................................70

BIO GR A PH ICA L SK ETCH ................................................................................................... 77







6









LIST OF TABLES


Table Page

4-1 Ankle torque week 1- Percentage (%) change (%i) compared to pre-injury. ..................41

4-2 Electromyeloencephalogram week 1 Percentage (%) change (T ) compared to pre-
inju ry ........ ........ ............................................................................ 4 1

4-3 Ankle torque week 4- Percentage (%) change (1 ) compared to pre-injury. ..................43

4-4 Electromyeloencephalogram week 4- Percentage (%) change (T ) compared to pre-
inju ry ........ ........ ............................................................................ 4 3

4-5 Ankle torque week 8 Percentage (%) change (% ) compared to pre-injury ..................45

4-6 Electromyeloencephalogram week 8 Percentage (%) change (T ) compared to pre-
inju ry ........ ........ ............................................................................ 4 5









LIST OF FIGURES


Figure Page

3-1 The experimental set up and procedures done in chart form.................. ......... .......30

3-2 Motorized bicycle used for training the rats....................................................................32

3-3 Ankle torque, displacement and EMGs are simultaneously recorded and time-
locked to onset of dorsiflexion....................... ......... ....................... ............... 34

4-1 Pre-injury ankle torque graph. ................................................ ............................... 38

4-2 P re-injury E M G graph. ........................................................................... ..................... 38

4-3 Ankle torque graph at post-op week 1 with significant differences ...............................39

4-4 Electromyeloencephalogram graph at post-op week 1 with significant differences. ........39

4-5 Ankle torque graph at post-op week 4 with significant differences ...............................41

4-6 Electromyeloencephalogram graph at post-op week 4 with significant differences. ........42

4-7 Ankle torque graph at post-op week 8 with significant differences ...............................43

4-8 Electromyeloencephalogram graph at post-op week 8 with significant differences. ........44

4-9 Axial rotation graph of all 4 groups at different time points with significant
d iffe re n c e s ...................................... ..................................... ................ 4 6

4-10 Base of support graph of all 4 groups at different time points with significant
d iffe re n c e s ...................................... ..................................... ................ 4 6

4-11 Graph of BBB showing percentage change in score at post-op week compared to
post-op w eek 4. .............................................................................47

4-12 Open field locomotor graph showing BBB score of all 4 groups at different time
points with significant differences. ...... ....................................................................... 48

4-13 Immunohistochemistry of GABAb receptors in lumbar spinal cord tissues.....................50

4-14 Immunohistochemistry of 5-HT (Serotonin) fibers in thoracic spinal cord tissues...........51

4-15 Immunohistochemistry of BDNF (Brain derived neurotropic factor) in lumbar spinal
c o rd tissue e s ................................................... .......................................... 5 1









4-16 Immunohistochemistry of GAP43 fibers in central canal of thoracic spinal cord
tissue es ......................................................... ....................................52

4-17 Avidine-Biotine Complex (ABC) figures of GAD67 fibers in thoracic spinal cord
tissue es ......................................................... ....................................52













ANOVA

BBB

BDNF

Ca2+ channels

C1l channels

CPG

Deg/sec

EMG

EPSP's

GABAb

GAP43

IgG

IHC

ITB

Kdynes

MASCIS

Na+ channels

PAD

PIC

SCI

VDAT

5-HT


LIST OF ACRONYMS AND ABBREVIATIONS

Analysis Of Variance

Basso, Beattie and Bresnahan scale

Brain Derived Neurotropic Factor

Calcium channels

Chloride channels

Central Pattern Generator

Degrees/second

Electromyeloencephalogram

Excitatory Postsynaptic Potentials

Gamma Amino Butyric Acid

Growth Assosiated Protein 43

Immunoglobulin G

Imunohi stochemi stry

Intrathecal Baclofen

Kilodynes

Multicenter Animal Spinal Cord Injury Studies

Sodium channels

Primary Afferent Depolarization

Persistent Inward Currents

Spinal Cord Injury

Velocity Dependent Ankle torque

5-Hydroxytryptamine (Serotonin)









Abstract of Thesis Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Master of Science

EFFECTS OF ACUTE LOCOMOTOR TRAINING WITH OR WITHOUT BACLOFEN
THERAPY ON SPASTICITY FOLLOWING CONTUSION SPINAL CORD INJURY (SCI)

By

Rita I. Jain

December 2006

Chair: Floyd Thompson
Cochair: Prodip Bose
Major Department: Medical Sciences

Progress has been made in the therapeutic treatment of spasticity, especially utilizing

intrathecal baclofen (ITB). In addition to medical treatment, two decades of studies in animals

and humans report exciting possibilities for locomotor training to improve locomotor recovery.

However, many unknowns remain regarding the timing of treatments, and whether they interact

to facilitate or inhibit rehabilitation directed at recovery of voluntary motor activity. Since

spasticity development is progressive, we explored the potential for acute initiation of therapies

to influence the development of spasticity. These studies were performed to evaluate the safety,

feasibility, and efficacy of two early intervention treatments (performed alone, or in

combination) on measures of spasticity and long term functional outcome measures following

midthoracic contusion SCI. Four groups of animals received contusion injuries to the

midthoracic spinal cord using the New York University (NYU) impounder and the Multicenter

Animal Spinal Cord Injury Studies (MASCIS) protocol for moderate injury (10 gm weight drop

at a distance of 12.5mm). Two groups of animals received ITB pumps (Azla Corp., Palo Alto,

CA) at the time of injury; the other two groups received pumps with saline vehicle. The tip of the

intrathecal cannula was placed in the subdural space of the L1-L2 lumbar spinal cord. Beginning









at day 1 following injury, two 20 minutes sessions of locomotor exercise were performed using a

custom made cycle locomotor trainer on one ITB and one vehicle control group. Velocity

dependent ankle torque, ankle extensor muscle EMGs, hindlimb axis, and open field locomotion

(BBB), were recorded in all groups. The data indicate that at 2 months following injury, cycle

training alone produced a greater reduction in ankle torque than baclofen alone. However, ankle

extensor spasticity was significantly lower, and rate of open field recovery was greatest in the

animals that received the combination of ITB and cycle training, compared with animals that

receiving no treatment or either of the treatments performed alone. No between group differences

were observed in footprint data for acute treatments alone or combined. Terminal

Immunohistochemistry of lumbar spinal cord segments revealed increased expression of markers

for GABA (GABAb, GAD67) & monoamines (DBH (NE), and 5-HT) in combined treated

animals. Treatment related upregulation of GABA/GABAb molecules might exert significant

roles in pre-synaptic inhibition and upragulated monomamines might prevent supersensitivity of

monoamine receptors (a-1 & 5-HT) and thus might block both the early onset and the late onset

of spasticity. These data indicate that acute treatments using locomotor exercise / ITB are safe

and feasible initiated as early as postcontusion day 1 and ITB and locomotor treatments

differentially influenced early and late onset of the development of spasticity.









CHAPTER 1
INTRODUCTION

Spinal Cord Injury

The spinal cord (SC) is a vital organ of the body, which is necessary for the maintenance

of posture and locomotion. Trauma to the SC leads to a series of events resulting in changes in

reflex excitability from spinal shock to a hyperreflexive condition known as spasticity. Frequent

causes of SC damage are trauma (car accident, gunshot, falls, etc.) or disease (polio, spina bifida,

Friedreich's Ataxia, etc.). The SC does not have to be severed in order for a loss of functioning to

occur. In fact, in most people with spinal cord injury (SCI), the SC is intact, but the damage to it

results in loss of functioning. About half of the patients with SC cord trauma have incomplete

injuries without any signs of voluntary motor or sensory perception below the level of the lesion.

The victims of cord trauma are mainly young men in their early 20s to 30s (National Spinal Cord

Injury Statistical Center). Efforts should be made to improve the quality of life of the young

victims and to reduce the clinical burden in society. To date, no suitable therapy for the victims

of SCI is available. New therapeutic strategies with the possibility of regeneration of the lesioned

SC axons are needed to improve the quality of life of patients with SCI. Spinal cord injury

induces secondary biochemical responses that include both neurotoxic and neuroprotective

processes (Tator,1995). It is believed that the balance between these reactions in part determines

ultimate tissue damage and the degree of associated neurological recovery. Although functional

regeneration appears to be limited after SCI in mammals, it is not unlikely that plasticity of

surviving cells contributes to functional recovery that is often observed.

Spasticity

Spasticity is a form of muscular hypertonia, due to a velocity-dependent increase in tonic

stretch reflexes during passive movement, which results from abnormal spinal processing of









proprioception after SCI. Spasticity, is a major health problem for patients with a SCI. It limits

patients' mobility and affects their independence in activities of daily living and work. Spasticity

may also cause pain, loss of range of motion, contractures, sleep disorders and impair ambulation

in patients with an incomplete lesion. The effectiveness of available drugs is still uncertain and

they may cause adverse effects. Assessing what works in this area is complicated by the lack of

valid and reliable measurement tools (Taricco et al. 2006).

The present treatment for spasticity is the use of anti-spastic drugs such as baclofen, which

is a GABAb agonist. But the appropriate dose and the time frame at which it should be started to

treat spasticity is not known. In humans, baclofen is given chronically i.e., 1 month after the

injury, as the FDA does not approve of its use acutely. Whether baclofen given acutely after SCI

can prevent the development of spasticity is not known. It is not known that acute administration

of baclofen administration is safe. In animals and humans following SCI, spasticity appears at a

chronic stage. One of the primary goals of my thesis was to obtain relevant information

regarding baclofen's safety and efficacy in the acute stage, before the onset of spasticity.

Rehabilitation in the form of treadmill training is widely used in human SCI cases to improve

their gait. Treadmill training in humans is laborious and requires a lot of manpower. Setups are

available in only a few centers across the nation, so the training is not accessible to all the

patients. The purpose of this study was also to test the hypothesis that a customized motorized

bicycle could be used instead of the treadmill to treat spasticity in the acute stage and to maintain

the feasibility and safety of the patient without any deterioration in his condition. This study

compared combination therapy of drug (baclofen) and locomotor training (bicycle) with

individual therapy, started acutely after midthoracic contusion SCI in animal model, and to test if









combination therapy, started acutely, is more useful in preventing the development of spasticity

and in improving gait.

The wide range of outcome measures of spasticity and gait and comparison of two

treatment modalities locomotorr training and drug therapy) proposed in this study might provide

translational data which ultimately influence the quality of SCI patients. These studies propose a

strategy to utilize quantitative measures of behavior, neurophysiology, IHC, and imaging (MRI)

techniques to further increase our understanding of neurobiology of spasticity and gait following

SCI and locomotor rehabilitation. Translation of these findings may provide safe, timely, and

effective intervention strategies and evidence-based resources for translatable therapeutic design,

which can ultimately benefit the SCI patient.









CHAPTER 2
BACKGROUND

This chapter presents the relevant background of SCI induced spasticity and gait problems

as well as present treatment modalities to deal with these clinical problems. The scientific society

studying SCI uses various types of injury models in animals to mimic the type of injury seen in

humans but the most commonly used method for SCI is mid-thoracic contusion SCI which has

documented to develop spasticity, even though there are various other types of injury models

which are presented below:

Recent studies of SCI models have indicated an increasing need for a more thorough

understanding of the multiple components of spinal cord segmental plasticity for which

therapeutic interventions can be rationally targeted by various interventions, including locomotor

training (Raineteau, & Schwab 2001). In that context, this thesis work was focused on spasticity

-a common aftermath of SCI that reflects maladaptive changes in spinal cord circuitry

affecting motoneuron excitability and output. Spasticity is often one of the most difficult

neurological consequences to manage. Therefore, studies that can lead to a more in-depth

appreciation of the underpinnings of spasticity and therapies that can attenuate its impact are of

great importance. Accordingly, this project addresses locomotor training rehabilitation with or

without drug (baclofen) therapy to test the effects) on spasticity using multidisciplinary

analytical approaches.

Spinal Cord Injury Animal Models

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

of recovery, and to develop therapeutic intervention. The experimental SCI animal models

include the transaction, isolation and contusion.









Spinal Cord Contusion Model (Incomplete Spinal Cord Injury)

Basically, human spinal cord injuries are caused by transient compression or contusion of

the spinal cord even though penetrating wounds of the spinal cord can result from a knife or

gunshot. So, we used animal spinal cord contusion models to study the pathophysiology of SCI

and its rehabilitation as it mimics natural human SCI. Most therapies that have gone to human

trial were first validated in spinal cord contusion models. Weight drop contusion model was

used by Wrathall (1985) and described morphological and behavioral changes in that model; she

also developed a combined behavioral score to assess motor, sensory and locomotor changes.

Patients with complete spinal cord lesions never improve up to the stage of being able to walk

without the assistance of the weight support system and consequently are not able to step on a

static floor (Van de Crommert et al. 1998). The inability of patients with complete transactions to

achieve unassisted walking, unlike the fully spinalized cat, suggests that the greater improvement

observed in subjects with incomplete lesions may not solely be attributable to spinal

mechanisms, since generation of stepping is probably more dependent on supraspinal and/or

proprioceptive inputs in humans than in cat (Edgerton et al. 2001; Van de Crommert et al. 1998).

Considering these clinical and functional data and knowing that the ratio of incomplete versus

complete spinal cord lesions is becoming increasing known in the population of paraplegics

(Tator et al. 1993), experimental studies using animals with incomplete spinal cord injuries

appear clinically and pathophysiologically relevant (Multon et al. 2003).

Spinal Transection Model (Complete Spinal Cord Injury)

In this model there is complete damage to both the descending and ascending fibers and

there is no connection between the caudal part of the spinal cord and the brain and disrupts all

the neurophil at the injury site. This type of injury is less common in humans as the human spinal

cord is surrounded by vertebras, tissues and muscles that provide protection to the spinal cord.









Spinal Isolation Model

In this model the lumbar region of the spinal cord is functionally isolated via complete

spinal cord transaction at two sites; this model eliminates supraspinal, infraspinal and peripheral

afferent input to motoneurons located in the isolated cord segments while leaving the

motoneuron skeletal muscle fiber connections intact. However this model is not used in

experimental set up, as cases of human isolation models are not seen frequently so the

observation in animal experiments cannot be implemented in human subjects as it can be done in

contusion animal models experiment.

Anatomical (Neuromodulatory) Changes Following SCI

Midthoracic spinal cord contusion injury is known to disrupt connectivity to and from the

lumbar spinal cord which hosts important segmental circuitry that:

1) initiates and sequences lower limb locomotor behavior (central pattern generator, CPG)

2) governs reflex excitability (Wrathall et al.1985, Thompson et al. 1992; 1998).

Impact injury of the thoracic spinal cord produces early increase followed by significant

decrease in the monoamines (dopamine, norepinephrine and serotonin), measured below the site

of injury. (Thompson et al. 1999).These neuromodulators play an important role individually and

interactively in regulation of sensory transmission and the excitability of intemeurons, fusimotor

and alpha motoneurons (Gladden et al. 1998; 2000). It is known that locomotor exercise

increases the expression of norepinephrine in the central nervous system (Dishman et al. 1997).

The unifying strategy of exercise based locomotor therapy relates to the hypothesis that

repetitive activity induced by training initiates neuronal activity in proper sequence that

optimizes the utilization of the diminished but residual central nervous system.











A Rodent Model of Post-SCI Spasticity

The availability of a reliable animal model of post-SCI spasticity is prerequisite to this

research. Ideally, the model should be amenable to applications of rigorous outcome measures

and test approaches that can have direct translational potential. Previous use of a rodent

midthoracic contusion injury paradigm has revealed significant neurophysiological, locomotor,

neuromuscular, and histological changes which have collectively demonstrated the feasibility of

reproducing significant features of human spasticity (Bose et al. 2002; Thompson et al. 1992,

1993, and 1998). Thompson-Bose lab developed a novel velocity-dependent ankle torque

assessment protocol to reveal bonafide features of spasticity in the rat (Bose et al. 2002;

Thompson et al. 1996, 2001a, 2002) and showed that changes in spasticity were time-locked to

neurophysiological events in relevant areas of the spinal cord. Further, a reflex test protocol (i.e.,

rate-depression) showed that a fundamental inhibitory process which controls sensory input to

hindlimb muscle stretch reflex pathways was significantly decreased following midthoracic

contusion injury (Thompson et al. 1992, 1993, 1998). These changes are progressive in onset,

severe in magnitude, permanent in duration, and are highly relevant to features observed

clinically in humans. Therefore, the methods and model developed in our lab and its employment

in this project collectively provide an important and clinically relevant opportunity to investigate

issues related to the neurobiology of spasticity and how experimental therapies may modulate

this hallmark feature of SCI in a very acute setting.

Mechanism of Spasticity

The onset of spasticity has been correlated in time and intensity to the progressive

development of several lasting changes in the excitability of monosynaptic reflexes that compose

the neural pathways from the affected muscle stretch receptors to those muscle motor neurons,









(Neilsen et al. 1993).Two premotoneuronal mechanisms have been postulated to account for

these changes:

1. multiplication of synaptic input by collateral sprouting of primary afferents (Krenz

and Weaver 1998).

2. decreased presynaptic inhibition (Calancie et al.1993).

Frank and Fuortes (1957) reported that stimulation of group I afferents from flexor muscles

was able to depress the Ia monosynaptic EPSPs, without changing the membrane potential, the

motoneuron input resistance, or the generation of antidromic action potentials. The depression of

the Ia EPSPs was due to presynaptic inhibition. Eccles and Krnjevic (1959) found that

stimulation of specific sensory nerves produced a long lasting depolarization of group I muscle

and cutaneous afferents and suggested that this primary afferent depolarization (PAD) was the

mechanism for presynaptic inhibition. Rudomin and colleagues localized a circuit of

interneurons that received afferent collaterals from primary afferent fibers and convergent inputs

from the terminals from descending fibers (Rudomin 1999). They showed the circuit for

presynaptic inhibition involved a minimum of two interneurons, the second or last order being

GABAergic that made axo-axonic synapses on GABAa receptors on primary afferent terminals.

The activation of these receptors induces an outward Cf current that produces long lasting PAD.

During the time course of the PAD (20-300msecs), the GABAa mediated conductance changes

along the primary afferent membrane decreases the effectiveness of afferent volleys to depolarize

the membrane and activate the voltage gated calcium channels that are essential for calcium

triggered release of neurotransmitters from the primary afferent terminals. The decrease in

transmitter release thereby decreased the amplitude of the excitatory postsynaptic potentials

produced during the PAD. The knowledge that descending systems modulated PAD, combined

with the demonstration that vibratory inhibition was reduced in spastic patients emphasized the









hypothesis that a major source of spasticity was due to the reduction of GABAa mediated

presynaptic inhibition following interruption of descending modulation of the segmental circuitry

that produced PAD. Neurotransmitters like GABAb, GAD67 also play an important role in the

development of spasticity which acts as inhibitory molecules preventing the development of

spasticity.

GABA

GABA is a spinal inhibitory interneuronal neurotransmitter. There is a high density of

GABAergic immunoreactive cell bodies and terminals predominantly within Lamina I through

III of the dorsal horn. Occasionally GABA cells were observed in layers IV, V, VI and X and in

the ventral horn. GABA functions in pre and postsynaptic inhibition in the spinal cord, via

axoaxonal and axosomatic or axodentritic synapses (Shapiro 1997). In vertebrates 2 major types

of GABA receptors (GABAa and GABAb) are found. GABAa contains a transmembrane ion

channel that conducts chloride ions and is gated by the binding of two agonist molecules.

Activation of GABAa opens the chloride channel, and the influx of the chloride ions inhibits the

neuron by causing hyperpolarization. GABAb is present at lower levels in the central nervous

system than is GABAa and is coupled to Ca2+ or K + channels via second messenger systems.

GABAb can be distinguished from GABAa by its affinity for the agonist baclofen and its lack of

affinity for muscinol and bicuculline. Activation of GABAb by baclofen decreases Ca

conductance and transmitter release and thus acts an inhibitory molecule in synaptic transmission

(Bowery et al. 1980, 1989; Shapiro 1997) and prevents the development of spasticity.

Since GABA is localized to interneurons, reactive synaptogenesis of interneuronal

GABAergic neurons can be seen in the spinal cord of spastic animals. Thus this molecule is very

important for the proposed research since we use baclofen as an antispastic drug and examine its

action on the presynaptic GABAb receptors.









Present Antispasticity Treatment for Spinal Cord Injury

Baclofen has been used widely as an anti-spasticity drug. Baclofen reduces muscle tone

and spasms with similar efficacy in patients with spasticity caused by complete or incomplete

SCI, or from cerebral origin (Davidoff 1985; Meythaler et al. 2001). A recent finding has proven

that the effect ofbaclofen is more potent when applied intrathecally than orally (Azouvi et al.

1996). GABAb receptors are distributed extensively in the spinal cord, especially presynaptically

on the primary sensory afferent terminals (Yang et al. 2001). Baclofen, as a potent GABAb

receptor agonist, has been shown to decrease synaptic transmission by binding to the presynaptic

GABAb receptors at the afferent terminal through a second-messenger pathway, ultimately

decreasing the calcium influx and neurotransmitter release (Batueva et al. 1999; Miller 1998). In

addition, baclofen's binding to the presynaptic GABAb receptors can also decrease the

neurotransmitter release by activating potassium channels (Gage 1992) causing hyperpolarization

of the post synaptic membrane, thus contributing to its presynaptic inhibitory effect (Li et al.

2004). In the paper by Li (2004) the author showed that in motor neurons of normal animals (or

humans) with intact spinal cord and brain stem, there are voltage-dependent persistent inward

currents (PICs) that, once activated, can remain active for many seconds after stimulation,

producing sustained depolarizations (plateau potentials) and firing (self-sustained firing), thus

greatly increasing their excitability (Gorassini et al. 2002; Lee and Heckman 1998a, b). A PIC is

a depolarizing current generated by voltage-sensitive channels; the voltage sensitive channels

stay open as long as the membrane potential remains above threshold for their activation

(Heckman et al. 2004). The PICs are composed of a low-threshold persistent calcium current,

carried by Cavl.3 L-type calcium channels, and a TTX-sensitive persistent sodium current (Lee

and Heckman 2001; Li and Bennett 2003). Large PICs are not present in motor neurons

immediately after spinal cord injury because of the massive loss of brain-stem-derived









monoamines that normally facilitate PICs (Hounsgaard et al. 1988). Exogenous application of

metabotropic receptor agonists (such as 5-HT) can enhance PICs and thus recover plateaus and

self-sustained firing after acute spinal transaction or in vitro slice injury (Lee and Heckman

1998a, b). Baclofen has been shown recently to decrease the amplitude of these enhanced PICs in

motor neurons of turtle spinal cord slices and to decrease spontaneously occurring PICs in deep

dorsal horn neurons of turtles and rats (Russo et al. 1998). Thus, raising the possibility that

baclofen's clinical action may be partly postsynapticby decreasing the PICs that play an

important role in the production of spasticity (Bennett et al. 2001a, b; Li et al. 2004a). It has

been found that GABAb decreased PIC only in acute SCI not in chronic SCI, it increased the

PIC, as chronic spinal rats have a moderately lower (30%) sensitivity to baclofen than do acute

spinal rats, which might be due to down regulation/desensitization of GABAb receptors or

decreased background GABA levels after chronic SCI (Li et al. 2004). This finding may justify

our hypothesis that baclofen started acutely after SCI can prevent the development of spasticity

by reducing the monosynaptic reflex mainly by decreasing presynaptic neurotransmitter release

and decreasing the PICs by reducing the Ca2+ PICs due to the up- regulation of the GABAb

receptors. Systemic baclofen depressed the monosynaptic excitation of Clarkes column neurons

by impulse in muscle and cutaneous afferent fibers (Shapiro 1997).

Baclofen, in addition to GABAb, can bind to a novel bicuculline-insensitive GABA

receptor site on primary afferents of the spinal cord and reduce the amount of transmitter

released (Bowery et al. 1980,1984). Baclofen is given in an intrathecal pump (ITB). Orally it

cannot easily cross the blood brain barrier and only 1% of the total dose reach the central

nervous system and so oral doses are generally twice or thrice the required amount which is not

feasible. ITB supplies the exact amount of drug to the appropriate area for the required amount

of time, thus enabling the effects of baclofen to be studied after immediate administration and to









see the development of tolerance after withdrawal. The major drawbrack of baclofen therapy in

patients today is that it is started much later after the injury as the Food and Drug Administration

does not approve baclofen for acute treatment. Spasticity is usually absent in acutely injured

animal preparations, and it only gradually develops with chronic injury (>1 month) (Bennett et al.

1999). Also it has been shown that Baclofen started even one week after injury helps in

improving spasticity (Wang et al. 2002). So the basic aim of this project is to acutely start

baclofen therapy along with bicycle training or alone to prevent the development of spasticity

and to see whether there is rebound spasticity after the baclofen treatment is stopped after a

certain period of time, in our case after 1 month.

Locomotor Rehabilitation

Rehabilitation in the form of treadmill or customised bicycle is widely used in human SCI

and in animal models. Customized bicycle developed as an alternative modality to treadmill, to

train SCI injured rats was used in our project to prove it is as effective as treadmill. But exactly

what time period after injury it should be started to prevent the development of spasticity and

improve the gait and whether training after injury may have other side effects on the patient's

body are not known. So we want to test the hypothesis that bicycle training acutely after injury

along with baclofen therapy prevents the development of spasticity. Exercise improves SCI

extensively due to its adeptness at enhancing sensory function which is mediated by molecular

systems dependent on neurotrophic actions. Voluntary wheel training and forced treadmill

exercise increased the expression of BDNF and other neurotropic molecules important in

synaptic function and neurite outgrowth in the spinal cord and innervated skeletal muscles

(Gomez-Pinilla et al. 2005). There is increasing evidence that the human spinal cord is capable

of a significant amount of plasticity and that this plasticity is, to a large extent, driven by

activity-dependent processes (Edgerton et al. 1997, 2001). This plasticity may occur at any of









many spinal cord regions or cell types such as motoneurons, premotor pattern-generating

neurons, and/or nonneuronal cell types. Studies have shown that locomotor training improves the

ability to perform full weight-bearing stepping on a treadmill in cats after a complete spinal cord

transaction (de Leon et al. 1998; Edgerton et al. 1997), which has supported the presence in the

lower spinal cord of a central pattern generator (CPG) able to generate rhythmic motor activities

in the absence of supraspinal descending inputs (Grillner et al. 1985). How the CPG can shape its

functional properties in response to the training is not clear, but it is thought that reinforcement

by sensory afferents of existing sensorimotor pathways, rather than generation of new

connections, might be responsible for the beneficial effects (de Leon et al. 1998, 1999, 2001).

There could also be anatomically altered synaptic connections, increased active zones of

synapses, altered sensitivities of neurotransmitter receptors, or altered production of

neurotransmitters. Sensory input provided during locomotor training are critical for driving the

plasticity that mediates locomotor recovery and pharmacological treatments can be used to excite

the spinal neurons that generate stepping (Edgerton 2001). So bicycle training along with

baclofen (GABAb agonist) can show a drastic improvement in locomotor pattern of SCI subjects

as the paddling movement of the bicycle gives a constant sensory input by activating the

receptors in the joints, muscles and tendons in the ankle, knee and hip joint of the hind limbs.

Locomotor training is beneficial in maintaining and even improving neural function

following insult or disease (Wernig et al. 1999). It is recognized that trophic factors such as

neurotransmitters are critical modifiers of the structure and function of neural networks such as

5-HT. Serotonin is a neurotransmitter of descending pathways from brain to spinal cord,

primarily in the ventral and lateral funiculi of the spinal cord influencing interneurons and motor

neurons via postsynaptic inhibition. The serotonergic cells of origin are in the raphe nuclei of the

brain stem and the reticular formation. The serotonergic cells in the nucleus raphe obscurus and









nucleus raphe pallidus project to the intermediolateral cell column and ventral horn of the spinal

cord. After complete or incomplete SCI, the descending 5-HT bulbospinal tracts in the lateral

and anterior funiculi undergo a slow wallerian degeneration because of the small fiber size and

lack of myelin, and this involves the axon terminals with the reduction of uptake, with the

development of spastic paraparesis. The motor scores have been significantly correlated with

changes in 5-HT staining in the ventral horn but not in the dorsal horn (Shapiro 1997). The 5-HT

system surrounds the corticospinal tract in the lateral funiculus, which explains the correlation

between losses of 5-HT and motor deficit after SCI. So an increase in the 5-HT in the lumbar

spinal cord after SCI with baclofen and locomotor training can correlate with the improvement in

the motor functions.

Physical activity even in an intact brain and spinal cord can induce the expression of

trophic factors in the hippocampus and other brain regions (Gomez-Pinilla et al. 1997; Gomez-

Pinilla et al. 1998),while exercise leads to the expression of trophic factors such as BDNF and

NT-3 specific neural networks (Gomez-Pinilla et al. 2002, Ying et al. 2003) which are important

in growth and neural function of the neurons. Exercise has been shown to induce BDNF in the

lumbar enlargements of the uninjured spinal cord (Gomez-Pinilla et al. 2001) and treadmill

walking increased labeling of BDNF, its receptor, trkB, and neurotrophin-4. Neurotropin

modulation induced by neuromuscular activity can play a role in facilitating functional recovery

following SCI .The injured spinal cord generally losses the ability to synchronize and interpret

the coordinated ensemble of afferent information that produces a predictable motor outcome in

an uninjured patient and so produces random motor pool activation. This deficit may be due to

the absence or rare occurrence of synchronized events normally associated with load-bearing

stepping. In the absence of these coordinating events, the spinal cord loses the ability to

synchronize input into functional movements of the limbs. A patient who is hypo responsive to









sensory input is less likely to respond to the proprioceptive input associated with load-bearing

stepping, so the presence of spasticity, which is exaggerated stretch reflex due to hyper

responsive sensory input, is a positive sign of the potential for a SCI patient to regain some

locomotor ability. Clearly, understanding the physiological mechanisms that underlie spasticity

will enhance our efforts to facilitate locomotor recovery in SCI patients. Activity-dependent

motor training facilitates the recovery of posture and locomotion after a complete SCI in

mammals. Because functionally recovered spinal animals i.e., SCI animals showed no evidence

of regeneration of descending pathways (Joynes et al. 1999) or showed minimal changes in hind

limb skeletal muscle properties (Roy et al. 1998, 1999) to account for the recovery

characteristics, the functional behavior exhibited by these animals must have been mediated by

the plasticity in existing spinal pathways. It is generally accepted in the literature that exogenous

application of BDNF and NT-3 on spinal motoneurons improves the regeneration of the fibers in

the spinal cord and thus can improve the outcome. Neurotrophins supplied by endogenous

sources may have an even greater effect on compromised cells and motor training have shown to

improve the levels of neurotropins in the injured spinal cord. The positive effects of motor

training have been documented in animals (Hodgson et al. 1994; de Leon et al. 1998) and

humans (Harkema et al.1997). The success of rehabilitative strategies is highly task specific,

which closely simulate the functional situation of walking are the most effective in promoting the

restoration of locomotion (Edgerton et al. 1997; de Leon et al. 1998.) Rehabilitative strategies

that stimulate walking, like treadmill or bicycling, are effective in improving locomotion in SCI

due to the phasic sensory input produced by repetitive foot contact with the ground or the foot

pad in the case of bicycle to result in the induction of activity dependent events such as increased

neurotropins levels in circuitry by repetitive loading of the hind limb (Gomez-Pinilla et al. 2005).









In summary, the above literature review shows that locomotor training whether it is

voluntary, wheel running or treadmill prevents the development of spasticity by modifying the

neurotransmitter levels and enforcing neuroplasticity in the surviving sensorimotor neurons so as

to develop a rhythmic reflex pattern that leads to improvement in the gait. Also baclofen therapy

started acutely can prevent the development of PICs which may be another cause of the late

onset spasticity and reinduce the presynaptic inhibition by acting on the GABAb receptors and

upregulating them.









CHAPTER 3
EXPERIMENTAL DESIGN AND THE STANDARD METHODOLOGY USED IN THE
PROJECT

Objective

The whole thesis work was conducted in a rodent animal model using established

techniques and protocols. Standard contusion SCI animal model was used in this project and the

injury device and the animals used are described below. The experimental set up for spasticity

measurement (Ankle torque and EMG), for locomotor gait assessment using footprints, and the

open field locomotor (BBB) scoring are all standard techniques. The bicycle training

methodology was developed in this lab and will be described in more detail. The following

segments will describe those techniques and protocols used in this project (Figure 3-1).

Animal Subject

Twenty four twelve week old female Sprague-Dawley rats (SPF) weighing 220-260 g

(Charles River Laboratories) at the start of this study were used in this project. Rats were housed

two per cage, in 12 hour light/dark cycle, and given food ad libitum. The total number represents

here is the actual experimental animals (24) not including the inadvertent losses: a) animals that

died during surgery (2 animals), b) those excluded based upon post-injury selection criteria, (9

animals) or c) animals that died unaccountably during the 2 month training and drug therapy

program (none). The total number of animals used was thirty five. An attending veterinarian

supervised care of these animals. All procedures were performed under the guidelines of Animal

Care and Use Committee.

































Figure 3-1 The experimental set up and procedures done in chart form

Contusion Injuries

Contusion injuries were produced using a standard New York University (NYU)

Multicenter Animal Spinal Cord Injury Studies (MASCIS) impactor (Basso et al. 1996). The

injury was performed under ketamine (100 mg/kg), xylazine (10 mg/kg, 1:3 with normal saline),

and glycopyrrolate (200 ul in each animal) anesthesia and previously reported in detail (Bose et

al. 2002, Thompson et al. 1992,1998). A laminectomy was performed at T8 segment, exposing

the underlying dura. The spinal column was stabilized with angled Allis clamps on the T7 and T9

spinous processes. An incomplete spinal contusion was made at the T8 segment of the spinal cord

using the impounder tip of the MASCIS 10-g weight impactor device (2.4 mm in diameter)

dropped from a height of 12.5 mm (computerized operation). The whole procedure was

performed under aseptic conditions. The animals were monitored routinely and were given


The experimental desig









postoperative care on a regular basis or as required until full bladder function was re-established

and no evidence of pain or other discomfort was detected.

Baclofen Pump Implantation

All rats were anesthetized with subcutaneous injection of xylazine (6.7 mg/kg) followed by

intraperitoneal ketamine (100 mg/kg)/glycopyrrolate (40 mg) (Reier et al. 1992; Thompson et al.

1992, 1993). Prior to implant, osmotic pumps (model 2004, 0.25 mL/h; Azla Corp., Palo Alto,

CA) were filled with baclofen (3.2 [g/igL; implanted in twelve animals) or 0.9% physiological

saline (implanted in twelve animals). Osmotic pumps were incubated overnight in 0.9% saline at

370C in sterile conditions for immediate delivery of drug or vehicle. The dose rate was 0.8 [g/hr.

Particular efforts were taken to avoid introducing air space within the infusion catheter. A 4cm

vertical skin incision was made spanning the thoraco-lumbar juncture. A subcutaneous pocket

was dissected to accommodate the osmotic pump. Musculature covering spinal processes was

retracted from T12-L1, and a T13-L1 laminectomy was performed. The osmotic pump was

maneuvered into the subcutaneous pocket and secured to surrounding musculature using 6.0 silk

sutures. The infusion catheter was tunneled through the rostral bank of dissected musculature,

and secured by polyacrylamide adhesion (VetbondTM tissue adhesive; 3M, St. Paul, MN) to

exposed spinal process. The wound margins of muscle and skin were infiltrated with a long-

lasting local anesthetic (lidocaine). With the aid of a dissecting microscope, the duramater was

cut and the silastic tubing inserted into the subarachnoid space of the lumbar enlargement.

Excess tubing was secured to surrounding musculature with 6.0 silk sutures. Muscle layers were

closed with absorbable sutures (Dexon II; Sherwood, Davis & Geck, Wayne, NJ) and the skin

closed using stainless steel wound clips (Autoclip; Becton Dickenson, Sparks, MD).

Postoperative care included a 4mL subcutaneous injection of warmed (370C) 0.9% physiological

saline and overnight assistance with body temperature regulation using a temperature controlled









heating pad. The day after surgery behavioral and neurophysiological assessment were started

(Figure 3-1). After 4 weeks of ITB treatment, the pump was removed by similar procedures, and

the infusion catheter was ligated with 6.0 silk sutures and left in situ.

Bicycle Training


















1



Figure 3-2 Motorized bicycle used for training the rats.

Careful attention was taken during training, as it was started acutely (PO day 1) after the

injury. The animals were trained over the course of 2 months. The training schedule was

performed 5 days a week using two 20 minute trials/day, starting from PO day 1. On the first day

of training, the rats were given five minutes of training. The bicycle exercise regimen (Figure 3-

2) involved immobilizing the rats in a custom made harness with the hind limbs suspended. The

hind feet were strapped onto the pedals using cotton tapes. The exercise consisted of a pedaling

motion, which fixed one limb while extending the other without overstretching the limbs. The

cycling speed was 31 rotations/minutes (around 11 meters/min, distance wise). During the first

week of training, the rat tail was attached to the aluminum support boom by surgical tape to









maintain the trunk stability during exercise. However, following second week of training,

gradually the load on the pedals was increased by positioning the body harness towards the chest,

so that the hind portion of the body was more extended over the pedal. The basic mechanism and

principle of bicycle training is that of locomotor activity-initiated sensory input derived from

weight bearing, as well as from the receptors of the skin, bones and muscles of the ankle, knee

and hip joints of the hind limbs (Figure 3-2).

Footprints

To document hind limb gait abnormalities, footprints were acquired while rats walked at

11 m/min, along a 20 x 40 cm surface of a treadmill (Columbus Instruments, OH, USA). Prior to

each trial run, hind limb footpads were coated with nontoxic blotter ink and the treadmill lined

with recording paper. Footprints were acquired from each animal with six consecutive steps

considered optimal for data analysis. Hind limb axis was measured using the angle of

intersection between left and right hind foot axis during coordinated stepping. Foot axis was

determined by a line passing through the third distal phalanges, metatarsophalangeal joints, and

between the cuboid bone and medial cuneiform bone. The base of support was determined by

measuring the horizontal distance between the central footpads of the hind feet. Prior to footprint

recordings, the animals walked for a few minutes to accommodate to and become familiar with

the treadmill walkway. Prior to the surgery preoperative footprints were taken to compare the

changes in the walking pattern after injury and after training (Figure 3-2).

BBB Score

An open field-testing procedure (Basso, Beattie, and Bresnahan (BBB) 21-point scale)

(Basso et al. 1995) was applied after the contusion injury at different time points to assess the

locomotor deficit and its improvement following exercise and or baclofen therapy. These

observations were made at POD 1, wks 4 and 8 in a blind fashion (Figure 3-2). The rats were










placed in a clean molded-plastic circular enclosure to walk freely to perform this procedure. It

rates behavior from individual joint movements of the hind limb, to plantar stepping, to

coordinate walking and finally the more subtle behaviors of locomotion, such as paw position,

trunk stability and tail position. Observations are recorded for 4 min in an open field and then

converted to a numerical score from the scale.

BBB Score Inclusion Criteria: to decrease injury variability of animals with in the study,

the animals were scored using the BBB, at PO day 1 after injury. Those animals that scored > 7

were considered too mildly injured and were excluded from the study.

Statistical analyses of the data (velocity-dependent ankle torque, footprints and open field

locomotor recovery) included between groups ANOVA and post hoc tests to achieve group

differences.

Ankle Torque


Analysis of Triceps Surae
Lengthening Resistance
Displace.
| ^Tension
Displacement -Force- EVIG
EMG .- EMG /
Triceps Surae 12deg
LVDT

Force F JJJcJJJJ\ F ce 259
S I Trans. Model
Ankle .FT V '40 408 RMS I150V
Rotation Displ. EG
Displacement Trans. 1\ \ \ \ I 50msec
12 Degrees Ling





Figure 3-3 Ankle torque, displacement and EMGs are simultaneously recorded and time-
locked to onset of dorsiflexion.

Pre-operative as well as postoperative recordings utilizing the ankle torques protocol were

taken. Details regarding instrumentation, animal set-up and recording procedures have been









previously reported (Bose et al. 2002; Thompson et al. 1996) but will be described below briefly.

Rats were immobilized in a custom designed trunk restraint, without trauma or apparent

agitation. All recordings were performed in awake animals. The proximal portion of the hind

limbs to the mid-shank, were secured in a foam fitted cast that immobilized the limb while

permitting normal range of ankle rotation (60 to 160 degrees). The lengthening resistance of the

triceps surae muscles was measured indirectly by quantitating ankle torque during 12-degree

dorsiflexion rotations of the ankle from 95 through 83 degrees. Contact with the foot was

achieved using a foam-fitted cradle aligned with the dorsal edge of the central footpad 2.6 cm

distal to the ankle joint. The angle of contact between the displacement shaft and the moment

arm was 95 degrees (Figure 3-3). The neural activity of the triceps surae muscle was measured

using transcutaneous EMG electrodes. The electrode was inserted in a skin-fold over the distal

soleus muscle just proximal to the aponeurotic convergence of the medial and lateral

gastrocnemii into the tendonocalcaneousus. A reference electrode was placed in a skin fold over

the greater trochanter. A xylocaine 2% jelly (Lidocaine HC1, Astra USA Inc.) was applied over

the electrode insertion points to minimize pain during recording. A topical antibiotic ointment (a

combination of Bacitracin, Neomycin and Polymyxin B; Fougera Altana Inc., Atlanta, GA) was

also applied on these areas after taking off the electrodes at the end of each trail to reduce any

chance of infection. Controlled dorsiflexion was achieved through the use of an

electromechanical shaker (model 405, Ling Dynamic systems, Royston Herts, U.K.). A force

transducer (LVDT) (model FT-03; Grass Instruments, Quincy, MA) was placed in series

between the output shaft of the ling shaker and the central footpad (figure 3.3). Root mean square

(RMS, i.e., a 0.707-DC equivalent of the full wave rectified AC signals) of EMG bursts was also

recorded on an additional channel of the signal acquisition system. EMG magnitudes are









reported as mean RMS magnitude of the EMG bursts time-locked to ankle dorsiflexion.

Collectively, this arrangement allows for simultaneous monitoring of triceps surae EMG,

resistive force, and velocity of shaft displacement. Recorded data were processed using Data

Wave Technologies signal acquisition system (model 32C; Data Wave Technologies, Denver,

CO) with an analogue to digital sampling rate of 200 KHz. Dorsiflexions of 12 degrees were

performed with 3-sec intervals at 49, 136, 204, 272, 350, 408, 490, and 612 deg/sec. At each test

velocity, five consecutive sets of waveforms, 5 waveforms per set, that is, 25 in total, were

recorded, signal averaged, and saved for subsequent analysis. An average of two time points

recording was used for data analysis. Regression of ankle torque against the ankle joint rotation

yielded resistance (torque) per degree of rotation for each velocity variable of the protocol with

torque values expressed in Kdynes (1000 dynes 5 1 Kdyne). The protocols were performed using

the fastest rotation first, then the slowest rotations

Immunocytochemistry of Spinal Cord Tissue

The standard Fluorescent and Avidine-Biotine Complex (ABC) immunohistochemical

techniques were utilized for visualizing neurotransmitters such as GABAb, 5-HT, GAD67, BDNF,

GAP43. These techniques allow the visualization of varicosities as well as fibers that are

interpreted as axons using 40rm thick cryostat sections of the lumbar and thoracic spinal cord.

Qualitative evaluation was done using a light microscope and bight field microscope.

In brief, spinal cord segments (thoracic segments caudal to the injuries, and lumbar spinal

cord, L3-L6) were dissected and removed after perfusion (4% paraformaldehyde in PBS) and kept

in the same fresh fixative mixture for 1 hour and was cryoprotected for at least 2 days in 30%

sucrose in 0.1 mol PB. The specimens were cut serially (cross section) by cryostat (40 [tm









thickness) and processed by Avidine-Biotine Complex (ABC) and Florescent

immunohistochemistry (IHC).

The immunoreactivity of GAD67, GABAb GAP43 and BDNF were identified in lumbar and

thoracic spinal cord. The cryostat cut sections were incubated with primary antibodies generated

against GAD67 (mouse mAb; 1:1,000, National Hybridoma Laboratory, St Luis, USA), GABAb

(guinea pig mAb, 1:4,000; Chemicon International), GAP43 (mouse mAb, 1:5000; Chemicon

International), and BDNF (rabbit Ab, Chemicon International) for 24-48 h at 40 C. The sections

were then washed in PBS and incubated for 1.5 h in alexa fluor-congugated appropriate anti-

mouse, anti-guinea pig or anti-rabbit IgG (1:1000, Molecular Probes). For ABC technique, anti-

guinea pig (1:200; Chemicon), anti-mouse, and anti-rabbit (1:200; mouse and rabbit Elite kits,

Vector Lab) secondary antibodies were used to bind with appropriate primary antibodies.

Sections were then washed again and mounted for microscopic analyses.











CHAPTER 4
RESULT


The following sections present results of velocity-dependent lengthening resistance due to


dorsiflexion of the ankle, open-field locomotion ability and gait (footprints) analyses.


Velocity-Dependent Ankle Torque and Associated EMG's


Pre-injury Ankle Torque and EMG Data


Baseline measures of velocity-dependent ankle torques and extensor EMG's were obtained


from all animals before injury at post-injury weeks 1, 4 and 8.


PREINJURY -ANKLE TORQUE

120
z 100
E80 I, SALINE CONTROL
Z SALINE CYCLE
W 60
[O BAC CONTROL
S40
:3 4o BAC CYCLE
a-
g 20
0
612 490 408 350 272 204 136 49
VELOCITY IN DEGS ISEC




Figure 4-1 Pre-injury ankle torque graph.


EMG- preinjury

1.400000

1.200000

1.000000

0.800000
0.600000

0.400000- saline control
0.200000- m saline cycle
O bac control
0.000000 o bac cycle
612 490 408 350 272 204 136 49



Figure 4-2 Pre-injury EMG graph.











Postoperative Week 1 Ankle Torque and EMG Data


ANKLE TORQUE-WI


0 150

z
w 100


i50

*<


612 490 408 350 272 204


VELOCITY IN DEGSISEC


EEK 1 *, p_0 05, compared to preinjury
$, p-0 05, compared to sal cycle
@, p-O 05, compared to bac cont
#, p-0 05, compared to bac cycle










SALINE CONTROL
SALINE CYCLE
136 49 o BAC CONTROL
o BAC CYCLE
PREINJURY


Figure 4-3 Ankle torque graph at post-op week


1 with significant differences.


EMG- weekly


*,p 05, compared to preinjury
#, p-O 05, compared to bac cycle


@, p-O 05, compared to bac cont








saline control
-# saline cycle
"" O bac control
[ lO bac cycle
preinjury recording
612 490 408 350 272 204 136 49


Velocity in degs /sec


Figure 4-4 Electromyeloencephalogram graph at post-op week 1 with significant differences.



When tested at one week following injury, baclofen cycle group revealed significantly


decreased magnitudes of ankle torque during rotation at each of the eight ankle rotation









velocities, even less than the control values recorded before injury (Figure 4-3). These were

about 11% lower at the highest velocity (612 deg/sec) and 0.8% lower at the lowest velocity (49

deg/sec). Similarly the mean values observed for the baclofen control group did not show

significant changes compared to the pre-injury group, only 7% increase at the highest test

velocity (612 deg/sec), and 28% increase at the lowest test velocity (49 deg/sec) (Table 4-1).

Mean values observed for the saline control group revealed significantly increased

magnitudes of ankle torque during rotation at each of the eight ankle rotation velocities when

compared to corresponding pre-injury values (Figure 4-3). Approximately a 52% increase was

observed at the highest velocity (612 deg/sec), and a 38% increase at the lowest test velocity (49

deg/sec) (Table 4-1). These values were significantly different when compared to comparable

measures in both baclofen cycle and baclofen control groups (Figure 4-3).

The saline cycle group also showed a significant increase in magnitude of ankle torque at

each of the eight ankle rotation velocities tested when compared to pre-injury values (Figure 4-

3), approximately 39% increase at the highest test velocity (612 deg/sec) and 37% increase at

the lowest test velocity (49 deg/sec) (Table 4-1).

There were significant differences in the velocity-dependent ankle torques between saline

control and saline cycle groups when compared to corresponding values obtained from baclofen

control, baclofen cycle and pre-injury groups; but no significant differences were observed

between baclofen control, baclofen cycle or pre-injury groups (Figure 4-3).

The EMG-RMS magnitudes recorded at each of the test velocities closely paralleled the

velocity dependent ankle torque measurements (Figure 4-4). Significant parallel increases in

ankle torque and EMG magnitude, respectively, were observed during ankle rotations at the










slowest four velocities at post-op week 1 in control and saline cycle group when compared to


baclofen cycle group


Table 4-1 Ankle torque week 1- percentage (%) change (T1) compared to pre-injury.

Wkl Ankle Torque Data% %1
Groups
612 490 408 350 272 204 136 49
Saline Control 52%" 43%" 43%" 41%" 41%T 39%0 35%0 38%"
Baclofen Cycle 11%i 7%[ 7%0 8%[ 7%1 8%[ 6%I 0.8%1
Saline Cycle 39%t 31%T 25%t 29%t 20%t 14%t 19%t 37%t

Baclofen Control 7%" 10%T 12%" 24%" 18%" 17%" 14%f 28%"


Table 4-2 Electromyeloencephalogram week 1 percentage (%) change (1 ) compared to pre-
injury.


Wkl EMG Data % i
Groups
612 490 408 350 272 204 136 49
Saline Control 62%" 89%" 101%" 108%" 168%" 289%" 289%" 695%"
Baclofen Cycle 42%i 52%1 50%[ 45%i 22%o 39%1 67%[ 8%1
Saline Cycle 35%0 40%T 20%t 49%0 73%0 143%" 29%o 109%"
Baclofen Control 38%i 5%t 41%[ 34%[ 6%[ 11% 50%T 103%t


Postoperative Week 4 Ankle Torque and EMG Data


ANKLE TORQUE-WEEK 4 psO 05, compared to preinjury
$, p0O 05, compared to sal cycle
@, p<0 05, compared to bac cont


2 5 0 .
200 -
150 .
100 --
50
0
612 490 408 350 272 204 136 49
VELOCITY IN DEGSISEC


* SALINE CONTROL
* SALINE CYCLE
o BAC CONTROL
O BAC CYCLE
m PREINJURY


Figure 4-5 Ankle torque graph at post-op week 4 with significant differences.


05, compared to b e













*, p:5O 05, compared to preinjury
EMG-WEEK 4 #, p0 05, compared to bac cycle
@, p-O 05, compared to bac cont
$, p-O 05, compared to sal cycle
2.5

2
2 m saline control
1.5 saline cycle
E $ O bac control
S1 T o bac cycle
Spreinjury recording
o 0.5

0
612 490 408 350 272 204 136 49
velocity in degslsec


Figure 4-6 Electromyeloencephalogram graph at post-op week 4 with significant differences.

At post-injury week 4, a significant decrease in the ankle torques was observed at all test

velocities in the saline cycle contused group compared to these values observed at weekly (Figure

4-5) for example, at the highest velocity (612 deg/sec), at week 1 the ankle torques were 39%

greater than control, but only 4% greater at week 4; a 35% decrease. And at the lowest test

velocity (49 deg/sec), the week-4 saline cycle animals revealed ankle-torques that were 5%

greater than controls, compared with 14 % at week 1 for these measures. On the other hand,

baclofen control and baclofen cycle showed the same pattern as week 1. The saline control group

ankle torques remained significantly elevated, and were similar to weekly data, a 61% increase at

highest test velocity (612 deg/sec) and a 9% increase at the lowest velocity (49 deg/sec) (Table

4-3). The ankle torque magnitude showed the same pattern in the lower velocities as that of

higher velocities.

The EMG magnitudes mirrored the pattern of the ankle torques in all the four groups at all

the velocities there was no significant difference in the EMG magnitude at all the lower 4

velocities when compared to higher velocities in post operative week 4 (Figure 4-6) (Table 4-4).









Table 4-3 Ankle torque week 4- percentage (%) change (T1 ) compared to pre-injury.

Wk 4 Ankle Torque Data % i
Groups
612 490 408 350 272 204 136 49
Saline Control 61%T 52%T 47%T 43%T 27%T 22%T 18%" 9%0
Baclofen Cycle 5%t 5%t 10%t 2%t 7%T 9%t 2%t 0.05%t
Saline Cycle 4%T 0.2%T 3%t 3%t 1%t 5%0 6%o 5%t
Baclofen Control 18%t 13%t 9%t 6%t 11%t 12%t 13%" 8%t
Table 4-4 Electromyeloencephalogram week 4- percentage (%) change (1 ) compared to pre-
injury.
Wk 4 EMG Data % 1
Groups
612 490 408 350 272 204 136 49
Saline Control 60%T 117%T 127%T 138%T 122%T 152%T 248%T 296%T
Baclofen Cycle 27%i 27%[ 8%t 15%t 49%t 20%t 14%[ 33%[
Saline Cycle 39%0 33%0 41%0 28%0 23%0 3%t 16%0 43%t
Baclofen Control 27%1 7%0 33%1 17%1 31%t 13%t 21%t 128%0

Postoperative Week 8 Ankle Torque Data and EMG Data



ANKLE TORQUE-WEEK8 *, pO 05, compared to preinjury
$, pSO 05, compared to sal cycle
#, pSO 05, compared to bac cycle


250 -





0#

0lll~iii


136 49


* SALINE CONTROL
* SALINE CYCLE
o BAC CONTROL
O BAC CYCLE
* PREINJURY


Figure 4-7 Ankle torque graph at post-op week 8 with significant differences.


612 490 408 350 272 204
VELOCITY IN DEGSISEC














*, pSO 05, compared to preinjury
EMG week 8 $, pSO 05, compared to sal cycle
+, pSO 05, compared to sal control
#, pSO 05, compared to bac cycle
2.5

2- T#

1.5


# # saline control
0.5 # $ saline cycle
05 bac control
0 2 #bac cycle
612 490 408 350 272 204 136 49 N preinjury data

Velocity in degs/sec

Figure 4-8 Electromyeloencephalogram graph at post-op week 8 with significant differences.

By post-op week 8, significant velocity-dependent ankle extensor spasticity re-appeared

in the baclofen untrained contused group, a 75% increase at the highest velocity (612 deg/sec)

compared to the corresponding value of pre-injury control group (Table 4-5). While only an 8%

increase was observed at the lowest test velocity (49 deg/sec) (Figure 4-8). The velocity

dependent ankle torques in saline control group appeared similar to those observed at post-op

week 1 and 4, suggesting that this significant velocity dependent increase in ankle torque was

enduring. Surprisingly, at this post-injury time point, this re-emergent spasticity was not

observed in baclofen cycle group; only a 10% increase in ankle torque was observed at the

highest test velocity (612 deg/sec) and only a 1% increase was observed at the lowest velocity

(49 deg/sec), even after removal of the baclofen pump. The saline cycle group showed a 41%

increase in magnitude in ankle torques at the highest velocity (612 deg/sec) and a 9% increase at

the lowest velocity (49 deg/sec), relative to these measures in the pre-injury control group. At

this point, EMG magnitudes were also observed to be increased significantly at the two fastest









ankle rotation velocities (490 and 612 deg/sec) in both saline and baclofen control and saline

cycle group as compared with the baclofen trained contused animals. Moreover, the EMG

magnitudes were observed to be decreased for the saline cycle group at the next 3 test velocities

(408-272 degs/sec) (Figure 4-8). No significant increases in ankle torque or EMG magnitude

were observed during ankle rotations at the slowest four velocities at post contusion week 4 and

post-op week 8. The EMG pattern for baclofen cycle remained similar to the ankle torque data

for the same week i.e., not much increase in magnitude as compared to pre-injury data (Table 4-

6).

Table 4-5 Ankle torque week 8 Percentage (%) change (1 ) compared to pre-injury.
Wk 8 Ankle Torque % Data 1
Groups
612 490 408 350 272 204 136 49
Baclofen Control 77%" 60%T 60%T 55%" 33%" 12%[ 14%" 4%"
Baclofen Cycle 10%0 15%" 11%o 2%T 5%t 6%T 7%0 1%l
Saline Cycle 41%T 24%t 20%T 22%t 19%t 5%[ 5%[ 9%t

Baclofen Control 75%0 63%t 57%t 61%T 34%t 12%t 8%[ 8%t

Table 4-6 Electromyeloencephalogram week 8 Percentage (%) change (1 ) compared to pre-
injury.

Wk 8 EMGData% ti
Groups
612 490 408 350 272 204 136 49
Saline Control 46%t 65%t 42%t 118%t 243%t 196%t 12%t 51%[
Baclofen Cycle 29%i 20%[ 24%o 33%0 53%t 1%[ 19%t 43%[
Saline Cycle 58%t 74%t 36%t 41%T 15%t 66%t 17%t 74%t

Baclofen Control 46%t 128%t 52%t 84%t 228%t 142%t 216%t 235%t










Gait and Open Field Locomotion


Footprint Analysis: Gait


Axial rotation


#, pSO.01, compared to bac cycle week 4
, pSO.01, compared to saline control preinjury
@, pSO.01, compared to bac control preinjury
$, psO.01, compared to sal cycle preinjury
*, pSO.01, compared to bac cycle preinjury


0 -


Pre-injury Post injury week 4


@

A $


Post injury week 8


m saline control
m saline cycle
o bac control
O bac cycle


Figure 4-9 Axial rotation graph of all 4 groups at different time points with significant
differences.


Base of support


#, pO0.01, compared to bac cycle week 4
+, pO0.01, compared to sal control week 8
", pO0.01, compared to saline control preinjury
@, pO.01, compared to bac control preinjury
$, pO.01, compared to sal cycle preinjury
*, pO0.01, compared to bac cycle preinjury

s ,


Post injuryweek 4


Post injuryweek 8


* saline control
* saline cycle
o bac control
O bac cycle


Figure 4-10 Base of support graph of all 4 groups at different time points with significant
differences.


LT ~rI


3

2

1

0


Pre-injury










Footprint analyses were performed pre-injury, post-op week 4 and week 8. .Before injury,

limb axis and base of support were measured to be 28.33 2.00 degs and 2.91 0.37 cms,

respectively. At post-op week 1, in saline control group the values for limb axis and base of

support were 44.45 degs and 3.79 cms respectively, whereas in baclofen controls group the

readings were 46.08 degs and 4.49 cms respectively. The baclofen training group showed the

least value 34.44 degs and 3.21 cms, whereas the saline cycle group showed 45.86 degs and 4.13

cms respectively. Thus after 1 month post-op saline control and baclofen cycle showed the least

change when compared to the pre-injury values (Figure 4-9 and 4-10). Compared with the pre-

injury control values, these measures revealed that limb axis and base of support were

significantly increased in the baclofen untrained contusion-injured animals and saline cycle

group during week 4. At post-op week 8, the trend was reversed with baclofen cycle showing

increase in the base of support and angle of axial rotation as compared to all the other 3 control

groups at same time point and post-op week 4 and also compared to pre-injury data. At post-op

week 8 ironically saline control showed the most improvement in all the 4 groups even without

treatment (Figure 4-9 and 4-10).

Open Field Locomotor Recovery

Open field locomotor recovery y=6.6428x+49.976
70R2=1
M6990
0 61 48 61 33
I54 6038
E 60 1 5 6 Saline control
0 __ 46 4
: 50 Saline cycle
$ 40 Baclofen control
y = 7.4524x + 38.976
30 R2 = 1 Baclofen cycle
S20 Linear (Saline control)
S10 -- Linear (Saline cycle)
S o Linear (Baclofen
IL- control)
PO week 4 PO week Linear (Baclofen cycle

Figure 4-11 Graph of BBB showing percentage change in score at post-op week compared to
post-op week 4.







*, pO.05, compared to bac contl po
OPEN FIELD LOCOMOTOR CHART+, p5o.05, compared to sal control
OPEN FIELD LOCOMOTOR CHART psomrdt sal v
$. p-O.05, compared to sal cycle
p 0.05, compared to sal control #, p50.05, compared to bac cycle
(., ps0.05, compared to bac control
25 &, ps0.05, compared to sal cycle
s0 0.5 co'mir, r.j i.: bac control poweE k8

20 $

015

m10



I5 saline control
0 I saline cycel
Pre PO Day PO PO n bac control
1 week week bac cycle


Figure 4-12 Open field locomotor graph showing BBB score of all 4 groups at different time
points with significant differences.

Open field locomotor capacity (BBB) was scaled in both trained and untrained animals

before injury, at post-op day 1, at post-op week 4, and at post-op week 8 to evaluate recovery

during the early, intermediate, and late phases of recovery. Before the start of the treatment

(bicycle and drug alone or combination), at postoperative day 1 the saline cycle group animals

had the highest score as compared to the 3 other groups.

At post-op week 4, saline cycle trained contused animals displayed extensive movement of

all three joints of the hind limb, (mean score, 17 2) i.e., about 61% recovery compared to post-

op day 1, but as a group, these data revealed significant variability in the scores among the

groups (Figure 4-11). Baclofen cycle trained animals also showed good improvement in their

score (mean, 13 2) with a total recovery approximately a 56% increase from post-op day 1

(Figure 4-11). In contrast, baclofen control group (mean score 12 1) showed about 46%

recovery while saline control animals (mean score 11 + 1) showed about 51% recovery when

compared to post-op day 1 scores respectively (Figure 4-11 and 4-15). Bicycle trained animals









displayed a frequent to consistent weight supported plantar stepping and occasional to frequent

FL-HL coordination (mean score, 15 2). This BBB score in the bicycle trained group was

significantly greater (p<0.05, ANOVA) than values observed in either of the untrained groups

(Figure 4-11).

At post-op week 8, baclofen cycle animals revealed scores that were significantly

increased (mean score, 161) when compared to values of the post-op week 4 and post-op day 1.

This mean represented a recovery of approximately 70% when compared to post-op week 1 and

14% improvement compared to post-op week 4. Whereas, saline cycle group did not show any

increase in its BBB score, instead revealed a drop (mean score, 16 1) when compared to values

of post-op week 4 score. In fact, its condition deteriorated by about 1.5% compared to post-op

week 4 (Figure 4-11). Saline untrained animals showed increase in their score (mean score, 13 +

2), about 15% improvement as compared to post-op week 4. Baclofen control group showed an

increase in its score (mean score, 15 0.2) i.e., about 15% improvement as compared to post-op

week 4. Both of the BBB scores in the locomotor trained groups were significantly greater

(p<0.05, ANOVA) than the scores recorded for the untrained groups. However, at this stage,

animals of both trained and untrained groups showed consistent FL-HL coordination and

consistent weight supported stepping (mean scores, bicycle, 14.25 1.4, control, 15.25 1.7).

Please note, the terminologies never (0%), occasional (less than or equal to half, <=50%),

frequent (more than half but not always, 51-94%), and consistent (nearly always or always, 95-

100%) used above (Basso et al. 1995)

The BBB score for an uninjured rat is 21 points.

In summary, open field locomotor recovery scores scaled at post-op weeks 4 and 8 were

significantly higher in both of the training groups compared with untrained controls. The









baclofen cycle group demonstrated the highest recovery at post-op 2 month, which was also

significantly higher than the untrained group. However, at post-op week 8, both baclofen and

saline training groups showed similar recovery (ANOVA), whereas in post-op week 4, the saline

cycle group showed more recovery than the baclofen cycle. The differences in the recovery may,

in part, be reflected by the relative starting point; the saline cycle group started at a very high

score compared to baclofen cycle group at post-op day 1.

Neurotransmitters

Immunocytochemistry results are shown in Figures 4-13 4-17.

GABAb


Figure 4-13 Immunohistochemistry of GABAb receptors in lumbar spinal cord tissues.

In this Figure there is an increased expression of GABAb receptors in the baclofen cycle as

compared to the other three groups, with saline cycle group showing more expression then

baclofen control and saline control showing the least expression


Baclofen cycle Baclofen control










Saline cycle GABA b Saline control









5-HT (Serotonin)


Figure 4-14 Immunohistochemistry of 5-HT (Serotonin) fibers in thoracic spinal cord tissues.

In this Figure there is an increased expression of 5-HT fibers in the baclofen cycle as

compared to the other three groups, with baclofen control group expressing more fibers then

saline cycle and saline control showing the least expression.

BDNF













Figure 4-15 Immunohistochemistry of BDNF (Brain derived neurotropic factor) in lumbar spinal
cord tissues.

In this Figure there is an increased expression of BDNF neuromolecules in Cycle Group's

both baclofen and saline as compared to the baclofen control and saline control, which may

point to the fact that exercise tends to increase the expression of BDNF.










GAP43


Figure 4-16 Immunohistochemistry of GAP43 fibers in central canal of thoracic spinal cord
tissues.

GAP43 a neurotropic factor having functions similar to BDNF showed an increased

expression in baclofen cycle and somewhat more in saline cycle then compared to baclofen

control and saline control.

GAD67
















Figure 4-17 Avidine-Biotine Complex (ABC) figures of GAD67 fibers in thoracic spinal cord
tissues.

In this Figure GAD67 showed an increased expression in baclofen cycle as compared to

saline cycle and also increased expression in baclofen control, while saline control showed

minimal expression.


Bwlofen cycle Baclofen control






Saline cycle Gap43 Saline control









Result Summary

The above results support the conclusion that baclofen cycle showed the greatest

improvement with the lowest torque amplitude in all the velocities tested for ankle torque and

EMG. The BBB score showed greatest percentage improvement in post-op week 8 compared to

post-op day 1. Immunocytochemistry images showed an increased expression of the presynaptic

inhibitor molecule GABAb in baclofen cycle group as compared to all the other groups. Also

there was an increased expression of neurotrophic factors such as BDNF, GAP43, 5-HT and

GAD67, which is an indication of improvement in locomotion and increased growth of axonal

fibers. Baclofen alone and locomotor treatment groups did show some improvement until post-

op week 4 but later were not able to maintain the same pattern in post-op week 8. The saline

control group did not show any improvement in spasticity at all time points but an improvement

in BBB score which indicates that they have an intrinsic ability to initiate stepping without any

treatment.









CHAPTER 5
DISCUSSION

The purpose of these studies was to test the hypothesis that acute treatments using baclofen

and locomotor training, individually, and in combination, would significantly decrease the

development of hyper reflexive spasticity following SCI by comparing velocity dependent ankle

torque and time locked triceps surae EMG. Several behavioral tests (e.g. footprint analysis, and

open field locomotor assessment) were also performed to test the influence of these treatments

on aspects of locomotor functional outcome. In addition, to gain a better understanding of the

fundamental neurobiology associated with SCI and the treatments, the expression of certain

neurotransmitters that play an important role in spasticity and locomotion was compared in the

treated and non-treated groups.

Compared with non-treated animals, individual treatment using locomotor training or ITB

significantly reduced the development of early onset spasticity. However, ITB treatment during

the first month post-injury did not decrease the development of late onset spasticity, while cycle

training reduced the late onset spasticity by approximately 50%. The combination therapy (ITB

and cycle training) profoundly reduced the development of both early and late onset spasticity.

Ankle Extensor Stretch Reflex

Saline Control

In saline control group there was hyper reflexive pattern spasticityy) seen at all the

velocities tested and at all time periods (i.e., at post-op week 1, 4 and 8 with the EMG signals

showing the same pattern as that of the ankle torque). This shows that when no treatment is given

following SCI there is development of spasticity after the initial hypo reflexive period as the

repetitive proprioceptive signals from the sensory receptors are not controlled and it leads to









spasm of the flexor muscle. So some form of treatment is essential for the treatment of spasticity

following SCI.

Saline Cycle

The saline cycle group showed hyper reflexive pattern at post-op week 1. These data

suggest that only exercise sufficiently addressed the mechanism that led to the hyper reflexia at

this time point. These presumably include hyperactivity of the cells in the spinal cord after injury

due to the release of the excitatory neuromolecules and the membrane damage which results in

imbalance in the extracellular and intracellular components which is most common till week 1

post-injury. The EMG signals at this week also showed the same pattern.

In contrast, at post-op week 4, the ankle torque amplitude data of this group showed a

pattern of hypo reflexia. This decrease in excitability may be due to the exhaustion of excitatory

neurotransmitters after a certain time period after injury. Another possibility is that training

induced activity may initiate a balancing of the neurotransmitters and preserve descending

controls of segmental regulatory processes. As it has been reported in our lab studies, a pattern of

hypo reflexia was observed at post-op week 2 in untrained and trained animals without drug

treatment and continued up to week 6. Immediately after SCI motoneurons receive unusually

large EPSPs from cutaneous stimulation consistent with the acute loss of descending brainstem

innervations of the dorsal horn. These EPSPs do not easily cause reflexes immediately after

injury, because of the profound loss of motoneuron excitability that occurs after injury from

other mechanisms, such as decreased dendritic PICs. Recent studies indicate that a significant

decrease in these postsynaptic, dendritic excitatory mechanisms may play an important role in

the hypo reflexia at this time (Li et al. 2004), due to the loss of intrinsic persistent inward

calcium and sodium currents (PICs) that normally prolong and amplify synaptic inputs. In

addition, these investigators' studies have revealed a spontaneous re-emergence of PICs that may









significantly contribute to late onset spasticity that develops around post contusion week 6.

Accordingly, at post-op week 8, we observed a hyper reflexive pattern in all the velocities tested.

These observations indicate that training alone cannot maintain the state of hypo reflexia without

any complimentary drug treatment. A similar pattern was seen in the EMG signal at all the

different time periods tested and at all the different velocities tested.

Baclofen Control

At post-op week 1, the baclofen control group did not show any hyper reflexive pattern in

all the velocities tested. These data suggest that baclofen alone can stabilize the excitatory

environment that influences motoneuron excitability immediately after injury, such as cell

membrane disruption and an imbalance of inhibitory and excitatory neuromolecules. It is

proposed that baclofen maintained an increased concentration of inhibitory neuromolecules by

activating the GABAb receptors and their subsequent contributions to controlling excitation. At

post-op week 4, a hypo reflexive pattern was observed due to the above described actions

combined with a reduction in availability of unregulated release of excitatory neurotransmitters.

However, at post-op week 8, a hyper reflexive pattern was observed in all the velocities tested.

This may be due, in part, to the withdrawal effect of baclofen as the baclofen pump was removed

at post-op week 4. The withdraw-induced hyper reflexia may be correlated with an increased

stretch reflex excitability associated with a significant ligand-mediated down-regulation of

GABAb receptors in the ITB treated spinal cord, a previously reported result of the chronic

exposure to baclofen (Kroin et al. 1993). This change also relates to the abrupt loss of the

previously described GABAb associated inhibitory processes. In addition, it may be due to

baclofen modulation of the PIC (Heckman et al. 2004), which plays an important role in the

development of spasticity; baclofen blocks the Ca2+ channels which play an important role in

generating the PIC's, and it may have generated PIC's in the absence of baclofen at post-op









week 8. The EMG signals showed the same pattern as that of velocity dependent ankle torque,

and it was time locked at all the different time periods tested.

Baclofen Cycle

ITB treatment along with exercise decreased ankle extensor stretch reflex excitability as

indicated by significant decreases in velocity dependent ankle torque and time-locked EMG

magnitude. During the Iweek period of ITB treatment with bicycle of the lumbar spinal cord, the

low-velocity ankle torque was unchanged, whereas torque recorded during high-velocity ankle

rotations were significantly decreased compared to pre-treatment values. The reduction in torque

was also accompanied by significant reduction in the short-latency EMG that was time-locked to

ankle rotation. The reduction in EMG activation is consistent with the increase in the threshold

for activation associated with the presumed mechanism of action of GABAb receptor mediated

decrease in transmitter release at the primary afferent terminals (Peshori et al. 1998). A similar

finding was reported previously from our lab (Wang et al. 2002). Similarly during post-op week

4 and week 8 the ankle torque and EMG activation were near the pre-treatment values at all the

velocities tested.

Current evidence suggests that following the initial trauma, many secondary events

including membrane damage, systemic and local vascular effects, altered energy metabolism,

oxidative stress, inflammation, electrolyte imbalances, unregulated release of neurotransmitters,

and a cascade of biochemical changes affect cellular survival, integrity, and excitability (Tator

1995). From the above data we can see that baclofen, which is a GABAb agonist, inhibited the

release of the excitatory neurotransmitters, which prevented the membrane damage and cellular

imbalance in the intracellular and extracellular compartment during the first week post-op,

therefore preventing the development of spasticity. During post-op week 4, exercise and baclofen

maintained an inhibitory environment and prevented the development of spasticity most likely by









blocking the PIC, which develop in the motoneurons after injury after a brief period of hypo

reflexia due to the activation ofNa+ and Ca2+ channels, and baclofen prevented it by blocking the

Ca2+ channels.

At post-op week 8, even after the removal of the drug there was no development of hyper

reflexia. This may be due to exercise, which must have maintained the inhibitory environment

and stabilized the firing pattern from the motoneurons. It remains to be seen how baclofen works

in presence of exercise and how exercise maintains the hypo reflexive state even after removal of

the drug. This can be done in future studies by doing single cell motoneuron recordings using

patch clamp.

Thus it proves that without baclofen and locomotor exercise treatment spasticity develops

immediately after injury and thus proves our hypothesis that acute baclofen and bicycle training

is the most effective in preventing the development of the spasticity without deteriorating the

condition of the animal as we see from the data that neither alone baclofen or locomotor

prevented the development of spasticity at all post-op week 1, 4 and 8.

Open Field Locomotor Assessment

Saline Control

Animals with surgical lesions of the dorsal spinal cord at T8 that preserved ventral funiculi,

demonstrated sufficient self-training that no detectable difference was observed in their

locomotor recovery compared with animals that were systematically trained using a treadmill

(Fouad et al. 2000). This is true as we also saw a 51% improvement in this group at post-op

week 4 when compared to post-op day 1. At post-op week 8 group means BBB scores showed a

slight improvement of about 4% from post-op week-4, which is attributed to their intrinsic ability

to self-train. Even though the ankle torque data showed spasticity starting from post-op week 1, 4

and 8 this group showed gait improvement without any treatment.









Saline Cycle

This group of animals started with a slightly higher score compared to the other entire

group on post-op day 1. It showed an improvement of about 61% at post-op week 4, which is

better than the baclofen cycle group. Also, the ankle torque showed hypo reflexive pattern at this

time period, which may be due to the exhaustion of the excitatory neurotransmitters as reported

earlier or stabilization of the neuronal circuits.

While at post-op week 8, a decrease in BBB score was seen of about 2% from post-op

week 4, and also the ankle torque showed hyper reflexive pattern going along with this finding.

This may be due to the fact that bicycle training maintained a pattern of uniformity and

efficiency, which resulted in the improvement of the BBB score at post-op week 4.Training

alone may not be efficient to have a presynaptic inhibitory effect like that of baclofen on the

ventral horn motoneurons which may continue to fire continuously leading to hyper reflexia in

post-op week 8. After certain period of exercise the improvement in the segmental circuit sub

serving the muscle and joints may reach the plateau level from where further improvement is not

possible. Thus exercise alone may be unable to maintain an inhibitory environment for

preventing the development of late onset spasticity and also the gait.

Baclofen Control

The BBB score for baclofen control group improved about 47% from post-op day 1 to

post-op week 4, and also it improved about 15% from post-op week 4 to post-op week 8.The

score for post-op week 4 corresponds to the ankle torque and EMG data; we observed no

spasticity in this group at that period of time. Thus baclofen may have stabilized the cell

membrane of the cells after injury and prevented the imbalance in the intracellular and









extracellular environment and thus prevented the development of spasticity and also improved

the gait pattern as the BBB score improved.

But, in contrast to ankle torque week 8 data; there was a resurge of hyperreflexia after the

withdrawal of the drug at week 4. The BBB score improved about 14% from post-op week 4.

This may be attributable to the preservation of fibers diffusely located in the ventral caudal and

ventro-lateral funiculi of the rat spinal cord (Basso et al. 2002; Brustein and Rossignol 1998), or

gray matter of the T13-L2 spinal segments (Magnuson et al. 1999). Even after injury, baclofen

may have stabilized them, and after withdrawal of the drug, their connectivity to the muscles and

joints of the hind limbs may have improved. Collectively, these may have contributed to the

improvement of the BBB score. The ankle torque may be increased due to the withdrawal effect

on segmental excitability processes as explained above in the ankle torque data.

Baclofen Cycle

In this group we saw an improvement in the BBB score from post-op day 1 to post-op

week 4, a total improvement of 57%. This data corresponds to the ankle torque data where there

is no hyper reflexia at post-op week 4, thus it proves that acute locomotor training and baclofen

drug prevented the development of spasticity and also improved the gait of the injured animals

instead of deteriorating them. In animals and humans with SCI, previous studies have shown

improvements in gait parameters following locomotor training using body weight support on the

treadmill and manual assistance (Behrman and Harkema 2000; Harkema et al. 1997; Dietz and

Harkema 2004) but have not concurrently evaluated effects of bicycle locomotor training

following animal with SCI. The findings of the present study are consistent with the suggestions

that as therapy, the locomotor training regimen using bicycle, promotes the recovery of walking

by optimizing the activity-dependent neuroplasticity of the nervous system (Muir & Steeves









1997; Bose et al. 2005). Neuronal circuits, stimulated by task appropriate activation of peripheral

and central afferents via locomotor training, may also reorganize by strengthening existing and

previously inactive descending connections and local neural circuits (Muir & Steeves 1997)

(Bose et al. 2005).

At post-op week 8, the BBB score improved from post-op week 4, even after removal of

the baclofen drug. This proves that the bicycle training may have prolonged the effects of

baclofen or it might itself have prevented any secondary damaging effects that may occur after

withdrawal of the drug.

Thus, it proves that acute drug therapy and locomotor treatment improves gait and

maintains the reflex excitability to the pre-injury values.

Footprints

Hind limb axial rotation and base of support were assessed by footprints, collected while

walking along a treadmill, and were measured to compare between cycle trained and control

animals. Changes in these parameters in humans have been correlated with dysfunction of

descending long tract and propriospinal systems (Kunkel-Bagden et al. 1993).

Saline Control

Saline control group, which received no treatment, did show deterioration in the base of

support and axial rotation at post-op week 4, but at post-op week 8, the base of support showed

near pre-injury values, while the axial rotation remained similar to post-op week 4 values. As

these groups of animals were not bicycle trained and therefore not handled frequently they

retained their pre-injury parameters. It has also been shown that control SCI animals have the

intrinsic ability to self train.









Saline Cycle

Saline cycle group showed a pattern similar to that of baclofen control with deterioration at

post-op week 4 and maintaining the same pattern at post-op week 8. Thus exercise alone may not

be beneficial to improve the gait parameters without the drug.

This may lead to one conclusion that whatever changes occur in the segmental circuit that

maintain the foot placement and locomotion occur within the first 4 weeks after the injury and it

remains the same and does not change later.

Baclofen Control

In this group we saw an increase in base of support and axial rotation at post-op week 4,

but at post-op week 8, the values remained more or less similar to post-op week.

This may point out that baclofen alone may not be effective in improving the gait

parameters and that some form of locomotor training is required as we see an improvement in

baclofen cycle at post-op week 4.

Baclofen Cycle

At post-op week 4, the base of support and the axial rotation showed pre-injury values, but

at post-op week 8 the base of support and axial rotation showed much increased value compared

to the other group. The deficits observed in the baclofen treated animals of the present study

could have occurred through changes in the activity of these long tract or propriospinal systems:

either at synaptic terminals, cell body of origin, or upon spinal intemeurons' modulating posture.

It is known that baclofen modulation of the synaptic actions of spinal ventromedial funicular

fibers mediated presumably by GABAb receptors on or near axon terminals and last order spinal

interneurons (Jimenez et al. 1991; Quevedo et al. 1992). In addition, GABAb sensitive sites have

been reported in vestibular and functional companion nuclei that regulate the gain of the

vestibulospinal reflex (Manzoni et al. 1994). Therefore, it is possible that the ITB treatment









induced changes in posture and limb axis via actions within the spinal cord or within neuronal

sites in the brainstem that regulate posture and equilibrium. However, the differences in the time

course for the changes in hind limb axis and base of support with improvement in post-op week

4 and deterioration at post-op week 8 may be that after the withdrawal of the drug the GABAb

receptors must have up regulated but there is no ligand (baclofen) present to bind them. But this

finding goes against the fact that in baclofen cycle animals the ankle torque showed hypo

reflexia at all the time points tested. This could mean that the drug must be acting at different

anatomical sites (Wang et al. 2002) to have different effect on ankle torque and footprints. Saline

cycle group also did not show much improvement in the footprint parameters at post-op week 8.

One of the possibilities may be that after baclofen pump removal, the toll of exercise may

have caused some unwanted stress on the hind limb paws that might have led to external

deviation of the paw leading to anatomical defect and thus increase in base of support and axial

rotation in post-op week 8. As baclofen pump was removed after post-op week 4, but training

was continued up to post-op week 8, the residual effect of baclofen must have prevented the bad

effects of exercise. As for the baclofen cycle group, the combination of drug and locomotor

therapy prevented the early changes, but once the drug was removed, exercise alone could not

maintain the inhibitory environment to prevent the changes from taking place.

Neurotransmitters

Saline Control

Neurotransmitters like 5-HT, GAD67, GABAb and neurotropins like BDNF and GAP43

showed a decreased expression when compared to all the other groups. This may be due to the

fact that these groups did not receive any locomotor training and baclofen drug. Baclofen, which

is a GABAb receptor agonist, is primarily used as an anti-spastic drug and also the ankle torque

and EMG data did not show any decrease in spasticity when their torque amplitude was









measured. Thus these animals showed the least improvement when compared to all the other

groups.

Saline Cycle

Neurotransmitters like 5-HT, GAD67, GABAb receptors and neurotropins like BDNF and

GAP43 did show an increased expression when compared to saline control group. Locomotor

training may have facilitated the increased expression of neurotropins like BDNF and GAP43. As

predicted from Cotman's paper (2002) exercise increases the expression of the neurotropins,

which have an effect on the growth of the axons and improves the neuronal plasticity. Since

there is little increase in GABAb receptor expression this may be correlated to the improvement

in spasticity seen during post-op week 4. Also, increased expression of 5-HT and GAD67 may

correlate with the improvement to the locomotion observed during post-op week 4 on the BBB

scale.

Baclofen Control

In this group we also saw similar neurotransmitter expression as that of saline cycle group.

As this group received baclofen drug only with no locomotor training, the drug must have

influenced the expression of GABAb receptor till post-op week 4, which also corresponds to the

improvement of spasticity during that period. Also, there must be some correlation between 5-

HT and GAD67 expression to that of baclofen drug as there was increased expression of the

former neurotransmitters. Neurotropins like BDNF and GAP43 also showed an increased

expression to that of saline control group, which co-relates to the improvement in the BBB score

observed at post-op week 4. But this group was not able to maintain the improvement till post-op

week 8 as the baclofen pump was removed at post-op week 4.









Baclofen Cycle

This group showed the maximum neurotransmitter expression when compared to all the

other groups. There was an increased expression of all the neurotransmitters and neurotropins

and also hypo reflexive pattern when tested for spasticity. This may be due to the fact that this

group received both baclofen drug and locomotor training. As we have discussed above, exercise

improves the expression of neurotropins like BDNF and GAP43, which promotes neuronal

plasticity and improved neuronal growth in case of injury, thus leading to improvement in gait

and spasticity. Baclofen drug increased the pre-synaptic inhibition by increasing the expression

of GABAb receptors, which was responsible for the hypo reflexive response seen during

measurement of spasticity.

Thus, all the above data show that if baclofen drug and locomotor training in the form of

bicycle, if started acutely after spinal cord injury, may prevent the development of spasticity and

help in improving the gait of the animal. By contrast, baclofen alone or cycle alone did not show

a continuous maintenance of hypo reflexive pattern, with later development of spasticity and not

much improvement in the gait.









CHAPTER 6
CONCLUSION

In humans, one of the most devastating chronic effects following SCIs is the development

of spasticity. Spasticity leads to velocity dependent lengthening resistance in the muscles

resulting in contractures and secondary biochemical changes which eventually cause destruction

of the muscles. In humans the present antispastic treatment is the continuous infusion of ITB, a

GABAb agonist that acts on the presynaptic receptors leading to inhibition of both monosynaptic

and polysynaptic reflexes. At present, there are no approved guidelines to administer this drug

acutely before the symptoms of spasticity become evident. Locomotor training in the form of

weight bearing stepping using treadmill has been shown to improve the gait of the SCI patients.

Treadmill training requires extensive use of manpower to hold the patient while training and also

this facility is present only in few selected locations across the nation. Therefore, the aim of this

thesis was to provide preclinical data to test the efficacy of baclofen therapy with or without

locomotor training (bicycle locomotor training) which has already been shown to be as effective

as treadmill (Bose et al. 2004).

To test the above hypothesis spinal cord injured rats were used as their spinal cord circuitry

resembles to that of human. Moreover, contusion injury mimics most common SCI in humans

and produces spasticity. The animals were divided into 4 groups after contusion spinal cord

injury:

Saline control group saline pump, no bicycle training

Saline cycle group saline pump, bicycle training

Baclofen control group baclofen pump, but no bicycle training

Baclofen cycle group baclofen pump + bicycle training.









The animals were selected only if their open field locomotor (BBB) score fell below 3

when tested at post-op day 1. The animals were trained over the course of 2 months. The training

schedule was performed 5 days a week using two 20 minute trials/day, starting from post-op day

1. On the first day of training, the rats were given five minutes training.

The baclofen and saline pump were removed at post-op week 4. The animals were tested

for spasticity at post-op weeks 1, 4 and 8 using the velocity dependent ankle torque set up

developed in our lab (Thompson et al. 1996; Bose et al. 2002) and also measuring the EMG

signals from the muscles in the same set-up. The animals were tested for gait improvement using

the BBB scale and footprints at post-op weeks 4 and 8. The animals were then sacrificed using

4% freshly prepared paraformaldehyde using an accepted protocol to remove the thoracic,

injured and lumbar spinal cord. The spinal cords were then used to do immunofluorescent

experiments to test the presence of various neuromolecules such as GABAb, GAD67, 5-HT,

BDNF and GAP43 which have documented roles in the motoneuron excitability and

neuroplasticity following injury and therapy.

As per the results we got from all the above experimental procedures we can come to the

conclusion that acute baclofen drug therapy with bicycle training prevented the development of

spasticity measured by velocity dependent ankle torque and associated EMG data. Moreover, this

combined treatment showed an increment in BBB score compared to post-op day 1 when

compared to other 3 experimental groups. The immunofluorescent images showed qualitative

increase of GABAb receptors as well as other neuromolecules such as 5-HT, GAD67, BDNF and

GAP43 as compared to all the other 3 groups. Hypo reflexive pattern seen in baclofen cycle group

might be related to GABAb receptors mediated presynaptic inhibition of the stretch reflexes.

However, interestingly, the base of support and axial rotation did not show improvement when









compared to other groups. While the control groups such as baclofen drug alone or saline cycle

alone did show some decrease in spasticity at post-op week 4, they could not retain the same

improvement at post-op week 8. Thus to conclude, we found that acute baclofen drug therapy

along with bicycle training prevented the development of spasticity. Moreover, the acute training

and drug therapy did not deteriorate the condition of the injured animals. Therefore, these

preclinical data have the potential to translate in human clinical trail.









CHAPTER 7
FUTURE WORK

In this research project, we have shown that combination therapy of baclofen drug and

locomotor training started acutely after spinal cord injury prevented the development of

spasticity and improved the gait, and overall locomotor capacity. Surprisingly, the individual

therapy of either drug (ITB) or locomotor training (customized bicycle) did not show benefit in

improving these condition. Baclofen and locomotor exercise possibly work synergistically and

thus, the mechanism underlies these benefits need to be unfolded. Therefore, a detailed study

involving GABAb receptor profile as well as properties of the motoneuron has to be investigated

by intracellular motoneuron recording using in vivo patch clamp during different time periods of

the treatment. As this research only conducted qualitative analysis of the GABAb receptors by

immunofluroscent technique, molecular techniques such as Western Blot or ELISA can be used

to quantitatively measure the expression of receptors in the spinal cord tissue to test differences

in expression in different groups. Moreover, neurotropic factor, such as BDNF, is an important

indicator of regeneration of injured axons which mediates neuroplasticity. Therefore, a detailed

molecular study to investigate the profile of BDNF and its receptor trkB can provide a better

understanding of neuroplasticity mediated by BDNF following locomotor training. Furthermore,

in vivo longitudinal MRI study using volumetric measurement of the lesion can be done to

further predict the benefits of this combined therapy. At present, I am planning to work on post

fixed spinal cord tissue using T2-wieghed MRI imaging to study the effects of locomotor and

drug therapies. These multi-dimensional studies may further enhance our understanding in the

mechanism of the recovery/benefits we have observed following locomotor and drug therapies.










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BIOGRAPHICAL SKETCH

Rita Jain was born in Mumbai, India. She completed her bachelor's degree in medicine in

2001. After her graduation she worked as a resident medical officer in Niramaya Hospital in

Pune, India. After working there for 6 months, she realized that her knowledge was not sufficient

to bring a breakthrough in the medicine world and to decrease the suffering of the patients. She

decided to go for higher education. She came to United States in 2003, to pursue her master's

degree in medical sciences at the University Of Florida. She chose to specialize in neuroscience.

For the next 3 years, Thompson's lab was her home. There she worked under the guidance of Dr.

Prodip Bose and Dr. Floyd Thompson on various research projects.


































































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