Assessment and Promotion of Plasticity and Locomotor Recovery following Spinal Cord Injury


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Assessment and Promotion of Plasticity and Locomotor Recovery following Spinal Cord Injury
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1 online resource (149 p.)
Mondello, Sarah E
University of Florida
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Gainesville, Fla.
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Doctorate ( Ph.D.)
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University of Florida
Degree Disciplines:
Medical Sciences, Neuroscience (IDP)
Committee Chair:
Howland, Dena R
Committee Members:
Reier, Paul J
Thompson, Floyd J
Lewis, Mark H
Behrman, Andrea L


Subjects / Keywords:
chondroitinase -- corticospinal -- plasticity -- rehabilitation -- rubrospinal -- sci
Neuroscience (IDP) -- Dissertations, Academic -- UF
Medical Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation


Damage to the spinal cord causes sensorimotor loss that is permanent. The resulting functional losses are debilitating, may be life threatening, and affect an individual’s ability to be independent in the home and community. Unfortunately, there are no effective therapeutics to reduce the functional deficits caused by spinal cord injury (SCI).  Identification of effective treatments has been complicated by the limited regenerative capacity of the central nervous system in addition to SCI being a multifarious problem likely to require a complex, combinatorial treatment approach.  Some individuals with incomplete SCIs may recover basic walking abilities but continue to have difficulty with more challenging forms of locomotion including those that require greater balance and alterations in leg trajectories.  In the current studies, a cat low thoracic hemisection model was used.  This incomplete, asymmetrical injury is similar to Brown Sequard Syndrome (BSS) described in a subpopulation of humans with SCIs.  In the first set of studies, a battery of gait features were assessed to compare performances during a basic locomotor task (flat overground walking) and an adaptive locomotor task (horizontal ladder crossing).  Gait features critical for successful performance of adaptive locomotion pre- and post-SCI were identified.  In the second study, results from previous chondroitinase abc (ch’abc) studies in the lab were extended to determine the effects of different treatment durations on anatomical plasticity and functional recovery.  The results from this study contribute important information relative to treatment duration for the ultimate translation of ch’abc to a clinical setting.  In the final study, retrograde tract tracing with Fluorogold (FG) was optimized for use in a large animal model and will contribute to future assessments of circuitry disruption and plasticity in the injured spinal cord. Collectively, the body of work presented in this dissertation contributes to our understanding of the anatomical and behavioral changes that occur following SCI, how functional performance might be enhanced with a promising therapeutic treatment, and how methods to assess anatomical plasticity can be improved to enhance future studies.
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by Sarah E Mondello.
Thesis (Ph.D.)--University of Florida, 2012.
Adviser: Howland, Dena R.
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2 2012 Sarah Elizabeth Mondello


3 To my Parents, who gave me the space and opportunity to determine my genuine goals, as well as the encouragement and resources to achieve them


4 ACKNOWLEDGMENTS Obtaining my doctoral degree was a long and challenging journey that could not have been completed without the support and guidance of several specific individuals. First and foremost, I would like to th ank my mentor Dr. Dena Howland, as she donated massive amounts of her time to offer critical advice, guidance, and support through each portion of this project. Her understanding and patience throughout the earlier learning stages of my doctoral degree we re invaluable, and I can only hope she will continue to be a source of support in the years to come. I can not Stephanie Jefferson, Adele Blum, Eric Brunk, Megan Black, Kenny Maskel l, Rhea Chattopadhayay, and Valerie Tainsh. As lab manager, Wilbur helped with absolutely every part of my project, from ordering supplies necessary for experiments, helping with surgeries, and being an overall handy man, to also helping maintain my sanit y through humor and kind words. I can not imagine getting through this process without his help. Nicole Tester, Stephanie Jefferson and Adele Blum took the time to teach me the majority of technical skills necessary for this project and as the more senior graduate students, never stopped offering valuable advise despite having moved on to different laboratories. Eric Brunk, Megan Black, Kenny Maskell, Rhea Chattopadhayay, Valerie Tainsh, and Martina Spiess were past laboratory team members that help ed with various experiments and analyses. Additionally, their humor acted as essential comedic relief during stressful times Thompson, Andrea Behrman, and Mark Lewis, offered ex tremely helpful guidance and advice throughout this entire process. They have helped me understand the process of


5 research and science overall, and have pushed me to become a strong scientist. They each deserve many thanks for their priceless time and efforts. My family ha s been extremely supportive and helpful as well. My parents spent hours of their lives listening to me vent and offering guidance whenever they could. My older sister Donna and brother in law Chuck always opened up their home in NY to me, providing critical breaks and opportunities to recharge. Both my younger sister Amanda and best friend Annie have lent strong, yet sensitive ears over the past few years, primarily in regards to non academic issues. Their support helped me to s tay on task. Lastly, and so importantly I would like to thank all of the priceless friends I have made along the way outside the laboratory. My happiness, sanity, and maintained perseverance throughout the majority of my time spent on this project was a result of their constant understanding, caring, and thoughtful words offered throughout every success and failure along the way.


6 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF FIGURES ................................ ................................ ................................ ........ 10 ABSTRACT ................................ ................................ ................................ ................... 11 1 BACKGROUND ................................ ................................ ................................ ...... 13 The Spinal Cord Is Inhibitory to Axonal Growth After Injury ................................ .... 13 The Multiple Motor Pathways that Control Locomotion ................................ ........... 15 Central Pattern Generator ................................ ................................ ................ 15 Propriospinal System ................................ ................................ ....................... 15 Sup raspinal Motor Pathways ................................ ................................ ............ 16 Locomotor Recovery following Spinal Cord Injury ................................ .................. 17 Complete versus Incomplete Spinal Cord Injury ................................ ............... 18 The E ffect of Spared Tissue on Locomotor Recovery ................................ ...... 20 Pathway Specific Potential for Plasticity ................................ ................................ 21 Axonal Die Back ................................ ................................ ............................... 21 Responses to Trophic Factor Application ................................ ......................... 22 Propriospinal Plasticity ................................ ................................ ..................... 23 Enhancing Propriospinal Plasticity ................................ ................................ ... 25 The Effects of Training on Functional Recovery after Injury ................................ ... 26 Cats as a Translational Model of Spinal Cord Injury ................................ ............... 27 The Pathology of Spinal Cord Injury ................................ ................................ ....... 28 The Glial Scar ................................ ................................ ................................ ... 30 The inhibitory nature of chondroitin sulfate proteoglycans ......................... 30 Chondroitin sulfate proteoglycan temporal expression patterns after spinal cord injury ................................ ................................ ..................... 33 Chondroitin sulfate glycosaminoglycans are the primary inhibitory component of chondroitin sulfate proteogl ycans ................................ ..... 34 Chondroitinase ABC as a Potential Therapeutic ................................ ..................... 35 Chondroitinase ABC Mediated Tract Plasticity ................................ ................. 36 Potential Mechanisms Underlying Chondroitinase ABC Mediated Effects ....... 38 Determining Optimal Chondroitinase ABC Application Paradigms ................... 39 Biological stability ................................ ................................ ....................... 39 Chondroitin sulfate glycosaminoglycan turnover rate ................................ 40 Delivery period ................................ ................................ ........................... 41 The Important Role of Tract Tracing ................................ ................................ ....... 42 2 DIFFERENTIAL RECOVERY OF GAIT FEATURES DURING BASIC AND ADAPTIVE LOCOMOTION FOLLOWING SPINAL HEMISECTION IN THE CAT .. 44 Introduction ................................ ................................ ................................ ............. 44


7 Methods ................................ ................................ ................................ .................. 45 Subjects ................................ ................................ ................................ ............ 45 Surgical Procedures ................................ ................................ ......................... 46 T10 spinal hemisection ................................ ................................ .............. 46 Behavioral Tasks Training Paradigm and Gait Features ................................ 46 Overall assessment of locomotor recovery ................................ ................ 46 Onset of reco very ................................ ................................ ....................... 47 Limb accuracy ................................ ................................ ............................ 47 Crossing speed ................................ ................................ .......................... 47 Percentage of abnormal foot positioning on rungs ................................ ..... 48 Step cycle duration ................................ ................................ .................... 48 Stride length ................................ ................................ ............................... 48 Footfall patterns ................................ ................................ ......................... 48 Stance percentage ................................ ................................ ..................... 49 Double support period ................................ ................................ ................ 49 Iliac crest mediolateral moveme nt ................................ .............................. 49 Duration to support after toe down ................................ ............................. 49 Ankle flexion during yield after toe down ................................ .................... 49 Tissue Processing ................................ ................................ ............................ 50 Perfusions and tissue preparation ................................ .............................. 50 Histology: Cresyl violet and myelin staining ................................ ............... 50 Statistical analysis ................................ ................................ ...................... 51 Results ................................ ................................ ................................ .................... 51 Lesion Magnitudes ................................ ................................ ........................... 51 Recovery Onset and Effects of Injury on Crossing Speed ................................ 51 Phalangeal versus Abnormal Metatarsal Placement onto Ladder Rungs Post Spinal Cord Injury ................................ ................................ ................. 53 Injury Effects on Cycle Duration and Stride Length ................................ .......... 54 Effects of Injury on Interlimb Coordination, Stance Duration, and Double Su pport ................................ ................................ ................................ .......... 55 Mediolateral Movement ................................ ................................ .................... 58 Distal Joint Stability ................................ ................................ .......................... 59 Discussion ................................ ................................ ................................ .............. 60 Task Specific Spatiotemporal Gait Changes after Injury ................................ .. 61 Trunk Support on Skilled Tasks after Injury ................................ ...................... 62 Task Specific Changes in Distal Joint Positioning after Injury .......................... 63 Conclusions ................................ ................................ ................................ ...... 64 3 THE EFFECT OF DIFFERENT CHONDROITINASE ABC TREATMENT DURATIONS ON LOCOMOTOR RECOVERY AND SUPRASPINAL TRACT PLASTICITY iN A FELINE MODEL OF SCI ................................ ........................... 74 Introduction ................................ ................................ ................................ ............. 74 Materials and Methods ................................ ................................ ............................ 76 Subjects ................................ ................................ ................................ ............ 76 Surgical Procedures ................................ ................................ ......................... 77 Low thoracic spinal hemisection ................................ ................................ 77


8 Chondroitinase ABC ad ministration ................................ ........................... 77 Fluorogold spinal injections ................................ ................................ ........ 78 Behavioral Procedures ................................ ................................ ..................... 78 Behavioral tasks and training paradigm ................................ ..................... 78 Assessment of l ocomotor recovery ................................ ............................ 79 Horizontal ladder, peg walkway, and narrow beam assessment: Limb accuracy ................................ ................................ ................................ 79 Tissue Processing ................................ ................................ ............................ 80 Perfusions and tissue preparation ................................ .............................. 80 Cresyl violet and m yelin staining ................................ ................................ 80 Assessment of injection site ................................ ................................ ....... 81 Immunohistochemistry ................................ ................................ ............... 81 Fluorogold labeled cell counts ................................ ................................ ... 82 Lesion ranking ................................ ................................ ............................ 82 Statistical Analysis ................................ ................................ ............................ 83 Results ................................ ................................ ................................ .................... 83 Lesion Magnitudes ................................ ................................ ........................... 83 Axonal Projections below the Lesion Site ................................ ......................... 83 Rate of Recovery ................................ ................................ .............................. 85 Ipsilateral Hindlimb Accuracy ................................ ................................ ........... 87 Horizontal ladder ................................ ................................ ........................ 87 Narrow beam ................................ ................................ ............................. 88 Peg walkway ................................ ................................ .............................. 89 The Relationship between Spared Tissue and Functional Recovery ............... 89 Onset of task recovery versus lesion size ................................ .................. 90 Ipsilateral hindlimb accurate targeting versus lesion size .......................... 91 Discussion ................................ ................................ ................................ .............. 92 Summary of Results ................................ ................................ ......................... 92 Supraspinal Connectivity below the Lesion ................................ ...................... 92 Locomotor Recovery and Supraspinal Plasticity ................................ .............. 93 Lesion Size ................................ ................................ ................................ ....... 96 Conclusion ................................ ................................ ................................ ........ 97 4 OPTIMIZATION OF FLUOROGOLD RETROGRADE TRACING ......................... 105 Introduction ................................ ................................ ................................ ........... 105 Materials and Methods ................................ ................................ .......................... 106 Subjects ................................ ................................ ................................ .......... 107 Surgical Procedures ................................ ................................ ....................... 107 Fluorogold spinal injections ................................ ................................ ...... 107 Tissue Processing: Histology and Immunohistochemistry .............................. 108 Perfusions ................................ ................................ ................................ 108 Cresyl violet and myelin staining ................................ .............................. 108 Fluorogold immunohi stochemistry ................................ ........................... 109 Assessment of injection site completeness and fluorogold autofluorescence ................................ ................................ .................. 110 Results ................................ ................................ ................................ .................. 110


9 Tissue Damage at the Injection Site ................................ ............................... 110 Fluorogold Detection Techniques ................................ ................................ ... 111 Triton Increases Tracer Travel Time ................................ .............................. 112 Discussion ................................ ................................ ................................ ............ 114 5 SUMMARY ................................ ................................ ................................ ........... 121 Defining Recovery ................................ ................................ ................................ 121 Advancing Cho ndroitinase ABC to the Clinic ................................ ........................ 122 Conclusions ................................ ................................ ................................ .......... 124 LIST OF REFERENCES ................................ ................................ ............................. 125 BIOGRAPHICAL SKETCH ................................ ................................ .......................... 149


10 LIST OF FIGURES Figure page 2 1 Lesion magnitudes ................................ ................................ ............................. 66 2 2 Tasks, onset of recovery, and crossing speeds ................................ .................. 67 2 3 Phalangeal versus met atarsal placement onto ladder rungs .............................. 68 2 4 Changes in cycle duration and stride length ................................ ....................... 69 2 5 Change in hindlimb footfall pa tterns and hind to hindlimb coordination .............. 70 2 6 Change in hindlimb stance and double support periods ................................ ..... 71 2 7 Mediolateral mov ement of the iliac crest ................................ ............................ 72 2 8 Swing to stance transition ................................ ................................ ................... 73 3 1 Range of spinal hemisections ................................ ................................ ............. 99 3 2 Supraspinal connections below the lesion ................................ ........................ 100 3 3 Onset of basic and skilled locomotor recovery ................................ ................. 101 3 4 Ipsilateral hindlimb accuracy ................................ ................................ ............ 102 3 5 Spared tissue and task onset correlations ................................ ........................ 103 3 6 Spared tiss ue and accurate ipsilateral hindlimb targeting correlations ............. 104 4 1 Fluorogold injection schematic ................................ ................................ ......... 116 4 2 Effects of differ ent f luorogold concentrations, volumes, and survival periods on tissue damage at the injection site ................................ ............................... 117 4 3 Anti fluorogold immunohistochemical processing leads to greater fluorogold detecti on compared to native autofluorescent detection ................................ ... 118 4 4 Long term fluorogold detection using anti fluorogold immunohistochemical processing ................................ ................................ ................................ ........ 119 4 5 Triton enhances fluorogold tracing speed ................................ ......................... 120


11 Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy ASSESSMENT AND PROMOTION OF PLASTICITY AND LOCOMOTOR RECOVERY FOLLOWING SPINAL CORD INJURY By Sarah Elizabe th Mondello August 2012 Chair: Dena R uth Howland Major: Medical Sciences Neuroscience Damage to the spinal cord causes sensorimotor loss that is permanent. The resulting functional losses are debilitating, may be life threatening and affe ct an are no effective therapeutics to reduce the functional deficits caused by spinal cord injury (SCI). Identification of effective treatments has been complicated b y the limited regenerative capacity of the central nervous system in addition to SCI being a multifarious problem likely to require a complex, combinatorial treatment approach. Some individuals with incomplete SCIs may recover basic walking abilities but continue to have difficulty with more challenging forms of locomotion including those that require greater balance and alterations in leg trajectories. In the current studies, a cat low thoracic hemisection model was used. This incomplete, asymmetrical injury is similar to Brown Sequard Syndrome (BSS) described in a subpopulation of humans with SCIs. In the first set of studies, a battery of gait features were assessed to compare performances during a basic locomotor task (flat overground walking) and a n adaptive locomotor task (horizontal ladder crossing). Gait features critical for successful performance of adaptive locomotion pre and post SCI were identified. In the second


12 were extended to determine the effects of different treatment durations on anatomical plasticity and functional recovery. The results from this study contribute important a clinical setting. In the final study, retrograde tract tracing with Fluorogold (FG) was optimized for use in a large animal model and will contribute to future assessments of circuitry disruption and plasticity in the injured spinal cord. Collectively the body of work presented in this dissertation contributes to our understanding of the anatomical and behavioral changes that occur following SCI, how functional performance might be enhanced with a promising therapeutic treatment, and how methods to as sess anatomical plasticity can be improved to enhance future studies.


13 CHAPTER 1 BACKGROUND The S pinal C ord I s I nhibitory to A xonal G rowth A fter I njury The devastating consequences of spinal cord injury (SCI) and the permanence of the resulting effects in human s are well documented. The earliest documentation of SCI dates back to 1550 B .C. in the Egypt i a In that document oth arms and both legs, and unable to move vertebra. This is a condition which cannot the most notable neuroscientists, Ra paths are something fixed, ended, immutable. Everything may die, nothing may be regenerated (Cajal, 1928) Th ese concept s of the impossibility of functional recovery and the absence of axonal growth after SCI dominated the scientific community until the f post SCI growth was discovered Collateral sprouting, the growth of nerve fibers from intact axons af ter injury was first identified in experiments using nerve fibers supplying the skin in rabbits (Weddell et al., 1941 ) Similar growth was subsequently identified in the spinal cord by Liu and Chambers in 1958 Using a spared root prep, they denervated several dorsal roots from one side of the cord, waited several months, then transected the remaining roots from the c hronically injured side in addition to the matching roots from the non injured side. Two weeks later tissue was assessed with a neurodegeneration stain, which showed a greater number of degenerating profiles in the chronic compared to the acute side. This was attributed to connections between the chronic and acute sides by way of collateral sprouting (Liu and


14 Chambers, 1958; For Review See Guth, 1975) Subsequently, t he concept that regeneration of axotomized fibe rs does not occur was disputed by s everal studies which reported that severed axons returned to active states if stimulated properly (Li and Raisman, 1995; Kobayashi et al., 1997) For example, a study completed by Yi and colleagues determined that damaged rubrospinal tract fibers one year after injury could grow into a peripheral nerve graft if first treated with brain derived neurotrophic factor (BDNF) (Kobayashi et al., 1997) These combined findings, in addition to many others, suggest ed that the spinal cord has a much greater potential for axonal plasticity and functional recovery after inju ry than previously believed. T he greatest functional restoration most likely will be obtained through a combination of regeneration and collateral sprouting. Research to better understand and enhance these types of plasticity is being actively pursu ed using experimental animal models. A multitude of different approaches such as locomotor training (For review see Battistuzzo et al., 2012) cellular replacement, introduction of m a trix substrates as well as numerous different pharmacological interventions (For review see Jeffery and Blakemore, 1999; Lu and Tuszynski, 2008; Boulenguez and Vinay, 2009) all have shown some enhancement of axon al growth after injury. For this new growth to be functionally effective after an injury, it must make functional connections. The motor system is an extremely complex combination of m any tracts each p laying their own independent, as well as co mplementary roles in locomotion. Understanding the function of these pathways during locomotion in an uninjured system, as well as their contributions to locomotor recovery after injury is important for designing approaches to enhance locomotor recovery post SCI.


15 The Multiple Motor Pathways t hat Control Locomotion In 2004, the report of a survey taken by 681 SCI individuals ranked seven specific functions in order of their importance for quality of life T he ability to walk ranked four th highest, in dicating the need to determine methods for recovering locomotor function after injury (Anderson, 2004) The underlying neural control of walking and other types of locomotion ha s been well studied over the course of several centuries and provide s a strong foundation for guiding present and future investigators towards promoting recover y of locomotor function after SCI (For review see Clarac, 2008) Central Pattern Generator The underlying circuitry responsible for locomotion is complex and involves all levels of the neural axis spanning from intraspinal networks to supraspinal tracts (D.A, 1975; Eidelberg and Yu, 1981; Yu and Eidelberg, 1981) T he most notable locomotor related intraspinal system s are the central pattern generator s (CPG s ); a well organized circuit of interneurons and motoneurons located in the cervical and lumbar areas of the spinal cord. Th e s e circuit s fire in specific patte rns leading to alternating bilateral flexion and extension of the arms and legs to produce stepping. This CPG activity can occur spontaneously without descending supraspinal or sensory inputs and is considered to be the neural basis for the basic stepping pattern Circuitry can be modulated in response to external stimuli through spinal interneurons allowing for some degree of adaption to changes in the environment (For review see Grillner and Zangger, 1979; Rossig nol and Frigon, 2011) Propriospinal System Propriospinals (PSNs) make up a large portion of spinal interneurons and play a significant role in locomotion. This system contributes to trunk control, inter and intra


16 limb integration, and modulates input from both the descending supraspinal systems and peripheral afferents. Additionally, they synchronize motor circuitry throughout the entire length of the spinal cord (For review see Flynn et al., 2011) The PSN system is primarily contained within the spinal cord, linking different spinal segments together allowing for complex movements. However, some projec t to supraspinal centers like the lateral reticular nucleus (Alstermark et al., 1981b; Skinner et al., 1989) and cerebellum (Skinner et al., 1989) Depending on their length PSN pathways are 1 6 (Conta Steencken and Stelzner, 2009) Thus, short PSNs modulate activity occurring relatively local to the cell of origin and long PSNs make connections to distant regions of the spinal cord. In fact, evidence indicates that the lo ng PSNs may be responsible for connecting the cervical and lumbar CPGs for quadrupedal stepping/synchrony across the fore limbs and hindlimbs in the cat (Miller et al., 1973) and possibly the arms and legs in humans (For review see Die tz, 2002; Huang and Ferris, 2009; Tester et al., 2012) PSN projections can occur either in the rostral or caudal plane from their cell of origin, located in the spinal cord gray matter (Skinner et al., 1989) This flexibility allows for the complex circuitry that underlies the production and control of elaborate multi segmental movements. Sup raspinal Motor Pathways Voluntary locomotor control is mediated by supraspinal tracts that originate in the brain or brainstem. Primary supraspinal motor tracts include the corticospinal (CST), rubrospinal (RuST), reticulospinal (ReST), and vestibulospina l (VST) tracts. These tracts are separated into a medial system: ReST, VST, and a l ateral system: RuST, CST. Approximately 87.6% of the CST is located dorsolaterally while 12.4% of the CST


17 ( anter ior CST) travels in the ventromedial funiculi of the spinal cord (Kwon et al., 2011) These two systems, the CST and RuST, are essential for producing fine tuned precise movements like stepping over an obstacle (Mohagheghi et al., 2004) grasping food (Alstermark et al., 1981a; Alstermark et al., 1987; Whishaw et al ., 1998) and paw placement (Batson and Amassian, 1986; For review see Drew et al., 2002) The loss of these s ystems causes difficulties maneuveri ng through environment s and completing day to day activities. The medial system is responsible for creating the necessary tone in postural musculature allowing for upright locomotion. Additionally, t he ReST has been sho wn to initiat e stepping (Jell et al. 1985) The VST, originating in the vestibular nuclei within the medulla, is primarily controlled by the utricles and saccules of the vestibular system and displacements (Markham, 1987) In this way, the VST is critical for maintaining balance. Disruption to either of the medial or lateral systems has detrimental effects that vary depending on the magnitude of the lesion itself. Locomotor Recovery f ollowing Spinal Cord Injury Complete transection of the spinal cord, which removes all supraspinal input below the level of the lesion, results in immediate and complete loss of motor function is present in experimental animals as well as humans. It lasts for approximately two weeks in humans after which some reflexes begin to return (For review see Riddoch, 1917; Eidelberg, 1981) Some investigators argue that the period of spinal shock is much shorter on the magnitude of minutes to hours (For review see Ditunno et al., 2004) Regardless, there is a n overall peri od of motor depression after an injury that lasts for


18 several weeks. Several months following complete spinal transection spasticity may occur, characterized by hypertonus, clonus, involuntary somatic reflexes, and muscle spasms that are oft en extremely painful (For review see Rabchevsky and Kitzman; Adams and Hicks, 2005) This secondary impairment as a result of SCI is believed to be due to aberrant persistent inward currents (PIC). In the uninjured nerv ous system, PICs are depolarizing inward currents intrinsic to motoneurons that can self sustain firing as long as the cell remains depolarized. Cessation requires inhibitory synaptic input, often led by supraspinal centers. PICs are controlled by the mo noamines 5HT and norepinephrine, in which their primary sources are the raphe nucleus and locus coeruleus. Acutely after an injury, motoneuron excitability is reduced due to a loss of supraspinal connectivity and thus, monoamines. However, over time moto neuronal excitability returns to somewhat normal levels, and is particularly monoamine sensitive. Residual monoamines present in the spinal cord and vasculature are able to activate PICs however the remaining lack of supraspinal connectivity prevents the se PICs from being inhibited, causing spasticity (For review see ElBasiouny et al., 2009) There are currently several treatment options available for spasticity including baclofen, tizanidine, and botulinum neurotoxin, however they have varying degrees of efficacy (Rabch evsky and Kitzman) Multiple animal models of spasticity are used in order to improve our understanding of this impairment and improve treatment options (Thompson et al., 2001) Complete v ersus Incomplete S pinal Cord Injury Although voluntary stepping does not recover after an anatomically complete SCI, CPG based spinal stepping can be elicited as seen in humans (Dietz and Colombo, 2004) cats (Grillner and Rossignol, 1978; Eidelberg et al., 1980) rats (references),


19 dogs (Hart, 1971) and possums (Hinsey and Cutting, 1936) Incomplete SCI, like anterior cord syndrome, central cord syndrome, posterior cord syndrome, and brown sequard syndrome (BSS), result in substantially greater locomotor recovery due to the sparing of some tissue (Eidelberg, 1981) Individuals with incomplete SCI typically have notable recovery of basic locomotion like walking overground or on a treadmill, however the presence of some common deficits remain. Often, these individuals have decreased gait speed (Knutsson and Richards, 1979; Dietz et al., 1981; Conrad et al., 1983; Wainberg et al., 1986; Wagenaar and Beek, 1992; Lajoie et al ., 1999) and cycle duration (Lajoie et al., 1999; Barbeau et al., 2002) Additionally, a variety of different joint alterations have been reported such as increased hip angular excursion, and increa sed knee flexion during either foot touchdown alone or the entire step cycle. The ankle also has been reported as either being dorsiflexed, or plantar flexed causing foot drag (Conrad et al., 1983) Changes in muscle activation during walking also have been shown to occur after injury Typically, an abnormal co activation of antagonist muscles occurs (Fung and Barbeau, 1989, 1994) in addition to altered shape and timing of activation patterns (Fung and Barbeau, 1989; Domingo et al., 2007) Individuals with incomplete SCI often have greater struggles with challenging types of locomotion. This can be illustrated by studies looking at functional recovery in cats with a hemisection injury sim ilar to BSS. Studies completed by the Howland laboratory have shown that following a T10 hemisection, cats were able to recover bipedal treadmill and crossing of a greater d ifficulties when performi ng challenging tasks like crossing of a hor izontal ladder, peg walkway, or (Tester and Howland, 2008; Jefferson et


20 al., 2011) The disparity between functio nal recovery in individuals with complete versus incomplete SCI suggests that these two populations require di fferent rehabilitation strategies. The Effect of Spared Tissue on Locomotor Recovery The degree of recovery in individuals with incomplete SCI va r ies greatly across individuals and is partially due to the different types of pathways spared by the injuries. Animal studies in which specific cuts were made in order to axotomize select tracts both reveal individual tract functions through the process of elimination, as well as determines the contributions of different tracts on locomotor recovery after SCI. Extensive damage to the medial systems often is accompanied by severe and permanent postural control and locomotor impairments (Lawrence and Kuypers, 1968; Brustein and Rossignol, 1998) This suggests a critical role in basic locomotion for these systems (Lawrence and Kuypers, 1968; Brustein and Rossignol, 1998) In c ontrast, studies in which injuries were isolated to the CST and RuST showed only transient locomotor deficits and a quick recovery of overground locomotion (Laursen and Wiesendanger, 1967; Muir and Whishaw, 2000; Ka nagal and Muir, 2009) food grasping (Blagovechtchenski et al., 2000) and other skilled finger movements (Hepp Reymond et al., 1970) However, components of these movements remained impaired, indicating that these lateral systems are most important for more skillful, precision based movements. D amage to these lateral pathways paired with s paring of the ventromedial cord has been associated with substantial locomotor recovery after SCI (Windle et al., 1958; Nathan and Smith, 1973; Afelt, 1974; Eidelberg et al ., 1981; Schucht et al., 2002; Krajacic et al., 2010) indicating that alternate pathways can contribute to functional recovery after injury.


21 Pathway Specific Potential for Plasticity The contributions of certain pathways to recovery after injury are larg ely due to their innate responses to injury and plastic properties. These properties differ across systems. Axonal die back is a major response to injury that occurs in all transected fibers (Busch et al., 2009) which affects their participation in new circuit formation after injury; the further an axon retracts, the more distance regenerating and/or sprouting fibers must cover to bridge the lesion site and restore function. Even if a fiber with significant retraction sprout s onto nearby spared circuit r y, that circuit itself will have further distance to cover, increasing its chances for failure. Comparing axonal die back properties across several supraspinal tract systems provides insight into their individual potential for meaningful plasticity. The applicati on of trophic factors like BDNF and neurotrophin 3 (NT 3) have been associated with increased axonal growth of multiple supraspinal pathways after injury (Kobayashi et al., 1997; Hiebert et al., 2002; Kwon et al., 2002; Plunet et al., 2002; Dolbeare and Houle, 2003) The responsiveness of individual tracts to trophic factor application also can provide a general indication of specific tr ability to grow after injury Axonal Die Back Significant evidence indicates that the CST has limited regenerative potential. For example, s everal studies looking at plasticity in multiple pathways have reported minimal CST growth, but significant growth in the RuST (Richardson et al., 1984a; Houle and Ye, 1999; Decherchi and Gauthier, 2000; Plunet et al., 2002) ReST and VST (Houle and Ye, 1999; Oudega et al., 1999) One reason CST growth is limited after injury is likely related to its significant and prolonged axonal die back as de picted in several studies Specifically, these studies found that f ollowing injury the proximal


22 ends of CST axons form ed dystrophic endbulbs and retract ed for sev eral millimeters and for up to eight weeks (Pallini e t al., 1988; Oudega et al., 1999) Meanwhile, a xonal dieback only lasted for ~ four weeks post injury in the VST, ReST and RuST tracts with axons measuring approximately 0.5 to 1.5 mm from the lesion site (Houle and Jin, 2001) In contrast to the above mentioned results, one study als o found continued die back for eight weeks following axotomy of the VST, suggesting this tract may share regenerative and sprouting difficulties similar to the CST (Oudega et al., 1999) Responses to Trophic Factor Application Both the RuST (Liu, 1999; Liu et al., 2002; Murray et al., 2002; Jones et al., 2003b) and ReST (Xu et al., 1995; Menei e t al., 1998) have shown enhanced post axotomy growth in response to trophic factor application. However, the CST and VST have been reported to respond less notably in several studies Specifically, two separate groups found that while the addition of BD NF to red nucleus cell bodies resulted in enhanced regeneration of the RuST into peripheral nerve grafts, the same treatment to motor cortex cell bodies did not cause CST regeneration. Interestingly, they did indicate enhanced collateral sprouting both ro stral (Hiebert et al., 2002; Plunet et al., 2002) and caudal (Plunet et al., 2002) to the injury. A similar study comparing regeneration of the RuST, ReST, CST, and VST after application of one of three different growth factors (ciliary neurotrophic factor (CNTF), BDNF, or neurotroph in 3 ( NT 3 ) ) found enhanced regeneration of the ReST and RuST in response to all growth factors. However, the VST responded to only one of the growth factors, CNT F, while the CST did not respond to any (Ye and Houle, 1997) Similar results have been found i n additional studies indicating that both the CST (Schnell et al., 1994; Tuszynski et al., 1997; Blesch, 1999; Lu et al., 2001) and VST (Xu et al., 1995; Ye and Houle, 1997;


23 Menei et al., 1998) may be less apt for plasticity after axotom y However, despite these tracts underwhelming plastic responses to trophic factors, several studies utilizing different growth promoting methods have reported increased growth. For example administration of leukemia inhibitory factor (Blesch, 1999) and olfactory ensheathing cell transplants (Li et al., 1997) have both led to enhanced CST growth. Even more interesting is the study by Bareyre and colleagues who repo rted spontaneous sprouting of transected CST axons onto long PSN tracts in the cervical spinal cord following a thoracic SCI in rats. This new circuitry was capable of bridging the lesion site and enhancing functional recovery (Bareyre et al., 2004) Such findings indicate that CST growth may, in fact, be occurring in regions distant from the lesion where plasticity is not typically assessed. A similar phenomena was reported i n a study by Rozensweig and colleagues who showed substantial spontaneous CST growth in a primate model of SCI, citing species differences as a potential reason for the lack of CST plasticity commonly reported in rodents (Rosenzweig et al., 2010) Plasticity of the VST, though less well studied, has shown enhancements when growing into an embryonic tissue transplant (Ito et al., 1999) Overall, these findings indicate that the CST and VST have the potential for plasticity, but respond less readily to certain treatments compared to the RuSt and ReST. Alternatively, plasticity in the CST and VST may be occurring as readily as in the ReST and RuST, but in regions dista nt to the lesion that are not typically assessed. Proprio spinal Plasticity Although plasticity of the PSN system is currently not as well studied as the supraspinal systems, researchers are beginning to identify it as a key player in creating novel pathways that allow for neuronal signals to bypass the lesion a nd restore function.


24 In a study by Courtine and colleagues they determined that following several carefully timed and placed incomplete lesions, spared PSN axons were independently capable of mediating recovery of stepping without direct input fro m the brain (Courtine et al., 2008) Spontaneous PSN tract plasticity also has been shown to form novel circuitry after a high cervical injury resulting in recovery of chronic diaphragm activity (Darlot et al., 2012) These findings indicate incredible plastic potential in this PSN system that is capable of functional restoration However, based on detailed investigations by the Stelzner group the potential for plasticity appears to differ across the short and long PSNs. In a collection of studies, th ey showed that long and short PSNs respond differentially to axotomy in that there are fewer surviving short PSNs after injury compared to long PSNs (Conta and Stelzner, 2004; Siebert et al., 2010b; Conta Steencken et al., 2011) Furthermore, while short PSNs have an initial upregulation of growth factor receptor gene expression, as well as immune, inflammatory, and pro apoptotic gene ex pression transiently after injury, these same genes are downregulated in the long PSNs. This translates int o more short PSN cell death compared to long. However, surviving short PSNs have genetic pro files that return to normal by one month (Siebert et al., 2010a; Siebert et al., 2010b) and their neuronal size does no t significantly change between two week s and the course of the study (eight weeks). The authors sugges t that the surviving short PSNs could be a specialized population (Siebert et al., 2010a) This theory was validated in a separate study which reported significant short PSN axonal growth across a midline injury t o form functional synaptic connections with motoneurons after SCI (Fenrich and Rose, 2009)


25 Although the above mentioned set of st udies shows an initial and permanent decrease in the number of short PSNs over the course of a 16 week study (Conta Steencken and Stelzner, 2009) studies from my own lab have show n an initial yet transient decre ase in short PSNs followed by a significant increase by 16 weeks post injury (Blum, 2010) This difference in short PSN plasticity across studies may be a result of species differences, as our study was completed in feline and theirs in rat. However, it is most likely due to the extensive training our animals underwent and th e lack of training in their study as t raining increases BDNF production (Beaumont et al., 2008) which in turn increases plasticity (Girgis et al., 2007) Additionally, the Stelzner group used a contusion injury model, wh ile a a hemisection model was used in ours. Hemisection s typically have been assoc iated with greater axonal growth (Iseda et al., 2008) These contradictory findings indicate that the PSN system has plastic potential, however requires certain stimuli to activate this potential after injury. Enhancing Propriospinal Plasticity Although there are few studies that have specifically focused on the effects of trophic factors on PSN plasticity re sults following Schwann cell transplants into the lesion site show ed an enhancement in long and short PSN plasticity however, the greatest amount of plasticity was in the short PSNs (Xu et al., 1997; Takami et al., 2002; Doperalski et al., in preparation) Additionally, one study found that the combination of GDNF and Schwann cell seeded channels applied to the lesion site led to enhanced plasticity of short PSNs Collectively the above studies suggest that alth ough there is substantial PSN cell death after injury, those that survive are capable of substantial plasticity that can mediate and support recovery after SCI (Xu et al., 1995; Menei et al., 1998)


26 The Effects of Training on Functional Recovery after Injury Training itself has been shown to enhance plasticity and functional recovery after injury (For review see Marsh et al., 2010) Multiple st udies using training as the sole therapeutic after injury have report ed enhancements of the function being trained Examples of these functions include basic stepping (Harkema et al., 2011) treadmill stepping in both humans (Thomas and Gorassini, 2005) and cats (Lovely et al., 1986; Barbeau and Rossignol, 1987) wheel based stepping (Beaumont et al., 2008) staircase climbing (Singh et al., 2011) single pellet reaching and horizontal ladder walking in rats (Girgis et al., 2007; Starkey et al., 2011) Further assessments regarding how trai ning enhances functional recovery have identified training induced enhancemen ts of growth factor upregulation, specifically BDNF and Anti Growth Associated Protein 43 (GAP 43) (Girgis et al., 2007; Beaumont et al ., 2008; Ying et al., 2008) These growth factors enhance axonal plasticity (Liu, 1999; Murray et al., 2002) and help shape synaptic plasticity (Ying et al., 2008) Training also has been shown to enhance motoneuronal electrophysiol ogical properties like resting membrane potential, spike trigger level s (Beaumont et al., 2008) a nd increase excitability of the CST underlying leg muscle activity (Thomas and Gorassini, 2005) The way training is conducted after injury has drastic affects on functional outcomes Task specific training of one type of task has been shown to enhance recovery o f that task, but in some cases does not transfer to other tasks. For example, swim training was shown to enhance swimming kinematics, but had no e ffect on overground walking (Magnuson et al., 2009) Additionally, s tair climbing ascent training led to enhanced recovery of this specific task, but caused only partial improvements on overground and grid stepping (Singh et al., 2011)


27 Task specific training of one task also has been shown to have detrimental and/or positive effects on the recov ery of other tasks. For example, r ats that were trained specifically on single pellet reaching had significant functiona l improvements o n this task after injury, but were significantly worse at crossing a horizontal ladde r compared to their untrained rat counterparts (Girgis et al., 2007) Another example is one study completed by Garcia Alias and colleagues who found that r ats trained to walk on a grid had significantly enhanced recovery of skilled pellet reaching, but were significantly worse at horizontal ladder stepping compared to their untrained counterparts (Garcia Alias et al., 2009) The potential for task specific training on one task to transfer to oth er tasks most likely relates to the kinem atic similarities across tasks as s pecific neuromuscular activity patterns lead to differential muscle responses. This concept was assessed and confirmed in a st udy that compared t he muscle activity of spinalized cats trained to either step on a treadmill or stand after injury Those cats trained to stand had increased maximum rate of shortening in the ir medial gastrocnemius muscles, as well as greater muscle ma ss when compared to cats that were only trained to step on a treadmill. These same cats also had an overall greater shift of muscle fiber type towards fast fibers (Roy et al., 1999) Overall, these findings demon strate that the neuromuscular system is extremely sensitive to different training regimens, and that there is a strong need to enhance our understanding of how different training paradigms affect functional recovery after injury in order to maximize recovery Cats a s a T ranslational M odel of S pinal C ord I njury Cats are considered to be a highly translational SCI model as they share numerous similarities with humans in regards to neuro anatomical control of movement, gait m echanics, and interlimb coordination (For review see Vilensky, 1987; Dietz, 2002;


28 Majczynski and Slawinska, 2007) The overall organization of the large ascending and descending tracts within the spinal cord are s imilar across species (Majczynski and Slawinska, 2007; Watson, 2009) Additionally, t he presence of a central pattern generator, which underlies stepping patterning, has been confirmed in the cat (Miller et al., 1973; Smith et al., 1983) and indirectly identified in humans (Harkema et al.; Dimitrijevic et al., 1998; Jilge et al., 2004; Calancie, 2006) Evidence from both species also has indicated the presence of flexible networks in the spinal cord that are responsible for independent movement of each limb, suggesting that both species have (For r eview see Brown, 1914; Prokop et al., 1995) In regards to reflex characteristics, both c ats and humans share certain reflexive responses to afferent stimuli (Lisin et al., 1973) and also have several shared speed related gait change s (Vilensky, 1987) Postural control in response to perturbations also was identified as being similar across cats and humans when humans were positioned in a quadrupedal stance (Macpherson et al., 1989) D espite the more extreme differences in overall body size, the average c at spinal cord length is only 9 cm shorter than the average human spinal cord. Thus, the distance necessary for axonal plasticity to render functional changes is similar across cats and humans. These species similarities make the cat a valuable animal mod el for SCI research, th erefore the studies to be described in the experimental chapters ( 2 4 ) of this dissertation were completed in a cat model of SCI. The Pathology of Spinal Cord Injury The peripheral nervous system (PNS) is substantially more conduc ive to axonal growth after injury than the central nervous system (CNS) and It ha s been determined that the CNS environment is responsible for this disparity (Richardson et al., 1980;


29 Benfey and Aguayo, 198 2; Richardson et al., 1984a) Injury to the CNS, specifically the spinal cord, creates an upregulation of multiple different physiological responses that further inhibit this sys SCI occurs in a biphasic manner in which there i s an initial mechanical injury followed immediately by a period of ongoing damage This period is commonly known as secondary in jury and can continue for months after the mechanical injury (Rowland et al., 2008; Flynn et al., 2011; Kuzhandaivel et al., 2011) Secondary injury be gins immediately after insult, in which there is substantial hemorrhagic necrosis, microglial activation (Donnelly and Popovich, 2008) and an upregulation of pro inflammatory cytokines like interleukin tumor necrosis factor (Pineau and Lacroix, 2007) Within the ne xt 48 hours the Blood Brain Barrier reaches its peak level of permeability (Noble and Wrathall, 1989) ; n eutrophils invade the lesion site, and vasogenic and cytotoxic edema start to set in (For review see Rowland et al., 2008) Additionally h e morrhaging continues which leads to free radical product ion and a dysregulation of Ca++ ion concentrations followed by calpain activation and mitochondrial dysfunction (Schanne et al., 1979) E xtracellular glutamate eventually r eaches toxic levels at the lesion site (Wrathall et al., 1996) and collectively these components will result in cell death (Schanne et al., 1979) Studies indicate that neuronal death is primarily by way of necrosis, although there have been some reports of neuronal apoptosis (Liu et al., 1997; Beattie et al., 2000; Lu et al., 2000) Oligodendrocytes are more prone to apoptosis than neurons (Crowe et al., 1997) Within the next several days, multiple different inflammatory related cell types infiltrate the lesion site, including re active astrocytes, monocytes, and macrophages


30 (Popovich et al., 1997; Fleming et al., 2006; Donnelly and Popovich, 2008) The a xonal death that has been occurring throughout this entire injury period leads to a b reakdown of the myelin sheaths that once surrounded them. This myelin debris acts as a strong inhibitor to axonal growth and is more slowly removed by the immune system in the CNS than in the PNS (Filbin, 2003) In addition to the above mentioned responses to injury, a glial scar begin s to form. This scar is one of the primary inhibitory components of the lesion environment. The Glial Scar The glial scar is primarily formed by reactive astrocytes that are characterized by many intermeshing cytoplasmic processes. Additionally, it c onsists of reactive microglia, macrophages, fibroblasts, oligodendrocytes, oligodendrocyte precursor cells, S chwann cells, and meningeal cells (Fawcett and Asher, 1999) The fibrous qualities of this scar present as a physical barrier that is extremely difficult for axons to penetrate An even g reater obstacle against axonal growth within the scar is the upregulation of chondroitin sulfate proteoglycans (CSPG). Following SCI, these CSPGs are increased at the lesion site primari ly by reactive astrocytes however reactive microglia, macrophages, fibroblasts and oligodendrocyte precursor cells also play a role (McKeon et al., 1991; Dou and Levine, 1994; Smith Thomas et al., 1994; Fitch and S ilver, 1997; Fawcett and Asher, 1999; Dawson et al., 2000) These CSPGs create a strong chemical inhibition against axonal growth (Rudge and Silver, 1990; McKeon et al., 1991) The inhibitory n ature of c hondroitin s ulfate p roteoglycans CSPGs are one of the largest and most abundant proteoglycan families in t he normal, uninjured nervous system. They make up a large portion of the extracellular matrix present in the intercellular spaces between neurons and glial cells, forming a


31 tight meshwork with hyaluronate, tenascin, and link proteins (Hook et al., 1984; For review see Kwok et al., 2008; Zimmermann and Dours Zimmermann, 2008; Hyatt et al., 2010) They also are an important component of the dense perineuronal nets (PNN) t hat surround neurons and regulate plasticity, neuroprotection, and homeostasis (Deepa et al., 2006) CSP Gs also are important during neurodevelopment by interact ing with tenascin to help guide axons to the appropria te locations through inhibition and the formation of inhibitory PNNs (Snow et al., 1991; Brckner et al., 2000; Pizzorusso et al., 2002) External to the nervous system, CSPGs are present in cartilage by binding strongly to, and sta bilizing its components: Laminin, fibronectin, and collagen (Oldberg and Ruoslahti, 1982; Snowden, 1982) CSPGs are a large family consisting of seven different members: Brevi can, decorin, neurocan, aggrecan, versican, phosphacan and neuron glial antigen 2 ( NG2 ) Each member has a different core prot ein with unique characteristics and multiple attachment sites for chondroitin sulfated glycosaminoglycan sugar side chains ( CS GAGs) (Herndon and Lander, 1990) Thes e CS GAG chains are sulfated repeats of hexonate dissacharides (glucoronate or iduronate) and hexosamines (glucosamine and galactosamine) (Silbert and Sugumaran, 2002) Sulfation occurs primarily on C 2, C 4 and C 6 and the amount of this sulfation varies greatly across core proteins, thus there is substantial heterogeneity within this family (For review see Iozzo, 1998; Kwok et al., 2008) In additio n to the CSPGs there are several other families of proteoglycans including Keratan Sulfate (KSPG), and Heparan Sulfate (HSPG). These families consist of the same core proteins but in lieu of or in addition to CS GAGs have keratan or heparan sulfate chain attachments. KSPGs have recently been reported to have


32 inhibitory properties similar to CSPGs (Imagama et al., 2011; Hilton et al., 2012) while HSPGs have been shown to promote axonal growth (Mammadov et al.; Riopelle and Dow, 1990) Over the past decade CSPGs have become an area of intense research in regards to their inhibition of axonal growth following nervous system injury. Upre gulation of some CSPGs begins within hours following injury and develops over several weeks to months (Fitch and Silver, 1997) leading to the formation of a mature glial scar (McKeon et al., 1991; Levin e, 1994; Fawcett and Asher, 1999; Haas et al., 1999; Lemons et al., 1999; McKeon et al., 1999) Not only does this scar act as a physical barrier to axonal growth, but also as a chemical barrier mediated by the CSPGs. The inhibitory properties of CSPGs have been extensively studied and confirmed across many laboratories (Rudge and Silver, 1990; Snow et al ., 1990; McKeon et al., 1991; Dou and Levine, 1994; Milev et al., 1994; Davies et al., 1997; Fidler et al., 199 9; Hynds and Snow, 1999; Schmalfeldt et al., 2000; Becker and Becker, 2002) One of the initial in vitro studies to investigate this idea performed a stripe assay which determined that neurite outgrowth of chick dorsal root ganglia would grow abundantly onto a laminin stripe, but would stop or grow along the border of a k eratan sulfate (KS)/CSPG stripe. These findings indicated that neurite outgrowth is inhibited by KS/CSPGs (Snow et al., 1990) Extension of these results to a CNS trauma environment was completed in a study by McKeon and colleagues In this study, they found that onl y adult, P 30 rats had CSPGs and cytotactin/tenascin (CT) present in their gliotic scar following brain injury, while the neonates with a similar injury did not.


33 Additionally, it was only these adult rats that could not properly support axonal gro wth, indicating that the presence of CSPGs and CT correlates with inhibition of axonal growth (McKeon et al., 1991) A more detailed examination of CSPGs in vitro, determined that CSPG inhibition is gradient dependent with neurite gro wth inhibition being greatest in the presence of higher CSPG concentrations (Snow and Letourneau, 1992) These findings indicate that the removal of these CSPGs after an SCI may create an en vironment more conducive to axonal growth and act as a promising potential therapeutic. Chondroitin sulfate proteoglycan temporal expression patterns after s pinal c ord i njury The realization that CSPGs inhibit axonal growth after injury led to a mu ch closer examination of their upregulation pattern post SCI. While CSPGs are considered to be injury expression of each individual CSPG core protein is unique from one another. There are six different types of CSPGs: ne urocan, brevican, versican, phosphacan, NG2, and aggrecan (For review see Kwok et al., 2008) Currently, all studies regarding the expressions of each individual CSPG have been completed in rats. In an uninjured spinal cord, all CSPGs are expressed at low levels. As quickly as 24 hours post injury there is moderate upregulation of neuro can, brevican, versican (Jones et al., 2003a) and NG2 (Jones et al., 2002; Jones et al., 2003a) Notably, pho sphacan expression significantly decreases during this period, possibly because of an increase in proteolytic enzymes that degrade phosphacan, like plasmin (Wu et al., 2005) Expression of aggrecan also wa s reported to decrease (Lemons et al., 2001; Andrews et al., 2011) Peak expression of neurocan, brevican, versican occurs at 2


34 weeks post injury. NG2 expression peaks earlier at 1 week post injury and remains elevated for at least 7 weeks (Jones et al., 2002) Phosphacan expression does not begin to increase until 4 weeks post inj ury after which it remains elevated for at least 8 weeks and most likely play s a large role in axonal inhibition during chronic injuries. Expression of brevican, versican, and NG2 also were still elevated at 8 weeks post injury, bu t only in regions closely surrounding the lesion site. Neurocan expression was reduced to basal levels by 8 weeks (Jones et al., 2002; Jones et al., 2003a) and recovery of aggrecan expression was shown to begin at 2 weeks after hemisection injury (Lemons et al., 2001) but remained decreased after a contusion, as determined by a combination of western blot analysis and immunohistochemistry (Andrews et al., 2011) Overall, these findings demonstrate considerable diversity across the CSPGs in regards to the timing of their upregulation after injury. This is an important consideration in regards to the timing of administration of potential therapeutics that may act on these inhibitory components. Chondroitin sulfate glycosaminoglycans are the primary inhibitory component of chondroitin sulfate proteoglycans As previously described, CSPGs consist of two main components: Core protein, and CS GAG chains. By removing the CS GAGs from the core proteins using the bacterial enzyme cho ndroitinase abc (c CS GAGs contain (Snow et al., 1990) bacterial enzyme purified from Proteus Vulgaris a gram negative bacteria normally present in the intestinal tracts of humans and other animals It consists of two enzyme components capable of degrading chondroitin sulfate proteoglycans: an endoeliminase, which depolymerises CSPGs, and an exoeliminase which degrades tetra and hexa


35 saccharides resulting in disaccharides (For review see Crespo et al., 2007) has been used extensively as an in vitro tool for understanding the role of CS GAGs in axonal growth inhibition. Snow and colleagues was one of the first investigators to utilize this new tool and found that the removal of CS enhanced growth of chick dorsal root gan glia neurites onto KS/CS PG substrate suggesting that the CS GAG chains are the primary inhibitory portion of CSPGs (Snow et al., 1990) These studies were later confirmed by numerous others prior to advancing mediated CS GAG removal to an in vivo setting (Snow et al., 1990; Smith Thomas et al., 1994; McKeon et al., 1995; Zuo et al., 1998; Chung et al., 2000; Yu and Bellamkonda, 2001) Chon droitinase ABC as a P otential Therapeutic The confirmation that CS outgrowth in vitro sparked an interest among many investigators to determine this Previous studies comp leted in the Howland laboratory CSPG cleavage (Lemons et al., 1999) Following this study Yick and colleagues were to the spinal cord after injury led to enhanced axon al growth. In this study, a peripheral nerve graft implantation was paired with either BDNF applications did not enhance the graft, while did lead to enhanced growth (Yick et al., 2000) Further investigation s by numerous other groups also found enhanced plasticity within mu ltiple pathways, in addition to functional recovery. Bradbury and colleagues were the first c administration led not only to enhanced axonal growth and a


36 restoration of post synaptic activity caudal to the lesion, but al so enhanced recovery of multiple different functions (Bradbury et al., 2002) Following this study, a multitude of others were completed which also reported enhanced plasticity and functional recovery after injury (Tropea et al., 2003; Barritt et al., 2006; Houle et al., 2006; Massey et al., 2006; Galtrey et al., 2007; Vavrek et al., 2007; Cafferty et al., 2008; Iseda et al., 2008; Massey et al., 2008; Tester and Howland, 2008; Tom and Houl, 2008; Garcia Alias et al., 2009; Lee et al., 2009; Tom et al., 2009b; Bai et al., 2010; Karimi Abdolrezaee et al., 2010; Jefferson et al., 2011) Each of these studies utilized different injury models and treatment administration methods suggesting the robustn ess of this treatment and its potential as a future therapeutic. Chondroitinase ABC Mediated Tract P lasticity after injury, however the exact systems underlying this recove ry are currently unknown. M ul tiple groups have assessed the e ffect s o f application on the plasticity of several different pathways The most widely studied system has been the CST, whose plast icity seems to be enhanced with this enzyme. Some st udies, which used anterograde tracing techniques showed increased CST growth rostral to the lesion site (Karimi Abdolrezaee et al., 2010) paralleling the results of the trophic factor studies previously describe d. However, many studies also have found enhanced CST growth into the lesion site (Bradbury et al., 2002) or into tissue bridges (Iseda et al., 2008) a ffect on enhancing CST growth compared to me diated CST growth traveling closely around the lesion site and connecting caudal to the injury (Barritt et al., 2006; Garcia Alias et al., 2009) In fact, it has been shown that


37 inhibition of glycogen synthase kin ase 3, a component activated by CSPGs, led to enhances CST growth (Dill et al., 2008) Interestingly, the one study to a ssess CST plasticity that utilized a retrograde tracing technique did not find any FG labeled neurons the Rest, VST, and RuST (Bai et al., 2010) In this study, rats received a T10 transection and 12 weeks later, FG was placed into the transection site. The lack of motor cortex labeling in conjunction wi th enhanced labeling in the other aforementioned systems may be related to the CSTs tendency for extreme axonal dieback after axotomy. Additionally, it is possible that spared CST axons are more likely to sprout than axotomized axons are to regenerate proximal to the lesion site. As depicted by Bareyre and colleagues it i s also possible that sprouting of axotomized fibers might be occurring in regions distal from the lesion site (Bareyre et al., 2004) Sev eral studies have assessed the e supraspinal systems, all of which utilized a retrograde tracing technique. These studies reported c mediated enhancement of pla sticity in the ReST (Houle et al., 2006; Vavrek et al., 2007; Bai et al., 2010) and RuST (Houle et al., 2006; Vavrek et al., 2007; Bai et al., 2010; Jefferson et al., 2011) Additionally, the VST system also has shown (Vavrek et al., 2007; Bai et al., 2010) H owever one study did not find an enhan cement within this system despite seeing enhanced plasticity in the other previously described systems (Houle et al., 2006) These results, in conjunction with those previously described regarding the VST, suggest that this system may not be as plastic as the


38 other brainstem d erived descending path ways or, that plasticity primarily occurs at the terminal ends in this system. plasticity of the PSN system following injury has been minimally assessed mediated plas ticity within this system, further axonal growth into the peripheral nerve graft (Houle et al., 2006) Further be completed. Potential Mecha nisms U nder lying Chondroitinase ABC M ediated Effects Within the past several years, the first CSPG receptors have been identified. The transmembrane receptor protein tyrosine phosphatase sigma (RPTP) is one that has been shown to inhibit axonal gro wth through the CS GAG portion of the proteoglycan s (Shen et al., 2009) Several studies also have found that the disruption of the genes encoding this receptor results in enha nced axonal growth through CSPG regions after an SCI (Fry et al., 2009; Shen et al., 2009; Duan and Giger, 2010) In addition, both NgR1 and NgR3, two receptors known to mediat e myelin associated inhibitor (MAI) inhibition have been identified as binding with high affinity to the GAG moiety of CSPGs and thus may play a role in CSPG neurite growth inhibition (Dickendesher et al., 2012) Calcium and it s interaction with epidermal growth factor receptors (EGFR) and Protein Kinase C (PKC) also may be involved mediated plasticity though it has yet to be directly tested. Both kinase activity of EGFR and PKC activity lead to CSPG inh ibition of neurite outgrowth with t he blocking of either one of these components causing increa s ed neurite outgrowth. Subsequently, both E GFR phosphorylation and PKC activity are activated by calcium which has been shown to increase transiently in the presence of CSPGs (Snow et al., 1994; Sivasankaran et al.,


39 2004; Koprivica et al., 2005) Thus, the digestion of CS decrease the amount of calcium within the lesion site, decreasing the activity of these inhibitory factors. Lastly, the Rho/ ROCK pathway has been affiliated with neuronal growth cone collapse that is assoc ia ted with CSPGs, though the exact connection between the two is not well understood (Borisoff et al., 2003; Monnier et al., 2003; Duffy et al., 2009) Determining Optimal Chondroitinase ABC Application P aradigms Across the full range of studies, in a number of different way s : D uration of treatment, the period after injury at which treatment begins, volume, concentration location, and delivery device Interestingly, the majority of these study paradigms have reported some degree of mediated enhanced functional recovery and/or plasticity c was not effective was in a study completed by Jakeman and colleagues In this study, a single dose of thoracic contusion injury in mice. This treatment, paired with vol untary wheel running as training, did not lead to enhanced functional recovery (Jakeman et al., 2010) These findings suggest that it is important to determine the optimal administration paradigm(s) in order to Biological s tability One critical factor to consider is that c temperatu re (Tester et al., 2007) Therefore, following injury in order to ensure continued cleavage. Since direct application to the spinal cord is critical, many g roups implant a catheter system with tubing placed within the lesion site, as well as an injectable po rt implanted externally for ease of delivery


40 (Bradbury et al., 2002; Barritt et al., 2006; Houle et al., 2006; Ia ci et al., 2007; Vavrek et al., 2007; Carter et al., 2008; Garca Alas et al., 2008; Garcia Alias et al., 2009; Karimi Abdolrezaee et al., 2010; Carter et al., 2011) Groups utilizing this method tend to use an osmotic mini pump for injections in order to slowly inject solution to the cord and limit further exacerbation of the injury site Alternatively, multiple groups have created other term invasive catheter system. Examples o f this include a slow delivery (Hyatt et al., 2010) and and a hydrogel microtube scaffold system (Lee et al., 2009) All effectively prolong the GAG cleavage. Additionally, an adeno viral tet (Curinga et al., 2007) and mammalian cells modified (Muir et al., 2010; Kluppel, 2011) appear to be pro mising alternatives to a multiple injection delivery paradigm though they have yet to be tested in vivo. CS in vitro and in vivo (Jin et al., 2011) Results found both enhanced CS GAG cleavage and neurite outgrowth in vitro and substantial CS GAG cleavage in v ivo. Although this study did not address in vivo axonal growth, these results suggest that vector administration have potential therapeutic implications Chondroitin sulfate glycosaminoglycan turnover rate Understandin g the turnover rat e for CS consideration regarding treatment duration ; t he quicker the turnover rate, the shorter issue is only partially understood as there is no study that has focus ed specifically on this issue. However, multiple papers have partially addressed it through assessment of


41 immunoreactivity of either intact CSPGs (CS56 antibody) or cleavage byproduct) at the lesion site Multiple investigators have looked 10 14 days continued decrease in the amount of intact CSPGs present (Yick et al., 2000) as well as the continued presence of 2B6 sugar stubs (Cafferty et al., 2008; Tom et al., 2009a; Siebert et al., 2011) These combined findings suggest that CS GAGs remain cleaved and do not co mpletely turnover within a two week period. However, by three weeks post injury intact CSPG reactivity appears to return back to levels comparable to a vehicle treated animal (Hyatt et al., 2010) While these studies provide valuable clues, it is difficult to make conclusions from these results alone. The continued presence of 2B6 sugar stubs at the lesion site two weeks after injury does not strongly confirm that CSPG turnover has yet to occur, but instead indicates that the stubs from previous c Meanwhile, new CSPGs also may be present, but not detectable with the 2B6 antibody. Determining the immunoreactivity for intact CSPGs, as was done by Yick and colleagues would allow for more accurate assessment. Delivery period Determining the op there have been several investigators who have assessed delivery at more clinically relevant, chronic time points. A study completed by Garcia Alias and colleagues was one of the few and no additional interventions. CST regeneration, recovery of contact placing, and stride length were all similar acro ss the acute rats treated at the time of injury, and the delay ed rats treated at two four, or


42 seven days post injury. However, skilled reaching performance was significantly better in the acutely treated group. e potential to be effective in chronic injuries, but that recovery of certain behaviors respond differentially to different treatment periods (Garca Alas et al., 2008) four weeks after an incomplete injury has shown enhanced CST axonal growth (Iseda et al., 2008) and rescue of injured red nucleus neurons (Carter et al., 2011) However, enhanced axonal growth was not present following a larger contusion suggesting that (Iseda et al., 2008) line derived neurotrophic factor (GDNF) at eight weeks post injury (Tom et al., 2009b) or stem cell/progenitor cell transplants and GD NF at six weeks post injury (Karimi Abdolrezaee et al., 2010) however show enhanced plasticity and recovery of both basic (Tom et al., 2009b) and skilled locomotion (Karimi Abdolrezaee et al., 2010) despite effective a t chronic time points and in larger lesions, a combinatorial therapy approach is ideal. The I mportant R ole of T ract T racing Much of what we know about nervous system anatomy and plasticity after injury is a result of tract tracing ; i t i s a powerful technique that allows researchers to understand both the anatomy of their tracts of interest, as well as the response s of those tracts to insult. In regards to enhancing recovery after SCI, this technique is necessary for understan ding the circuitry changes that underlie certain functional changes in order to identify what therapeutic and training methods are effective. The Nauta Silver degeneration stain


43 anterograde tra ct tracing (Nauta and Gygax, 1954) This was followed by the retrograde tracer horseradish peroxidase (Nauta et al., 1975) A wave of new tracers were then developed including but not limited to biotin dextran amines phaseolus vul garis leucoagglutinin fast blue and fluorogold Each tracer comes with its own advantages and disadvantages, and the choice regarding which one to use for a specific study is dependent on the study paradigms and goals (For review see Lanciego and Wouterlood, 2011) Tract tracing in larger animal models, as performed in this work, is difficult. The tracing distance is often longer and in some cases certain tracers do not work properly. For example, pseudorabies virus traces beautifully in rodents (Lane et al., 2011) and ferrets (Jian et al., 2005) but is unsuccessful in cats Due to the valuable nature of tract tracing, improving tracing techniques for both small and large animal models would be beneficial for future studies in SCI research, as we ll as other neur oscience fiel d.


44 CHAPTER 2 DIFFERENTIAL RECOVER Y OF GAIT FEATURES D URING BASIC AND ADAP TIVE LOCOMOTION FOLLOWING SPINAL HEMISECTION I N THE CAT Introduction Although it is generally understood that human incomplete spinal cord injuries (SCI) like Brown Sequard Syndrome (BSS) result in substantial functional recovery the assessment of this recovery is typically limited to basic locomotion such as walking on a flat, unimpeded surface at a comfortable speed (Schwab a nd Bartholdi, 1996; Dietz et al., 1998; Rossignol et al., 1999) Reports in both feline incomplete SCI models (Helgren and Goldberger, 1993; Tester and Howland, 2008; Jefferson et al., 2011) and some patients with incomplete SCI (Barbeau et al., 2002; Capaday, 2002; Ladouceur et al., 2003) indicate that more difficult locomotor tasks requiring adaptation s of limb trajectories and balance responses lead to poorer recovery G ait features necessary for the performance of these more complex locomotor tasks have not been thoroughly examined. There is no standard battery to assess the adaptive features necessary oals after an SCI. In the current study, spatiotemporal features, ipsilateral limb targeting and maintenance, hind to hindlimb coordination, and trunk and distal limb control were determined both before and after a low thoracic spinal hemisection in cats as they crossed a flat 30.5 cm wide overground walkway requiring minimal alterations to the basic intraspinally controlled stepping pattern. These features also were determined for the same animals as they were challenged to cross a 30.5 cm wide horizontal ladder requiring greater supraspinal input, limb accuracy, enhanced balance, and postural stability (Bolton et al., 2006; Beloozerova et al., 2010a) With different neural control mechanisms contributing to succes sful locomotion across 1) a wide level pathway and 2) a horizontal ladder, the


45 goal of this work was to determine the necessary components of recovery based upon specific task requirements and demands. Developing a test battery that can effectively identi fy differences in recovery patterns across locomotor tasks will be valuable in assessing new interventions and therapies promoting recov ery after motor incomplete SCI. Methods All procedures involving animals were performed in agreement with the NIH guide lines for the care and use of experimental animals, which were approved by both the M alcom Randall VA Medical Center and the University of Florida Institutional Animal Care and Use Committees. A total of six cats, with similar lesions, were used in this s tudy to characterize performance and recovery of basic walking on an overground walkway and adaptive locomotor features required for crossing of a horizontal ladder. Subjects Cats were purpose bred, SPF, spayed, adult, females Spays were performed to remove potential hormonal effects on lesion magnitude and behavioral performance (For review see Sribnick et al., 2003; Sribnick et al., 2005) Prior to injury, all animals we re trained to perform a number of task s as in our previous studies (Tester and Howland, 2008) Performance of two of these tasks, 30.5 cm wide, flat runway (overgr ound walkway), and a 30.5 cm wide horizontal ladder were assessed for the current study. Once cats were able to consistently perform all tasks baseline data was collected Following injuries, training continued and performances were recorded periodicall y across five months


46 Surgical Procedures T10 s pinal h emisection Detailed surgical procedures and post op care are detailed in prior reports (Howland et al., 1995b, a; Tester and Howland, 2008) In brief, fascia and musculature were cleared from T10, after which a laminectomy was performed. The left half of the spinal cord was cut using iridectomy scissors. D urafilm and gelfoam were placed over the lesion site and muscle and skin were closed with absorbable suture s Behavioral T asks T raining P aradigm and Gait Features C ats were conditioned to perform locomotor tasks for food r ewards. Tasks ranged from simple to challenging: Bipedal (hindlimb) stepping on a treadmill and crossing of a 30.5 cm wide overground walkway horizontal ladder, peg walkway, and 5 cm wide narrow beam (refer to Tester and Howland, 2008 for full description of tasks) Training continued post SCI, beginning the second day after injury Specifically, cats were trained to step bipedal ly (hindlimbs) on a treadmill in addition to on e of the other tasks, which alter nated equally every day f or 22 35 weeks after injury. The current study focused on performances on the basic overground runway (30.5 cm wide x 4.5m long), and the horizontal ladder (30.5 cm wide x 4.5 m long, with rungs 2.5 cm wide and spaced 15 cm apart ) (Figure 2 1). Overall assessment of locomotor r ecovery A daily log was kept to qualitatively monitor recovery and changes in locomotor characteristics. For quantitative assessment, animals were filmed on each of the tasks usin g a 3D pan and tilt system ( Peak Vicon ) at 60Hz. Baseline data was collected prior to injury. Post injury, performances were filmed at two weeks, 4 weeks and then month ly for at least 20 weeks Behavioral assessments included onset of task


47 recovery, limb accuracy, paw placement on ladder rungs (phalanges versus metatarsal), step cycle duration, stride length, footfall patterns, % of stepcycle spent in stance (%stance), double support period, iliac crest mediolateral movement, interval between toe down and positive support, and ma ximum ankle flexion at yield. These gait horizontal ladder at pre injury, four, and twenty weeks post injury. Spatiotemporal analyses were based on 10 steps taken from crossin gs of similar speeds and were assessed using Motus Software (Vicon Peak ). Overground runway analysis for one of the cats was collected at 22 weeks post injury due to technical issues with filming at 20 weeks. Onset of recovery Onset of recovery was defi ned as the number of days for cats to recover the ability to perform three crossings independently. Limb accuracy The number of times each cat w as able to accurately place and maintain its ipsilateral hindlimb on a from the three best crossings pre injury as well as four and twenty weeks post injury. These crossing were chosen based upon speed and best performance. Crossing speed Crossing speeds were calculated using markers set ~1/2 meter from the end of each walkway and the time required to traverse this known distance (meters/second; m/s). Exclusion of runway ends from these calculations removed acceleration and deceleration affects which occur at the beginning and end of each runway cr ossing,


48 respectively. Crossings used for this assessment were those in which the cat was crossing at its typical speed. Percentage of abnormal foot positioning on rungs Cats normally walk on their toes (phalanges) during crossing of the basic overground walkway and the horizontal ladder. However, after injury cats also positioned their ipsilateral paw abnormally on the rung, on the metatarsals. For each animal it was determined whether they used an abnormal paw placement and if so, the percentage of ti mes they abnormally placed was calculated. Step cycle d uration The number of fields for a single ipsilateral hindlimb step cycle to occur was determined, and then converted to seconds, with each field being equivalent to 0.0166 seconds. The cycle s /secon d was then calculated and averaged. Stride length Stride length was defined as the length between toe touch downs of the ipsilateral calculated in Peak Motus, which measu res movement in the horizontal axis with respect to the camera, located perpendicular to the walkway. Footfall patterns T he number of frames in which the contralateral and ipsilateral hindlimb s were in stance (limb in contact with the walking surface) and swing while walking across the overground walkway and horizontal ladder were determined and depicted in diagram form to determine changes in interlimb coordination For the horizontal ladder, each rung was assigned a number based on the first rung steppe


49 was recorded onto the footfall pattern diagram. This allowed for an understanding of both the rung skipping pattern of cats prior to and after injury, as well as how often or infr equently cats paired hindlimbs onto the same rung. Stance percentage The number of fields the ipsilateral and contralateral hindlimbs were in stance were separately determined for ten step cycles, then the total number of fields for each of the steps for each separate hindlimb was determined. The percentage of fields that were in stance as compared to the entire step was calculated for each hindlimb. Double support period The number of fields in which the right and left hindlimbs were in stance together was determined and conve rted to seconds, with each f ield equaling 0.0166 seconds. Iliac crest mediolateral movement the field of view plane, the greatest value of the ipsilateral iliac crest in the left direction iliac crest in the right direction for the swing and stance phases, separately. Duration to support after toe down The maximum ankle f lexion occurring after initiation of stance (toe down) was determined. Then, the number of fields taken to reach the point at which the limb began to extend after this maximum flexion was determined. This was then converted to seconds, with frames being e quivalent to 0.0166 seconds. Ankle flexion during yield after toe down The maximum ankle flexion occurring after initiation of stance (toe down) was determined.


50 Tissue Processing Details of these procedures are described in our previous work (Tester and Howland, 2008) Perfusions and tissue preparation At 22 35 weeks after injury cats were deeply anesthetized with an overdo s e of sodium pentobarbital (>40mg/kg, i.p. ) Any supplement al doses were given I.V. H eparin (1cc;1000U/i.v.) was then administered and 20 minutes later 1cc of 1% sodium nitrite IV given Im mediately following injection of sodium nitrite, cats were transca rdially perfused with 0.9% saline, followed by 4% paraformaldehyde in 0.1M phosphate buffer (pH 7.4). T he spinal cords were dissected, blocked, and post fixed in 30% sucrose and 4% paraformaldehyde (pH 7.4). Lesion blocks were cut on a cryostat at 25 m s and stored in 0.1 M Phosphate Buffered Saline ( PBS ; pH 7.2, saline 0.9%) at 4 Celsius. Histology: Cresyl violet and myelin s taining Every 10 th section through the lesion block was mounted onto a Superfrost/Plus slide (Fisher Scientific ) subbed with chrom alum and poly L lysine ( c hromium potassium sulfate and poly L lysine, Sigma Aldrich ; gelatin, Fisher Scientific ). Sections were fume fixed on the slides with 4% paraformaldehyde. Tissue was rinsed in water, then placed in increasing alcohol concentrations (70 100%, 6 10 mins eac h) and finally, in xylene (10+ mins) Tissue then was rehydrated in decreasing alcohol concentrations, placed in myelin dye (Eriochrome Cyanine R; Fluka ; 10 min s) followed by dye differentiation in 1% ammonium hydr oxide. After extensive rinsing, slides w ere place d in 0.5% cresyl violet (cresyl violet with acetate, Sigma Aldrich ) for three minutes followed by differentiation with 1% glacial acetic acid in 75% alcohol Tissue was then


51 completely dehydrated in increasing alcohol concentrations, followed by xylene (10+ mins), and coverslipped with DPX (Fluka ). Statistical analysis Using Analyse It Software (Microsoft Excel ), the Kruskal Wallis test was bonferroni corrected in order to compare crossing speeds within tasks and across time points. The Friedman test was used to compare the task onsets for bo th tasks as w ell as the pre injury val ues across both tasks for crossing speed The Mann Whitney U test was used to compare metatarsal placement percentage and success across the different groups of cats. A p value of <0.05 was set to determine significance. Results Lesion Magnitudes Cresyl violet and myelin stained sections through the lesion epicenters of each animal were used to determine the extent of tissue sparing and damage. The represent ative cross sectional drawing created for each animal from review of multiple sections, showed that the lesions of the six cats were very similar (Figure 2 1). Typically, complete disruption of the ipsilateral gray and white matter was seen with an except ion in one cat. Contralateral gray and white matter was completely spared with the exception of some superficial white matter dorsally in four of the six cats. Thus, lesion variability was minimal. The similarity in lesion magnitudes minimized the possi bility of differences in functional recovery being related to differences in spared tracts. Recovery Onset and Effects of Injury on Crossing Speed Performance both prior to, and after a thoracic hemisection was compared on the overground walkway (Figure 2 2A ) and horizontal ladder ( Figure 2 2B ). Acutely


52 following injury, all cats showed the expected ipsilateral hindlimb flaccidity followed by some voluntary movement within 48 hours. During this period, the contralateral hindlimb showed good range of motio n and substantial or full weight support. On average, cats began independently crossing the overground walkway by thre e days after injury (Figure 2 2 C ). During these crossings, the ipsilateral hindlimb began showing partial stepping movements. Collectiv ely, these results are consistent with our prior works and the reports of others (Eidelberg et al., 1986; Helgren and Goldberger, 1993; Tester and Howland, 2008; Jefferson et al., 2011) The ability to independentl y cross the ladder occurred significantly later than on the overground walkway ( Figure 2 2C; p=0. 014, Friedman t est) On average, cats began crossing the horizontal ladder without assistance at 18 days post SCI. This was two weeks later t han on the overg round walkway. The range in initiation of horizontal ladder crossing (15 25 days) also was much greater tha n seen for overground walkway ( two eight days). Prior to injury, the typical comfortable crossing speed for all cats w ere significan tly faster on the overground walkway than th e horizontal ladder (Figure 2 2 D ; p=0.0 1 4 F riedman test) which is consistent with studies of normal cats (Beloozerova et al. 2010a) After injury, all crossing speeds were significantly slower from pre injury on the overground walkway at both four (Figure 2 2D; p=0.0 30 Kruskal Wallis test ) and 20 weeks after injury (Figure 2 2 D ; p=0.0 03 Kruskal Wallis test), and on the horizontal ladder at both four (Figure 2 2D; p= 006, Kruskal Wallis test) and 20 weeks after injury (Fig ure 2 2 D ; p=0.0 03 Kruskal Wallis test). Evaluation of the absolute changes in crossing speeds from pre injury values was not significantly different between the locomotor tasks at either post injury time points (Figure 2 2 E ).


53 Ph alangeal v ersus Abnormal Metatarsal Placement onto Ladder Rungs Post Spinal Cord Injury Pre injury, all cats effectively placed their hindlimbs on the ladder rungs. This ability was disrupted for the ipsilateral hindlimb following injury. Over time, the ability to place the ipsilateral hindlimb began to recover. By 20 weeks cats showed an average contactt rate (contact with rung) of 69.3% and an average effective placement rate (contact and maintenance of limb positio n on rung) of 56.7% (Figure 2 3 A ). S imilar to crossing of the peg walkway described in our prior work (Jefferson et al., 2011) cats crossed the horizontal ladder using either a three or four limb strategy. Typically, a three limb strate gy was employed early after injury when cats lacked the ability to accurately target with their ipsilateral hindlimbs. However, as recovery continued, animals began to incorporate their ipsilateral limbs more frequently and a four limb strategy emerged in some animals. Half of the cats evaluated (3/6) were capable of effectively placing and maintaining their ipsilateral hindlimb >75% of the time on the ladder rungs by 20 weeks (75%, 100%, 100%) T he other three cats were only able to contact and maintain their ipsilateral hindlimb onto a ladder rung <40% of the time at this chronic time point (12%, 13%, 40%). Thus, the cats fell out into two groups; Group 1 with ineffective limb placements and Group 2 with effective limb placements. To determine potential differences that might contribute to accurate paw placement and maintenance, frame by frame analyses of toe touch down through weight bearing was done for both groups of cats. P rior to injury, animals stepped onto and weight supported on ladder rungs wit h their phalanges ( on their toes; Figure 2 3 B ). After injury two alternate patterns emerged. The first pattern, which occurred less often, consisted of the paw not being brought forward enough and placing on the tip of a


54 single phalanx. The second, mor e predominant pattern was placement onto the metatarsals (MT) (Figure 2 3 C ). The percent of paw placements characterized by positioning on the metatarsals (% MT) of all placements showed a similar range in the lower (Group 1) and higher (Group 2) paw placement performing groups at four (group 1=36 100%, group 2=23 69%) and 20 weeks (group 1=38 85%, group 2=17 95%) (Fi gure 2 3 D,E ) However, there was a distinct difference in the maintain this abnormal placement on the rung as the limb b egan to weight bear. At four weeks Group 2 cats were able to maintain their ipsilateral hind paws on the rung s during a significantly greater percent of the placements with abnormal metatarsal positioning (% MT Success) compared to Group 1 cats (Figure 2 3 ; p=0.05, Mann Whitney U test ). This effect continued to the 20 week post injury period. These results suggest greater deficits, possibly proprioceptive, in the Group 1 cats. To further understand crossing recovery of more highly functioning animals ch aracterized by effective paw placement on the horizontal ladder, the remaining analyses focused on the Group 2 cats as they provided a sufficient number of contiguous ipsilateral steps for evaluation. The lesion epicenters of the Group 2 cats are shown in the second row of F igure 2 1. Injury Effects on Cycle Duration and Stride Length To understand what step cycle features might contribute to slower crossing speeds, cycle duration and stride length of both the contralateral and ipsilateral hindlimbs wer e assessed. Prior to injury, and consistent with normal control results from Beloozerova and colleagues, there were no differences in cycle duration between crossings of the overground walkway an d horizontal ladder (Figure 2 4 A ) (Beloozerova et al., 2010a)


55 reported to be longer on the ladder compared to overground (Bolton et al., 2006) At four weeks after injury, all three cats showed changes in thei r cycle durations (Figure 2 4 B ). During overground the direction of this change (faster or slower) wa s mixed across cats (Figure 2 4 B ). In contrast, all had slower cycle durations on the horizontal ladder. At 20 weeks on overground walkway, cycle duration had almost returned to normal with the absolute change in cycle duration approaching zero. The direction of even this minimal change continued to be different across cats at this time point. In contrast on the horizontal ladder, all cats continued to show slower cycle durations compared to pr e injury and the average absolute change in cycle duration from pre injury was greater on horizontal ladder compared to on the overground walkway (Figure 2 4 B ). Pre injury, stride length was shorter on horizontal ladder compared to overground walkway (Fig ure 2 4 C ). This was most likely a result of rung spacing on horizontal ladder. At four weeks after SCI, some cats had shorter stride lengths, while others had longer ones on both tasks. At 20 weeks, all cats had shorter stride lengths on overground walk way, while on horizontal ladder there continued to be variability in the direction of change. There was no difference in absolute change from pre injury across tasks (Figure 2 4 D ). Effects of Injury on Interlimb Coordination, Stance Duration, and Double Support Pre injury, stepping patterns of the hindlimbs on the overground walkway and horizontal la dder were similar (Figure 2 5 A,B ) which is consistent with reports in a normal, uninjured system (Beloozerova et al., 2010b) Both right and left hindlimbs alternate in a 1:1 fashion. Ho wever, after an SCI distinct differences were seen between overground and ladder crossing patterns. On overground walkway, the 1:1 pattern primarily was preserved at both four (Figure 2 5C) and 20 w eeks after injury


56 (Figure 2 5 D ); however this pattern was disrupt ed on the ladder (Figure 2 5 E J ). At four weeks the contralateral hindlimb of all three cats had a higher stepping frequency relative to the ipsilateral hindlimb. The stance phases of both limbs were generally increased and there was a greater ov erlap of th e stance phases (Figure 2 5 E G ). By 20 weeks, a 1:1 stepping ratio was recovered in on e of the three cats (Figure 2 5 H ), while the other two adopted a 2 contralateral:1 ipsilateral ratio (Figure 2 5 I,J ). The stance phase of the contralateral h indlimb remained incr eased in all cats (Figure 2 5 H J ). Assessment of the rungs used during horizontal ladder crossing identified further alterations in hind to hindlimb coordination after injury. Prior to injury, each cat placed their hindlimbs onto ever y third rung, skipping over two with each swing phase. Additionally, the ipsilateral and contralateral hindlimbs were placed on different rungs (F igure 2 5 B ). After injury, this pattern was lost. At four weeks, the contralateral hindlimb switched to a r elatively consistent pattern of stepping on every second rung, skipping over one. This pattern was seen in each o f the three cats (Figure 2 5 E G ). The ipsilateral hindlimb in all three animals inconsistently made contact on every three to five rungs, skip ping over two to four rungs, but primarily missed the rungs duri ng these attempts (Figure 2 5 E G ). Cats were most successful at accurately placing when their ipsilateral hindlimb targeted every other rung, skipping only a single rung. Cats frequently step ped onto the same rung with both hindlimbs which was not seen pre injury (Figure 2 5 B ). By 20 weeks post injury, unique patterns in regards to hind to hindlimb coordination and rung placements were seen in each cat. In the first example shown (Figure 2 5 H ), the cat uses a 1:1 stepping ratio similar to what was seen pre injury.


57 However, the hind to hindlimb coordination differed greatly from pre injury; the contralateral and ipsilateral hindlimbs placed onto every other rung, used the same rungs, and typ ically shared a rung during part of the support phase. The contralateral hindlimb strategy seen at four weeks continued to be used at 20 weeks post injury, placing on every other rung, skipping one. The two other cats used a different approach for crossi ng the horizontal ladder (Figure 2 5 I,J ). Their approach showed similarities to the pattern seen at four weeks post injury. Between two and four rungs were skipped by the ipsilateral hindimbs (placing onto every third to sixth rung). However, in contra st to similar attempts made at four weeks post injury, they were successful in accurately placing their ipsilateral hindlimb onto the rungs most of the time. The footfall pattern of the contralateral hindlimb remained identical to the pattern exhibited at four weeks post injury, placing onto every second rung and skipping only one rung. This footfall pattern showed frequent sharing of a rung with the contralateral hindlimb. Collectively, these results show that novel, yet consistent foot placement strateg ies and hind to hindlimb coordination emerged during recovery of horizontal ladder crossing in these three cats. Different cats used different strategies demonstrating that there are multiple ways this task can be successfully completed after injury. Qual itatively, the footfall pattern analysis suggested there were changes in the support (stance) phase on both locomotor tasks. To assess this, stance as a percentage of the step cycle and hindlimb double support periods were determined. Prior to injury, th e percent of time each hindlimb spent in stance over the course of an entire step was similar within a task, but different across the tasks. During horizontal ladder crossing, both hindlimbs showed increased periods of stance relative to


58 overground walkwa y in nor mal, uninjured cats (Figure 2 6 A ). This increase was consistent with a greater double support period during horizontal ladder compared to crossings of the overground walkway (Figure 2 6 C ). Four weeks post injury, the contralateral hindlimb stance period increased in all three animals during both overground and ladder compared to pre injury ( Figure 2 6 B ). This was in contrast to the ipsilateral hindlimb which showed relatively little absolute change on either task ( Figure 2 6 B ). The direction of t hat change from pre injury was increased in all cats in the contralateral hindlimb, but was increased in some cats and decreased in others in the ipsilateral hindlimb. At 20 weeks post injury, this trend continues with the absolute change in stance perio d from pre injury remaining higher in the contralateral hindlimb compared to the ipsilateral hindlimb. Pre injury, the period of double support was greater during ladder crossing than on th e overground runway (Figure 2 6 C ). Following injury, the absolute change in the double support period of the hindlimbs showed a very small increase during overground walking. The double support period and absolute change was much greater during ladder crossings compared to overground walkway locomotion at both four and 20 weeks post injury (Figure 2 6 D ). Mediolateral Movement To determine the effects of SCI on proximal stability, and how this differs across basic and skilled tasks, the change in the range of mediolateral movement of the top of iliac crest was assessed d uring both overground walkway and horizontal ladder crossings. Prior to injury, mediolateral movement was similar across the two tasks during both the stanc e and swing periods (Figure 2 7 A ). Movement was tightly controlled within a ~1.5 2 cm range. Foll owing injury, a ~25% and 50% change in the


59 range was seen during stance and swing respectively on o verground walkway (Figure 2 7 A ). While all cats had increased mediolateral movement at 4 weeks during stance, the change in direction from pre injury varied across cats during swing at 4 weeks post injury, and both swing and stance at 20 weeks after injury. In contrast, mediolateral movement was greater during horizontal ladder crossing compared to overground walkway. This suggests that the cats have much g reater difficulty controlling the midline position of their hindquarters during horizontal ladder crossing post injury in comparison to traversing the overground walkway. Additionally, in contrast to behaviors on the overground walkway, all cats had the same direction of change from pre injury on horizontal ladder at both timepoints and phases of the step cycle ( Figure 2 7B). Interestingly, while the greatest change from pre injury occurred during the swing phase on overground walkway, it occurred durin g the stance phase on horizontal ladder ( Figure 2 7B). Closer, frame by frame examination of this effect on horizontal ladder showed that the majority of iliac crest movement occurred as the contralateral hindlimb was in swing; the entire trunk would shif t to the contralateral side. These findings further suggest the importance of the contralateral hindlimb during the performance of the horizontal ladder after injury. Distal Joint Stability Distal control of the limb was assessed in two ways: 1) assessme nt of the period duration between toe touchdown (stance initiation) and initiation of positive support (indicated by ankle extension) and; 2) maximum ankle flexion at yield. Prior to injury, there was no significant difference in the period duration from toe touchdown to positive support between the overground walkway and horizontal ladder tasks (Figure 2 8A). After injury, some cats increase d the duration to weight support and others decreased


60 this period on overground walkway, while on horizontal ladder all cats increased this duration. The greatest average absolute changes however, were seen during overground walkway crossing. The magnitude of the changes was greater at 20 weeks post injury on the overground walkway compared to the horizontal ladder ( Figure 2 8B). Prior to injury, maximum ankle flexion during yield just prior to weight support was similar during the overground walkway and horizontal ladder tasks (Figure 2 8C). After injury, ankle angular degrees increased in some cats while decreasing in others during crossing of the overground walkway. In contrast, all cats showed an increase in ankle angular degrees while crossing the horizontal ladder at both four and 20 weeks after injury, indicating a decrease in ankle flexion at these time point s. The change from pre injury was decreased on horizontal ladder compared to the overground walkway at 20 weeks (Figure 2 8D). Discussion Overall, results from this study demonstrate that there are several differences across the performance of overgroun d walkway and horizontal ladder. Several features were different across these two tasks in the normal cat prior to injury. These features include crossing speed, stride length, stance percentage, and double support period. For some of these features, li ke crossing speed and double support period, SCI resulted in an amplification of these differences across tasks. The differences seen in stride length prior to injury remained similar after injury. Some of the greatest changes seen after injury occurred in the contralateral hindlimb. Prior to injury, stance percentage on horizontal ladder was longer in both hindlimbs compared to overground walkway. This effect was lost after injury in the ipsilateral hindlimb, but magnified in the contralateral


61 hindlimb resulting in extreme asymmetry across these two limbs. This asymmetry was amplified on the horizontal ladder task. There also were several gait features that were similar across tasks prior to injury but became different after injury. These features include the cycle period, footfall patterns, mediolateral movement, and distal joint stability. After SCI, the cycle period became decreased in all cats on horizontal ladder but had a less drastic change on the overground walkway, with the cycle period in creasing in some and decreasing in others. Footfall patterns after injury remained similar to what they were pre injury for overground walkway, but changed drastically on the horizontal ladder. Mediolateral movement became more enhanced on the horizontal ladder after injury to a much greater extent than on the overground walkway, with a similar effect occurring for the duration to positive support in the ankle. While maximum ankle joint flexion during yield became less flexed on the horizontal ladder in all cats, it was differentially affected on overground walkway, with some cats increasing in flexion and other decreasing. Task Specific Spatiotemporal Gait Changes after Injury Crossing of the horizontal ladder is accomplished more slowly than on the ove rground walkway. This reflects the difficulty of the tasks and the greater supraspinal contributions. In particular, ladder requires both cortico (Metz and Whishaw, 2002; Beloozerova et al., 2010b) and rubro sp inal input (Webb and Muir, 2003) which are known to play a c ritical role in more skillful types of locomotion. Although, there were no significant differences between the mean cycle duration across tasks, a significant decrease in stride length was seen on ladder compared to overground, indicating that on the hor izontal ladder task, cats travel shorter distances in the same time period it took to travel longer distances on the overground walkway. This is most likely related to


62 the spacing of the ladder rungs, as they restrict the positioning of the paws, affectin g stride length After injury, both crossing speed and cycle duration decreased during both tasks, but were more pronounced on the horizontal ladder. These enhanced decreases on horizontal ladder were accompanied by variable changes (increases and decr eases) across cats in stride length on the horizontal ladder. In contrast, all cats had a decreased stride length on the overground walkway by 20 weeks. This decrease in stride length on overground walkway is a well documented affect after SCI (Barbeau et al., 1999) as well as other neural injuries (von Schroeder et al., 1995) and is typically associated with a decrease in crossing speed (Barbeau et al., 1999) The differential effects on speed and cycle duration across the two tasks most likely relates to the drastic changes in footfall patterns that occur in p arallel as footfall patterns have been shown to heavily affect step length and leg propulsion (Balasubramanian et al., 2010) The novel foot fall pattern that emerges after injury on ladder facilitates increased use of the uninjured limb for support, both singly, and in double support. Specifically, ladder crossings were characterized by significantly longer periods of support relative to swing, in addition to longer periods of double support compared to that seen during overground walking. These features would enhance balance and postural control more greatly during horizontal ladder than overground walkway performance (Hazime et al., 2012) Trunk Support on S killed Tasks after Injury The enhanced mediolateral movement of the ipsilateral iliac crest particularly during stance while the contralateral hindlimb is in swing, may be related to changes in muscle activity after injury. For example, several studies have reported that active flexio n of the hip during stance is responsible for increased hip and knee flexion of the


63 other limb in swing (Patla and Prentice, 1995) Furthermore, others have reported an increase in the activity of hip abductors and adductors activity, specifically the tensor fascia latae and adductor longus, as a way to provide additiona l pelvic stability and support of thigh acceleration during late stance and early swing of uninjured populations while ramp or stair walking (Gottsc hall et al., 2011) The rectus femoris, gluteus maximus, and contralateral obliques, which are hip and postural muscles, also have been shown to become significantly peaked during perturbed stance (Stanek et al., 2 011) In combination, these results suggesting that the increase in mediolateral movement to the contralateral side may be a result of contralateral hip overcompensation. By compensating for the weakened, affected muscles of the ipsilateral limb, the co ntralateral limb helps to produce appropriate gait features which allow successful completion of the ladder task. Indeed, it has been reported that the musculature of the contralateral side recovers substantially quicker than the ipsilateral side after in jury (Little and Halar, 1985) Task Specific Changes in Distal Joint Positioning after Injury Changes in ankle position after injury, particularly on t he horizontal ladder, also may be related to task specific changes in muscle activity after injury. For example, the lateral gastrocnemius, an ankle extensor and knee flexor, has been shown to have increased activity during ladder in uninjured cats just p rior to the beginning of stance, and early stance (Beloozerova et al., 2010b) Additionally, the ankle plantar flexors gas trocnemius and soleus, also have been found to provide trunk support during single leg stance (Neptune et al., 2001) suggesting that prolonged activity of the ankle joint immediately after toe touchdown may be representative of a heightened need to maintain balance and postural control for target accur acy on the more challenging


64 ladder task. The lack of maximum ankle flexion as seen during overground crossings may not necessarily suggest normalized ankle function, but instead may be a result of increased lateral gastrocnemius, ankle extensor activity n ecessary for proficient ladder performance. Alternatively, the significantly greater increase in maximum ankle flexion seen on overground after toe touchdown may be a speed related change as the cats cross overground faster than ladder. The enhanced capa bility for cats with a high ipsilateral hindlimb targeting percentage to maintain weight support on a ladder rung despite abnormal metatarsal paw placement compared to those with low targeting accuracy, may be related to some of the previously described ch anges in muscle activity. Specifically, enhanced activity of the lateral gastocnemius and soleus muscles, as described in Neptune et al., 2001, may be enhanced in these cats with higher accuracy. Furthermore, the inability to maintain weight support afte r abnormal paw placement in a subset of animals, suggests that these low accuracy animals may have issues with proprioception (Witchalls et al., 2012) Conclusions Collectively, the results from this study characterize several task specific spatiotemporal, trunk, and distal stability features prior to injury, as we ll as task specific changes to these features after injury. These findings suggest that maximization of functional recovery in incomplete SCI populations may require a strong focus on certain functional components of the hip and ankle, like increasing str ength of the plantarflexors as well as the musculature of the hip, like the rectus femoris and gluteus maximus. Subsequently, the increased use of the contralateral hindlimb during successful ladder accuracy suggests an important facilitator role of this limb; BSS and other incomplete


65 SCI populations may strongly benefit from therapeutic strategies that focus on the contralateral limb as much as they do the more greatly affected limb.


66 Figure 2 1. Lesion magnitudes. The amount of spared tissue a t the lesion epicenter for all six cats included in the study Scale bar is 1mm.


67 Figure 2 2. Tasks, onset of recovery, and crossing speeds. Images of the overground walkway (A) and horizontal ladder task (B), as well as the number of days required to re cover these tasks after injury (C) were assessed. Pre injury crossing speeds (D, gray bars), post injury crossing speeds at four and 20wks after injury (D, white bars), and the absolute change in cros sing speeds from pre injury at four and 20 weeks (E) wer e compared (p=<0.05). The black bars (E) indicate when all animals had decreased values from pre injury.


68 Figure 2 3. Phalangeal versus metatarsal placement onto ladder rungs. Post injury, cats lose the ability to place and maintain their ipsilateral p aw on the ladder rungs. Overtime, this shows some recovery (A). Normal, uninjured cats step onto ladder rungs with the phalangeal portion of their foot (B). However, after injury cats also used an abnormal placement approach, placing their foot onto the rung with the metatarsal (MT) portion of their foot (C). Cats were separated into groups based on their ability to accurately target and maintain their ipsilateral hindlimb on the rung (irrelevant of type of placement; phalangeal or metarsal). Group 1 consist ed of cats able to accurately target and maintain ipsilateral hindlimb placement between 12 40% of the time by 20 weeks post injury. Group 2 consisted of those cats able to accurately target and maintain ipsilateral hindlimb placement between 75 to 100% o f the time by 20 weeks post injury. The percentage of times cats used a metatarsal approach with their ipsilateral hindlimb when crossing the horizontal ladder (% MT) was compared across Group 1 and Group 2 cats at 4 (D) and 20 (E) weeks after injury. The percentage of times the ipsilateral hindlimb succes sfully remained on the rung despite a metatarsal placement (% MT Success) also was compared across these groups at both post injury time


69 Figure 2 4. Changes in cycle duration and stride length. The average step cycle durations (A ) and stride lengths (C) on overground walkway and horizontal ladder were determined prior to injury (gray bars), and at four and 20 weeks after injury (white bars). The absolute change f rom these pre injury values at four and 20 weeks post injury also wa s determined for the cycle duration (B) and stride length (D). For absolute change graphs, black bars indicate when all animals showed decreased values from pre injury, and bars with a black/white gradient indicate when some cats increased, and some decre ased from pre injury.


70 Figure 2 5. Change in hindlimb footfall patterns and hind to hindlimb coordination. The timing of left (ipsi) and right (contra) hindlimb foot placements on overground walkway are depicted for a representative cat at pre injury (A), 4 weeks post injury (C), and 20 weeks post injury (D). Black rectangles indicate when a limb is in stance, and spaces between these denote swing. Footfall patterns for a representative cat were depicted for the horizontal ladder crossing at pre injury (B) All cats performed similarly at this time point. The post injury footfall patterns for each of the three cats on horizontal ladder were depicted for 4 weeks post injury (E,F,G) and 20 weeks post injury (H,I,J). Numbers inside the black rectangles for the horizontal ladder task indicate the rung that cats were standing on. Numbers outside of the black rectangles indicate a rung that the cat attempted to place their hindlimb onto, but failed. White rectangles indicate a stance period in which both h indlimbs were on the same rung simultaneously.


71 Figure 2 6. Change in hindlimb stance and double support periods. The percentage of time/step that the cat was in stance for both tasks (A) was determined for pre injury on overground walkway and horizonta l ladder for the ipsilateral (Ipsi) and contralateral (Contra) hindlimbs (G, g ray bars). The same values at four weeks and 20 weeks post injury also were determined (white bars). The absolute change from pre injury was depicted for the ipsilateral a nd con tralateral hind limbs at four and 20 weeks after injury (B). For both tasks, the period at which the ipsilateral and contralateral hind limbs were in stance at the same time (double support period) were determined prior to i njury (C, gray bars), and also four and 20 weeks after injury (C, white bars). The abso lute change from pre injury at four and 20 weeks also was determined (D). For graphs showing absolute change (B,D) white bars indicate when all animals had an increased change from pre injury, and b ars with a black/white gradient indicate when some cats increased, and some decreased from pre injury.


72 Figure 2 7. deviations during stance and swing were compared across ov erground and horizontal ladder perform ance pre inury (A, gray bars), four and 20 weeks after injury (A, white bars). The absolute change from pre injury also was determined from 4 and 20 weeks after injury (B). For graphs showing absolute change, white bars indicate when all animals had an increased change from pre injury, and bars with a black/white gradient indicate when some cats increased, and some decreased from pre injury.


73 Figure 2 8. Swing to stance transition The duration to positive suppo rt at the beginning of stance (A), and maximum ankle flexion at yield (C) were determi ned at pre injury (gray bars), four and 20 weeks after injury (white bars). The abso lute change from pre injury at four and 20 weeks after injury also was determined (B, D). For graphs describing absolute change white bars indicate when all animals had an increased change from pre injury, and bars with a black/white gradient indicate when some cats increased, and some decreased from pre injury.


74 CHAPTER 3 THE EFFECT OF D IFFERENT CHONDROITIN ASE ABC TREATMENT DU RATIONS ON LOCOMOTOR RECOVERY A ND S UPRASPINAL TRACT PLA STICITY I N A FELINE MODEL OF SCI Introduction Many studies (Jones et al., 2002; For review see Morgenstern et al., 20 02; Jones et al., 2003a; For review see Busch and Silver, 2007; Galtrey and Fawcett, 2007) including our own (Lemons et al., 1999; Tester and Howland, 2008) have shown that after spinal cord injury (SCI) there i s an upregulation of chondroitin sulfate proteoglycans (CSPGs) at the lesion site. Chondroitin sulfate glycosaminoglycans (CS GAG), which are attached to these CSPGs, are extremely inhibitory to axonal growth and play a primary role in preventing the axona l plasticity necessary for functional recovery (Snow et al., 1990; Smith Thomas et al., 1994; Zuo et al., 1998) R emoval of these CS GAGs using the bacterial enzyme c in rod ent models enhances both behavioral recovery (Bradbury et al., 2002; Cafferty et al., 2008) and axonal growth (Grimpe and Silver, 2004; Barritt et al., 2006; Houle et al., 2 006) The Howland laboratory has of SCI (Jefferson et al.; Tester and Howland, 2008) These studies showed enhanced locomotor recovery and plasticity of red n ucleus projections following a hemisection A multitude of studies completed in rodent models of SCI have shown beneficial effects using similar (Vavrek e t al., 2007; Lee et al., 2009) durations rang ing from a single injection to two weeks (Bradbury et al., 2002; Barritt et al., 2006; Cafferty et al., 2008; Garca Alas et al., 2008; Iseda et al., 2008; Garcia Alias et al., 2009) It is currently unknown how a shortened administration period will affect


75 locomotor recovery and anatomical plasticity i n a feline model of SCI and if four weeks of treatment is necessary. Cat s are a valuable tran slational they have similar pre dominant CS humans (4S and 6S disaccharides) while the rat shows a single dominant (4S disaccharide) lyase product (Tester and Howland, 2008) Thus, results from this study have meaningful implications for treatment duration in regards to the advancement of this therapeutic to a clinical setting. The upregulated temporal expression of CSP Gs after injury suggests that two recovery. In rats, peak upregulation of neurocan, brevican, and vers ican CSPG expression occurs at two weeks post injury, with peak neuron glial antigen 2 ( NG2 ) ex pr ession occurring at one week. Both neurocan and versican production then steadily decrease to only minimal expression by four weeks (Jones et al., 2002; Jones et al., 2003a) It is currently unknown whether th is same trend occurs in cats, but if so, due to the natural decrease of a substantial proportion of CS GAGs beginning at two weeks. Furthermore, an unpublished finding from our previous work was greater recovery of some locomotor features, like accurate targeting on a horizontal ladder, by two weeks post has already taken place by this two week time p administration may be unnecessary for continued functional improvements. In the


76 administration on rubro and cortico spinal plasticity, locomot or recovery, and the interaction of lesion size with treatment effects. Materials and Methods All animal procedures were conducted in accordance with the NIH guidelines for the care and use of experimental animals and were approved by both the M alcom Ran dall VA Medical Center s and University of Florida's Institutional Animal Care and with similar lesion magnitudes were used in this study treatmen t. All were trained 5x/week on 2 3 of 5 tasks daily Some cats were used only in the behavioral aspects of the study as they did not receive Fluorogold ( FG ) injections, or their injections did not meet inclusion criteria. Inclusion criteria were as follows: 1) F G l abeling at the injection site must be in both the ipsilateral and cont ralateral dorsolateral funiculi where the rubrospinal tract (RuST) and corticospinal tract ( CST ) traverse 2) FG labeled neurons in the non axotomized red nucleus or motor cortex must be easily detectable at 2 0X in order to be used for th ese analys e s The lesion ranking section of this study uses a sample of convenience (laboratory data bank) which incorporates 35 cats with varying lesion size s Only those that were trained similarly to the paradigm described for the two week treatment groups were included. Subjects All cats were purpose bred, SPF, spayed, adult, female cats Spays were performed to prevent any hormonally based behavioral changes during the study (For re view see Sribnick et al., 2003; Sribnick et al., 2005) Cats were placed into one of two groups : two week control (N=6) or two w ee ning of the


77 study, animals we re train ed to perform five tasks that they were consistently train ed and assessed on throughout the study, similar to Tester and Howland, 2008: 30.5 cm overground runway, bipedal treadmill, horizontal ladder peg walkway and narrow beam. Once cats were able to consistently perform the tasks they were filmed for baselin e data collection. Following injury, cats continued to be trained and were filmed performing e ach of the tasks regularly for five months. Cats then received intraspinal FG injections caudal to the lesion site for retrograde tract tracing. Following a 13 day survival period animals were transcardially perfused and lesion morphology, as well as axonal plasticity assessed. Surgical Procedures Low thoracic spinal hemisection A detailed description can be found in Tester and Howland 2008. Briefly, all cats received a left, T10 spinal hemisection created by iridectomy scissors. Following hemisection an injectable port body (Solomon Scientific San Antonio, TX) was glued and sutured subcutaneously to left dorsum musculature. Port tubing was positioned ov er the lesion site and dura sutured closed around tubing to maintain proper positioning within the lesion. Protease free treatment (1U/200 L) ( Seikagaku Corp oration Tokyo, Japan), or vehicle (Sterile saline or Tris HCL) were injected immediately after port placement in order to visually confirm proper functionality of the port system. A layer of durafilm and gelfoam were then placed over the lesion followed by muscle and skin sutured closed in layers. Chondroitinase ABC administration Prior to u se, enzymatic activity was tested and confirmed with fluorophore assisted carbohydrate electrophoresis (FACE) as described in (Tester et al., 2007)


78 (0.25 U in 50 L of saline or Tris HCL) or vehicle (Saline or Tris HCL) injections began the day cats were injured and continued every other day for two weeks. 50 L injections were made slowly at 0.14cc/s with a syringe pump Fluorogold spinal i njections Between 2 2 and 35 weeks following injury, nine o ut of the twelve cats underwent a second surgery to receive intraspinal injections of the retrograde tracer F luorogold (FG) caudal to the lesion site. The details of this procedure are fully described in Jefferson et al ., 2011. In brief, 0.5% FG (F luoro chrome, LLC, CO) was mixed in sterile, de ionized water in an aseptic hood the morning of the procedure and kept on ice prior to use. The lesion site was re exposed, and regions of the spinal cord caudal to the lesion site also were exposed for injections The most caudal dural suture at the lesion site was located from the previous surgery and FG injections were made ~13mm caudal to this suture. Four injection sites were made in a staggered formation using a 33 gauge Hamilton syringe. Each injection s ite consisted of two FG dumps of 0.25 L in each, for a total of 2 L s. The exposed spinal cord was then protectively covered with durafilm and gelfoam, the muscle and skin closed in layers. A survival period of 13 days allowed for FG to travel r etrogradel y to the brainstem and cortex Post surgical care procedures are described in depth in previous studies (Howland et al., 199 5aa; 1995bb) Behavioral Procedures Behavioral tasks and training paradigm Prior to surgery, cats were conditioned to perform five tasks for a food r eward. Tasks range from simple to challenging: Bipedal treadmill (0.5m/s) 30.5 cm wide overground runwa y, horizontal ladder, peg walkway, and 5 cm wide narrow beam (for detailed


79 description of tasks go to Tester and Howland 2008) Cats were trained 5x/week. Specifically, each day consisted of training on bipedal treadmill as well as one of the other tasks, which were alternated equally. Within 48 hours following SCI, training continued in the same manner as previously described f or 22 35 weeks after injury. However, depending on motor function, the skilled tasks were exchanged for the 30.5 cm wide overgrou nd runway during the initial week after injury. Assessment of locomotor r ecovery For qualitative assessment s performance. : the number of days for cats to recover the ability to perform a task after injury was recorded for 30.5 cm overground runway, bipedal treadmill, horizontal ladder, peg walkway, and narrow beam. For bipedal treadmill, this was the first day they took three consecutive, independent steps even if at overground runway, horizontal ladder, peg walkway, and narrow beam, this referred to the first day animals were able to make three independent crossings, consecutive or non consec utive. For quantitative assessment, animals were filmed on each of the tasks usin g a 3D pan and tilt system (Peak Vicon ). Filming occurred once prior to injury, as well as multiple times following SCI. For the first two month s post SCI, cats were filme d on each task every two weeks, then once a month for the remainder of the study. Horizontal ladder, peg walkway, and narrow b eam assessment: L imb accuracy also assessed for their ipsilateral hindlimb targeting accuracy. The percentage of times cats were able to accurately place their ipsilateral hindlimb onto a rung or peg was


80 calculated for each crossing. The average percentage from the three best crossings were computed a nd used for this comparison. Tissue Processing Perfusions and tissue preparation The details of this procedure are described in our previous work (Tester and Howland, 2008) In brief, at t hirteen days following FG injections, cats were deeply anesthetized with an overdo se of sodium pentobarbital (>40mg/kg, i.p. ) S upplemented doses were given i.v. as needed to ensure that animal s were properly sedated. Cats also were intravenously injected with 1cc heparin (1000U/i.v.), and 20 minutes later with 1cc of 1% sodium nitrite i.v. Immediately after, cats were transcardially perfused with 0.9% saline, followed by 4% paraformaldehyde i n 0.1M phosphate buffer (pH 7.4). T he spinal cord and brain were dissected, blocked into segments, and post fixed in 30% sucrose and 4% paraformaldehyde. The lesion, injection site, rostral midbrain, and medulla were cut on a cryostat at 25 m thick sectio ns, while the motor cortex was cut at 40 m Sections were stored in 0.1 M phosphate buffered saline ( PBS ; pH 7.2, saline 0.9%) at 4 Celsius until processed. Cresyl violet and myelin s taining This procedure is described in detail in our previous work (Tester and Howland, 2008) In brief, e very 10 th section of the lesion site was mounted onto colorfrost slides (Fisher Scientific ) subbed with chrom alum and poly L lysine (chromium potassium sulfate and poly L lysine, Sigma Aldrich ; gelatin, Fisher Scientific ). Tissue was fume fixed on the slides with 4% paraformaldehyde to enhance adherence Tissue was first dipped in water, and then dehydrated in incr easing increments of alcohol (five minutes each) After immersing in xylene (10 minutes) tissue was rehydrated in decreasing


81 increments of alcoho l and placed in an aqueous myelin dye (Eriochrome Cyanine R; Fluka ) for 10 minutes. After washing in water, differentiation took plac e in 1% ammonium hydroxide (one minute ) Tissue was then place d in 0.5% cresyl violet (cresyl violet with acetate, Sigma Aldrich ) for three minutes, and washed thoroughly in water, 70% a lcohol, and then diff erentiated in 95% alcohol with glacial acetic acid. Following this, tissue was dehydrated again in increa sing increments of alcohol and then immersed in xylene. Tissue was coverslipped with DPX (Fluka ). Assessment of injection site Every 40 th section of the injection site from animals that received FG injections was mounted on Superfrost/Plus slides (Fisher Scientific ) and coverslipped with Prolong Gold Antifade Reagent ( Molecular Probes ) Sections were assessed using fluorescent m icroscopy to ensure that the injection site reached the dorsolateral funiculi carrying the RUST and CST for both hemispheres of the cord. Those that did not have sufficient labeling were excluded from the tract tracing portion of the study. Immunohistoche mistry One, 25 m section, every 200ms through the red nucleus, and every 40 th section, every 640m s through the motor cortex were processed using the polyclonal anti FG antibody (1:10,000, Fluorochrome, LLC). This procedure is described in our previous study (Jefferson et al., 2011) In brief, sections were washed in a quenching solution ( 30% H 2 O 2 and PBS) for 30 minutes, followed by 2, 1 0 minutes rinses in 1% goat serum in PBS containing 0.4% Trit on X 100 (1% S PBS T) Tissue was then blocked in 5% S PBS T for one hour and then incubated in anti FG antibody overnight at room temperature. The next day, tissue was rinsed in 1% S PBS T and incubated in 5% biotinylated anti rabbit secondary antibody made in goat (Vector Laboratories ) for 1


82 hour. After a second rinse with 1% S PBS T tissue was incubated in avidin, biotin, complex ( ABC ) method (Vector Laboratories ) Following this incubation tissue was rinsed with PBS, then reacted with Diamino benzidine (DAB) brownish reaction product (Sigma Aldrich ) for 9 minutes. The DAB reaction was stopped with a PBS rinse. Tissue was subsequently mounted onto chrom alum and poly L lysine coated slides, and fume fixed in 4% paraformaldehyde for at least o ne hour. Tissue was then dipped in water and dehydrated in alcohol at incr easing increments at 5 minutes each Tissue then was placed in xylene for 5 minutes and coverslipped with DPX (Fluka ). Fluorogold labeled cell counts FG labeled neurons of each stained section in the red nucleus, and motor cortex were counted using a 20x microscope objective. Only those neurons with visible soma were included in the counts. The non axotomized cell co unts were separated from the axotomized cell counts. Additionally, the axotomized cell counts were taken as a percentage of the non axotomized cell counts to control for internal differences between cats (uptake of tracer, tissue processing, etc). Lesion r anking The lesion epicenters from 35 cats were stained with cresyl violet and myelin dye as described above T he control group consisted of nineteen cats, the two w group had seven cats, and the four w cats. Th ree individuals unfamiliar with the cats, but well versed in spinal cord morphology, ranked lesions based on the amou nt of total spared tissue with one being the lowest and 35 being the greatest amount of spared tissue. Individuals were asked to rank lesi ons that they felt were extremely similar with the same value. Each of the three individual sets of ranks were compared to ensure that r ankings were relatively similar and that rankers


83 properly understood and performed the task. The three sets of ranks w ere then averaged for each lesion and correlated against the onset of each of the five tasks, as well as the percentage of accurate ipsilateral hindlimb targeting at four, eight and 16 weeks post injury. Statistical Analysis Statistics were performed usin it statistical software (Microsoft Excel ) The Kruskall Wallis test was Bonferroni corrected and used to compare accurate ipsilateral hindlimb targeting across time points The Mann Whitney U test was used to assess group dif ferences and the S pearman test was used to correlate lesion rank and behavior. Results Lesion Magnitude s The lesions of 12 cats were stained with cresyl violet and myelin dye to determine the extent of their injury. Only thos e with similarly hemisected lesions were included in the part of the study assessing the effects of two Figure 3 1 shows the range of lesion sizes for the control (Figure 3 1A) (F igure 3 1B) groups. In both groups there was slight sparing present within the gray matter and ventromedial white matter. Additionally, both groups had lesion s that extended into the contralateral gray matter and ventrom edial white matter. Overall, the minor variations across within each group was equal across groups and did should not have skewed the results. Axonal Projections below the Lesion Site Both the RuST and CST tracts have been implicated in controllin g adaptive types of locomotion similar to the peg walkway and narrow beam (Morris et al.; Beloozerova


84 and Sirota, 1993; Lavoie and Drew, 2002; Pettersson et al., 2007) In a previous study y below the level of the lesion (Jefferson et al., 2011) however we have yet to address the effects of two ined were taken from the same animals used in our previous publication, with the addition of ing protocols as others in that group (Jefferson et al., 2011) In that study, only the percentages of axotomized to non axotomized FG labeled neurons were reported. Here, we report absolute neuronal counts as well, due to recent findings from our lab showing enhanced neuronal counts in both the axotomized and non axotomized red nuclei in control cats (Blum, 2010) This finding indicates that the non axotomized red nuclei/motor cortex may undergo more plastic changes than previously thought, and should be assessed independently. As expected, there were significantly more FG labeled neurons in th e non axotomized, contralateral side (Figure 3 2B ,E ) compared to the axotomized, ipsilateral red nu cleus (Figure 3 2A ,E ) in the two week control (Figure 3 2E; Mann Whitney U test, (Mann Whitney U test, p=0.05), four week control (M ann Whitney U test, p=0.01) as a result of the hemisection. The same was true for the motor cortex (Figure 3 2C,D,E; Mann l, labeled neuronal counts from the non axot omized red


85 nucleus were similar across the two and four w ee and their control counterparts. Absolute numbers from the axotomized side we re significantly higher in the two w ee red to their control counterpart (Mann Whitney U test, p=0.03), and the four w ee compared to their controls (Mann Whitney U test, p=0.03 ). had a signif icantly greater percentage of FG labeled neurons in the axotomized as compared to the non axotomized red nucleus compared to controls (Mann Whitney U test, p=0.03) (Jefferson et al., 2011) T here were no significant differences in raw FG labeled neurons or the percentage of axotomized versus non axotomized neuronal counts in the motor cortex between the (two or four weeks) (Figure 3 2 E) Rate of Recovery As described in earlier studies completed by the lab, ipsilateral hindlimb motor function was complet ely disrupted during the first one to two days after injury (Tester and Howland, 2008; Jeff erson et al., 2011) The contralateral hindlimb retained most of its function during this period, however there were some animals that could not fully weight support with this limb right away, but regained this ability within several days. There were no significant difference s regarding the recovery of basic locomotion runway typically began between one and three days following injury, though there were several cats t hat took longer (Figure 3 3A) Typically cats began stepping primarily with th eir contralateral hindlimb, while their ipsilateral hindlimb flexed and extended in a manner reminis cent of stepping, but with an extremely muted range of motion around


86 the joi nts. While the contralateral hindlimb of most cats was capable of full weight support, the ipsilateral hindlimb could only support a small proportion of their weight Surprisingly, recovery of bipedal treadmill stepping, which is controlled primarily by intraspinal circuitry, took significantly stepping, which involves more voluntary components (Figure 3 3A; Mann Whitney U Recovery of the skilled locomotor tasks : horiz ontal ladder, peg walkway, and narrow beam, took longer than basic locomotion as they require multiple supraspinally controlled locomotor features, including but not limited to balance, interlimb coordination, and accurate limb targeting (Figure 3 3B) (Whishaw et al., 1998; Metz and Whishaw, 2002) In our previous studies, we found that four weeks significantly increased the rate of recovery on each skilled task (Tester and Howland, 2008). Here, an effect. Horizontal ladder performance i s the easiest of t he skilled tasks to perform and was recovered significantly quicker than the peg walkway (Figure 3 3B; Mann Whitney, U 3B; Mann between the 2 nd and 3 rd week after injury Cats treated with 2 weeks recover thi s task (horizontal ladder) significantl y quicker than controls (Figure 3 3B; Mann Whitney U test, p=0.03) The time frames for recovery of peg walkway and narrow beam were substantially rol group, cats were able to recover peg walkway between 16 and 55 days post injury, and narrow beam


87 between 26 and 71 days post injury (Figure 3 was high variability across cats. While four out of the six cats rec overed peg walkway between 15 and 18 days after injury, two animals were never able to recover this task (Figure 3 more variable across cats compared to the control groups, ranging from 16 to 113 days after injury (3 3C,D). Despite these differences in variability across treatment groups, there were no significant differences in their average rate of recovery for these two tasks. However, the removal of these outliers led to a sign ificant different when comparing task onset of peg walkway but not narrow beam (Figure 3 3E Mann Whitney U test p=0. 02) Ipsilateral Hindlimb Accuracy Limb accuracy of the ipsilateral hindlimb is particularly d ifficult for animals to recover following i njury, and in fact many fail to do so on the more skilled tasks like peg walkway. In previous studies done by the lab it was showed that four administration resulted in a significantly greater ability for cats to accurately target with thei r ipsilateral hindlimb o nto a peg compared to controls (Tester and Howland, 2008; Jefferson et al., 2011) Here, we see that a shortened two does not significantly enhance limb accuracy over controls Horizontal l adder Pre injury, all cats were able to accurately place with their ipsilateral hindlimb onto a ladder rung 100% of the time, however after injury all animals lost this ability for at least several days (Figure 3 4A). By two weeks p ost injury, several cats had recovered cats), while only one animal in the control group had regained ipsilateral hindlimb


88 trend towards significantly greater percentage of ipsilateral hindlimb accuracy compared to controls at two weeks after injury (Figure 3 4A; Mann accuracy was transient and by four weeks the control anim als were performing equal to substantial recovery of ipsilateral hindlimb accuracy by eight weeks and we re targeting significantly more than at two weeks post injury (Kruskal This trend continued at 16 (Kruskal (Kruskal Narrow b eam Prior to injury, all cats were able to place their ipsilateral hindlimb on to the beam 100% of the time. After injury, recovery of ipsilateral hindlimb accuracy on the narrow beam was an all or nothing skill (Figure 3 4B). The majority of animals in both control irst eight weeks after injury, with none recovering by two weeks and only one becoming successful at four majority of them had a 100% success rate. By eight weeks post inj ury the control group was accurately placing their ipsilateral hindlimb significantly more than what they were at two weeks (Figure 3 4A; Kruskal Wallis test, p=0.04). This trend continued at 16 (Kruskal Wallis test, p=0.005) and 20 weeks after injury (Kr uskal Wallis test, p=0.004). weeks after injury (Kruskal perform as well as controls on this task.


89 Peg walkw ay Similar to the horizontal ladder and narrow beam, all cats were able to accurately place their ipsilateral hindlimb onto pegs 100% of the time prior to injury (Figure 3 4C). However, for the first two weeks after injury, ipsilateral hindlimb accuracy w as completely lost. By four weeks post injury, there was one cat from the control and targeting substantially more than the control cat at 100% as opposed to 11%, resp ectively. Over the course of the study only two other cats from each group recovered ipsilateral hindlimb accuracy on peg walkway, suggesting the extreme difficulty of this task. Additionally, accuracy percentage remained low in both groups, with neither significantly improving from two weeks post injury at any time point in the while the other cats ranged from 7 to 23% ipsilateral hindlimb accuracy (Figure 3 4C). The Relatio nship between Spared Tissue and Functional Recovery While most of the 35 lesions obtained from the laboratory data bank were variations on a hemisection, there were a few that were notably larger with only ~25% of cross sectional sparing, or notably smalle r with ~75% of cross sectional sparing. Furthermore, the effect the relationship between lesion size and locomotor recovery has yet to be determined In the current study, u nbiased individuals ranked the amount of total spared tissue pre sent at the lesion epicenter from all 35 cats : Control ( 19 ), two w ee k c four w ee k c rank was determined for each cat based on the ranks assigned by the three individuals. Lesion sizes ranged from very over hemisected with sparing in only a quadrant of the spinal cord crossection (Figure 3 5A), to very under hemisected with sparing in 3/4t hs of


90 the spinal cord crossection (Figure 3 5C). Those that were ranked as having a medial amount of sparing were near perfect hemi sections (Figure 3 5B). Average ranks for each animal were correlated with onset of task recovery, as well as accurate ipsilateral hindlimb placement on the horizontal ladder, peg walkway, and narrow beam. Onset of task r ecovery versus lesion size T here was a significant correlation in the control animals between spared tissue rank and recovery onset of overground walkway (Figure 3 5D; Spearman test, p=0.0001), horizontal ladder (Spearman test, p=0.001), peg walkway (Spearman test, p=004), and narrow beam (Spearman test, p=0.0001). The only lack of a relationship was on bipeda l treadmill (Figure 3 5). The two w ee striking relationship in that their lesion rankings did not significantly correlate with recovery onset of o verground, or peg walkway. However, the recovery of horizontal ladder (Spearman test, p=0.03), and narrow beam (Spearman test, p=0.0004) did significantly correlate with spared tissue rank. The onset of overground walkway was close to significantly correl ating with a p there were no significant correlations between lesion size and recovery onset on either of the tasks except for bipedal treadmill (Spearman test, p=0.04). This lack of a relationship between spa re d tissue and task onset in the four w ee k ch indicates that four lesions and less spared tissue to recover tasks quicker than cats with a similarly sized lesion that were treated either with two looking at the individual values for each animal, using the onset of narrow beam as an example (Figure 3 5E). He re, the onset and spared tissue rank values show that the 4


91 muc h quicker t han the control and two w ee of sparing. This effect oc curs in all assessed tasks. Ipsilateral hindlimb accurate targeting versus lesion size groups either significantly correlated or had a p value of <0.1, with ipsilateral hindlimb ac curate targeting at four weeks post injury on horizontal ladder (Figure 3 6A; p=0.01; 2 w re 3 6A). The same is true at eight weeks post ), peg walkway (Spearman test, two (Spearman test, control, p=0. 0004; two this was the control group at eight weeks post injury on peg walkway. In direct contrast, there was no relationship between lesion size and accurate targeting on either of the tasks for the four w ee p. Interestingly, by 16 weeks post injury the relationship seen between lesion size and accurate targeting was no longer present on was still present in narrow beam (Spea p=0.01). At 16 weeks post injury there was still no relationship between the two in the decreases the relationship between lesion s ize and locomotor recovery. Similar to what was seen in the onset of task recovery, the individual values of each animal, using accurate ipsilateral hindlimb targeting on horizontal ladder at four weeks as an example,


92 indicates that four w ee mals with less tissue sparing (larger lesions) recover accurate tar geting better than control and two w ee similar amounts o f spared tissue (Figure 3 6B). Discussion Summary of Results In the current study we found that there was a significant enhancement in axonal enhanced rubrospinal tract growth below the lesion similar to four week treated animals (Jefferson et al., 2011) they did not share a similar enhancement of skilled locomotor recovery. In a larger group of animals with more diverse lesion magnitudes, it was functional recovery in larger lesions. Supraspinal C onnectivity below t he L esion studies attribute locomotor recovery after SCI to the regeneration and sprouting of the CST (Bradbury et al., 2002; Barritt et al., 2006; Garcia Alias et al., 2009) However, the current study did not find enhanced CST connectivity below the level of the lesion based on our retro particular pathway. In fact, mu ltiple studies have reported a failure in significant CST axonal growth despite seeing growth in other systems like the RuST (Richardson et al., 1984b; Houle and Ye, 1999; Decherchi and Gauthier, 2000; Plunet et al. 2002) More


93 site, but this sprouting does not enhance the number of motor cortex neurons with connections below the level of the lesion. Alternatively, CST plasticit y may be occurring in more rostral regions of the spinal cord like the cervical cord, as shown in Bareyre et al., 2004. Sprouting in those regions would not be detected by low thoracic FG injections. have reported enhanced CST sprouting proximal to the lesion site (Bradbury et al., 2002; Barritt et al., 2006; Iseda et al., 2008; Garcia Alias et al., 2009; Karimi Abdolrezaee et al., 2010) stud y to assess CST plasticity utilizing retrograde tracing, in addition to the current study, had similar findings in that there was enhanced connections below the level of the lesion in non CST pathways (ReST, VST, and RuST) but none in the CST itself (Bai et al., 2010) This further suggests that if CST plasticity is occurring, it most likely occurs at the terminal ends or in more rostral segments of the spinal cord. Locomotor Recovery and Supraspinal Plastici ty Previous studies performed by the Howland laboratory did not show a significantly (Te ster and Howland, 2008; Jefferson et al., 2011) In the current study, we also see this similar lack of an effect. This result was expected based on the aforementioned results as cats recover these tasks within the first two weeks after injury; a perio week locomotor recovery was most apparent on the skilled locomotor tasks. These tasks require much more adaptation and mimic the type of everyday tasks that


94 individuals with incomplete SCI, like Brown Sequard Syndrome, primarily struggle with (Little and Halar, 1985; Eidelberg et al., 1986) The recovery period for horizontal ladder, peg walkway and narrow beam were similar across cats in the control group, with cats typically recovering horizontal ladder first, followed closely by peg walkway, then narrow beam. All control cats recovered recovered horizontal ladder significantly quicker than the control cats after injury, however had substantially greater variability regardin g recovery period for peg walkway and narrow beam. On peg walkway, four cats recovered within 15 to 18 days, while two never recovered. On narrow beam, four different cats recovered within 16 to 41 days, while two cats, different from those who failed to recover peg walkway, did not recover narrow beam until 111 and 113 days after injury. This extreme variability may be an indicator of aberrant plasticity and nonfunctional circuit formation resulting from an This is further confirmed by a lack of (Tester and Howland, 2008; Jefferson et al., 2011) Furthermore, results from retrograde tracing show significantly greater RuST connections below the level of the lesion in both the two and four week group did not (Tester and Howland, 2008; Jefferson et al., 2011) It is possible that the plasticity necessary to make function ally appropriate connections capable of producing long term beneficial effects. Neuro developmental studies have shown that


95 experience based plasticity is dependent on a lack of CSPGs (Pizzorusso et al., 2002; Pizz orusso et al., 2006) After injury, the expression of some CSPGs, specifically phosphacan, NG2, brevican, and neurocan, remain upregulated for longer than two weeks after injury (Jones et al., 2002; Jones et al., 2 003a) Additionally, aggrecan, which initially decreases after injury, begins to recover expression at two weeks post injury (Lemons et al., 2001) These specific CSPGs have been s hown to inhibit axonal growth (Sango et al., 2003) especially NG2 (Dou and Levine, 1994; Fidler et al., 199 9; Jones et al., 2002; Ughrin et al., 2003) and aggrecan (Lemons et al., 2003) Although eir ongoing expression may continue after the causing inhibition of axonal growth. In addition to ongoing CSPG upregulation 2 weeks after injury, there are multiple post SCI inflammatory factors, such as microglia, reactive astrocytes and oligodendrocyte precursor cells that infiltrate the lesion site quickly after injury and continue to be present for longer than two weeks after injury (Hill et al., 2001; Velardo et al., 2004; Donnelly and Popovich, 2008) Additionally, some of the greatest changes to lesion morphology occur between two and four weeks after injury (Hill et al., 2001; Velard o et al., 2004) plasticity, the combinatorial presence of these factors at the lesion site after cessation of appropriate connecti ons. Previous studies from the Howland laboratory have shown that both human and feline CS 6S disaccharides, while digestion of rodent CS GAGs results in primarily 4S


96 disaccharide p roducts (Tester and Howland, 2008) This species difference suggests spinal cord. This may partially explain differences seen in functional recovery across due to the larger size of the cat spinal cord compared to rat, axonal regeneration and sprouts must travel farth er in order to reach appropriate connections. Combined results from the present study, as well as those previously published by the Howland laboratory locomotor recovery compared to two weeks of administration in a cat model of spinal cord injury. Lesion S ize Multiple studies have found that larger lesions significantly correlate with poorer functional recovery and that smaller lesions are paired with greater functional recovery (Byrnes et al.; Semler et al.; Molt et al., 1979; Norrie et al., 2005) Here, we have found a similar result in that spared tissue significantly correlates with onset of task recovery, and accurate targeting of the mo re greatly affected hindlimb on horizontal ladder, peg correlate with these same loco motor features. Further investigation of individual treated animals with less spared tissue had functional recovery that surpassed those in ted groups with similarly small amounts of spared tissue. recovery even in large lesions, but that a minimum of four weeks of treatment is


97 necessary to produce this result. W e believe the mechanism underlying this effect is beyond what was assessed by the tracing techniques used in the study. Although two n plasticity, it is not enough to produce new functional connections that can surpass the limitations of a larger lesion. Conclusio n model of SCI is not sufficient for enhanceme nt of skilled locomotor recovery. While it does lead to a significantly greater number of red nucleus neurons with axons below the level of the lesion, this increased growth may not make functionally relevant connections. As seen in our previous studies, nucleus plasticity, as well as skilled locomotor recovery suggesting that this longer made. Although we did not see increased motor cortex neuronal connectivity below the level of the lesion using retrograde tracing for either two or four week treated groups, anterograde tracing. These results suggest t hat there may be plasticity occurring at regions rostral to the injury, or at the terminal ends that cannot be detected using retrograde tracing. Our assessment of the spared tissue functional recovery relationship indicates a positive correlation with mor e sparing leading to greater recovery and vice relationship by causing substantial recovery in lesions with both high and lower amounts of spared tissue, further confirms of a treatment period as four weeks in feline SCI. The high translatability of felines to


98 humans suggests that a longer treatment duration should be considered for future translationally designed, an


99 Figure 3 1. Range of spinal hemisection s. Horizontal sections of lesions from each of the control (A) and ch ondroitinase abc treated (B) animals. The entire len gth of the lesion was sectioned at 25 m and stain ed with cresyl violet and myelin dye The greatest amount of damage in each of the ma in cord regions was determined in each section and collapsed into a single drawing. Scale bar, 1mm.

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100 Figure 3 2. Supraspinal connections below the lesion. Th e amount o f fluorogold labeled neurons were determined for the non axotomized and axotomized side of the red nucleus and motor cortex. Additionally, the percentage of axotomized fluorogold labeled neurons as a percentage of the non axotomized side were calculated. *,p<0.05. p=0.0628. The axotomized red nuclei (A), and motor cortex (C), had less labeling than the non axotomized side of the red nucleus (B) and motor cortex (D), 100 m. Average raw values, and percentage of axotomized to non axotomized values for the two, and four week control groups, as well as the two and four week chondroitinase abc groups were reported (E).

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101 Figure 3 3. Onset of basic and skilled locomotor recovery. The number of days it took for cats to recover basic locomotor tasks: Bipeda l treadmill and overground walkway (A) and skilled locomotor tasks: H orizontal ladder, peg walkway, and narrow beam (B), were compared across two week control and two week P=<0.05). T he specific day of recovery after injury for each co ntrol (C) and ch ondroitinase abc cat (D) were listed for recovery of ladder, peg walkway, and narrow beam. Rows highlighted in gray indicate those cats that had extreme variability in their recovery period across the three skilled tasks after injury. Remo led to significant group differences in the peg walkway but not the narrow beam (E) Variation was assess ed using s tandard error of the means.

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102 Figure 3 4. Ipsilateral hindlimb accuracy. The percen tage of times cats were able to accurately target and place their ipsilateral hindlimb onto a rung, beam, or peg while crossing the horizontal ladder (A), narrow beam (B), or peg walkway (C) were determined periodically over a 20 week period post injury an d compared between two week control and c hondroitinase abc groups. (*p=<0.05), (p=0.0660). Standard error of the means are depicted in A C.

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103 Figure 3 5. Spared tissue and task onset correlations. Lesion epicenters were stained wi th cresyl violet and myelin dye and then ranked from the least amount of spared tissue (rank=1) (A) to the greatest amount of spared tissue (rank=32) (C). Some lesions were ranked the same if they had extremely s imilar amounts of spared tissue therefore the highest ranked les ion is not equivalent to the total number of cats included in the study. The median lesion (rank=16) (B), was extremely close to a near perfect hemisection. Correlation coefficients were determined which compared lesion rank and the number of days it took for animals to recover overground walkway, bipedal treadmill, horizontal ladder, peg walkway, and narrow beam after injury (D). Many significant, or trending towards si gnificant correlations were found in the control and two week ch ondroitinse abc treate d grou ps, but none were found in the four week ch ondroitinase abc treated group except for o n bipedal treadmill. In order to better understand these results, the values for each individual animal were plotted and compared. Here, we use recovery of narrow beam as an example (E).The trend lines, and r 2 value for each group are included. Scale bar 1mm.

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104 Figure 3 6. Spared tissue and accurate ipsilateral hindlimb targeting correlations. Correlation coefficients were determined which compared the lesion ra nk and percentage of accurate ipsilateral hindlimb targeting on horizontal ladder, peg walkway, and narrow beam (A). Similar to recovery of ta sk onset, both the control and two week ch ondroitinase ab c treated animals either significantly correlated, or tr ended towards a significant correlation with accurate t argeting on all three tasks at four and eight weeks post injury. There we re minimal correlations at 16 weeks post injury. In contrast, the four week ch ondroitinase abc treated group ha d no significant correlations at either of the time points Closer analysis of this effect using the individual values of a ccurate targeting on ladder at four weeks as an example, shows that the four week chondroitinase abc cats with the lowest amounts of spared tissue w ere able to place substantially more than those animals with a similarly low amo unt of spared tissue (B).

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105 CHAPTER 4 OPTIMIZ ATION OF FLUOROGOLD RETROGRADE TRACING Introduction The use of the retrograde tracer Fluorogold (FG) is a common technique for determining axonal connections in multiple different experimental paradigms performed in a variety of animal models ranging from lamprey (McClellan et al., 2006) to primate (Gimenez Amaya and Graybiel, 1990; Hayashi et al., 2006) Tracer is placed into axons of interest and travels retrogradely to label the neuronal cell bodies o f origin. There are multiple benefits to using FG in addition to its compatibility with a wide range of species. It has the rare ability to transport to the distal dendrites (Maranto, 1982; Bentivoglio a nd Su, 1990; Buhl and Dann, 1990; Naumann et al., 1992) and appears to label all neuronal cell population s (Zabo rszky, 2006) Additionally, it has strong autofluorescent properties that make it detectable without additional processing, but its signal also can be amplified and detected using immunohistochemical (IHC) processes (Chang et al., 1990; Naumann et al., 2000; Akhavan et al., 2006) Despite these benefits, there are several downsides to using FG which need to be minimized for tracer optimization. One prime example is the requirement of tissu e damage at the injection site for tracer uptake (Schmued and Fallon, 1986) Furthermore, multiple investigators have reported that FG is cytotoxic to both motoneurons (Garrett et al., 1991; Nauma nn et al., 2000) and dorsal root ganglia neurons (Garrett et al., 1991) when left in vivo for several weeks. This neurotoxic feature has the potential to become a significant problem in larger species who may require longer survival period s depending u po n transport distance. One way to minimize tissue damage is by decreasing FG concentration and volume (Schmued and

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106 Fallon, 1986) however this translates to decreased aut o fluorescence that quenches more q uick ly An anti FG antibody is commercially available and allows for the detection of FG using conventional immunohistochemical (IHC) tech niques that lead to Diaminobenzidine (DAB) reaction product (Chang et al., 1990) Thi s may be a promising solution for FG detection when autofluorescence is not as bright due to low FG concentration and volume and/or permanent label is desired Decreasing the survival period following FG injections also will cyto toxic effects. The detergent Triton X 100 (triton) has been implicated as a speed enhancing supplement for tract tracing with oregon green and fluororuby dextran amines in Possums (Fry et al., 2003) Triton is a non ionic surfactant commonly used to permeabilize cell membranes. Since the rate of FG uptake is directly related to its ability to cross the cell membrane (Martin W, 1991) a mixture of FG and triton applied to the region of interest may enhance both tracer uptake and speed of transport which may result in minimizing cytotoxicity. Additionally, the benefits of shortening the survival period may expand i nto other area s specific to a study such as financial motives and time dependent iss u es. The objectives of the current study are to determine methods for minimizing tissue damage while maximizing FG tracing speed and detection. Materials and Methods The following data is based on FG tracing results from 19 cats. All procedures were conducted in accordance with NIH guidelines and were approved by both the nstitutional Animal Care and Use Committees. The injection sites of each of the four animals were processed and assessed in order to ensure adequate spread of the tracer, especially in the dorsolateral funiculi

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107 which contains the rubrospinal (RuST) and corticospinal (CST) tracts. These tracts acted as the tracts of interest for this study as their cell bodies are located a far distance from the injection site s and we are well experienced with tracing these systems (Jefferson et al., 2011) (Refer to Chapter 3). Subjects All cats were purpose bred, SPF adult, female cats (Liberty Vendors Inc. ) Animals were placed into one of five different groups: 13 day FG without triton (N=12), which was our commonly used protocol for previous tract tracing experiments (Jefferson et al.) as well as a seven day FG without triton group (N=1), seven d ay FG with triton group (N=2), three day FG wit hout triton group (N=1), and a three day FG with triton group (N=3). Surgical Procedures Fluorogold spinal i njections Trito n X 100 ( 2.5%, Sigma Aldrich ) was mixed with sterile saline in a sterile setting the morning of the procedure. F luorogold (0.5%, F luorochrome LLC ) was then either mixed in sterile saline or the 2.5% T riton X 100 solution and kept on ic e prior to use. The spinal cord s egment T11 was exposed and injections were made in four injection sites placed in a staggered formation using a 33 gauge Hamilton syringe. Each injection site consisted of three FG injections of 0.25 L in each, for a total of 3 L s (Figure 4 1) The exposed spinal cord was then protectively covered with durafilm and gelfoam and the muscle and skin closed in layers. Our previous studies describe post surgical procedures in detail (Howland et al., 1995b, a)

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108 Tissue Processing: Histology and Immunohistochemistry Perfusions The details of this procedure are described in our previous work (Tester and Howland, 2008) In brief, at three, seven or 13 days following FG injections, cats were deeply anesthetized with an overdo se of sodium pentobarbital (>40mg/kg, I.P ) and supplemented i v as needed to ensure that animals were properly sedated. Cats also were injected with heparin ( i v .;1000 U) and then 20 minutes later injected with sodium nitrite (I.V .;1%, 1cc) Immediately after, cats were then transcardially perfused with 0.9% saline followed by 4% paraformaldehyde in 0.1M phosphate buffer (pH 7.4). The injection site s were blocked and sectioned at 25 m while the red nuclei and motor cortices we re blocked and sectioned into 50 m section s. Cresyl violet and myelin s taining A full description is described in our previous work (Tester and Howland, 2008) In brief, e very 12 th section of the injection site was mounted onto Colorfrost slides (Fish er Scientific ) subbed with chrom alum and poly L lysine (chromium potassium sulfate and poly L lysine, Fisher Scientific ) and fume fixed with paraformaldehyde. Tissue was first dipped in water, and then in alcohol s at increasing concentr ations for dehydration for five minutes. After dipping in xylene, tissue was then rehydrated in decreasing concentrations of alcohol and placed in myelin dye for 10 minutes. After washing in water, differentiation then took place in 1% ammonium hydroxide (Sigma Aldrich ) for one minute. Tissue was then place d in 0.5% cresyl violet (Sigma Aldrich ) for 3 minutes and washed thoroughly in water, then 70% alcohol, then differentiated in 95% alcohol with glacial acetic acid. Fol lowing this, tissue continued to be dehydrated in

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109 increa sing increments of alcohol for five minutes, and then placed in xylene. Tissue was then cove rslipped with DPX (Fluka ) Fluorogold immunohistochemistry A full description of this procedure can be fou nd in Jefferson et al., 2011. In brief, m ultiple sections of the injection sites and red nuclei were processed using the monoclonal anti FG antibody (1:60,000, 1:10,000, respectively) (Fluorochrome, LLC). Sections were washed in a quenching solution ( 30% H 2 0 2 and phosphate buffered saline ( PBS ) ) for 30 minutes, followed by two 10 minute rinses in 1 % goat serum in PBS with 0.4% triton X 100 (1% S PBS T). After this, tissue was blocked in 5% S PBS T f or one hour. Tissue was then incubated in anti FG antibody overnight at room tempe rature. The following day tissue was rinsed in 1% S PBS T and incubated in 5% biotinylated anti rabbit secondary antibody made in goat for 1 hour (Vector Laboratories ) Following another rinse with 1% S PBS T, tissu e was incubated for two hours in ABC Reagent (Vector Laboratories ) Tissue was then rinsed with PBS and reacted with Diaminobenzidine for 9 minutes resulting in a brownish reaction product The DAB reaction was stopped with a PBS rinse once the reaction product was the appropriate da rkness Tissue was then mounted onto chrom alum and poly L lysine coated slides and fume fixed in 4% paraformaldehyde for ~ one hour Lastly, tissue was dehydrated in alcohol s at incre asing concentrations for 5 minutes, placed in xylene for f ive minutes a nd coverslipped with DPX (Fluka ) The sections that were s tained for FG immunoreactivity one and nine years ago underwent the same procedures as described above. These slides were stored at room temperature in hard cover slide boxes.

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110 Assessment of injection site completeness and flu orogold a utofluorescence Every 12 th section through the injection sites were mounted onto Superfrost/Plus slides (Fisher Scientific ) and were coverslipped using prolong gold anti fade mounting media (Molecular Probes ). Sections were assessed usi ng the UV cube with fluorescent microscopy. Red nucleus and motor cortex sections adjacent to those known to have heavy FG labeling based on results from the anti FG IHC analysis described above, were processed in a similar manner in order to compare auto fluoroscent and IHC based FG detection. Results Tissue Damage at the Injection Site It ha s been previously reported that FG causes tissue necrosis at the injection site and that this damage is necessary for FG uptake and subsequent retrograde tracing (Schmued and Fallon, 1986; Divac and Mogensen, 1990) H owever the extent of this necrosis as well as its sensitivity to FG volume and concentration, have yet to be described. Here we compare the injection sites for animals that received FG injections of different concentrations volumes and had different survival periods after FG injection s Additionally, the e ffects of triton mixed in the FG solution were assessed. Cats that und erwent a three (Figure 4 2C) or seven day (Figure 4 2E) survival period after injections of the same FG concentration (0.5%) and volume (3 L) had similar amounts of necrosis at the injection site. Cats that both had a 13 day survival period after receivin g FG injections of either 0.5% (Figure 4 2A) or 2.5% (Figure 4 2B) had different amounts of tissue damage at the injection site, with the 2.5% FG cat having a substantially greater amount of damage Thus, a small increase in FG concentration had drastic e ffects on the tissue necrosis. Increasing the volume of FG injections to the

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111 spinal cord by just 1 L also resulted in greater damage at the injec tion site Specifically, cats that received 2 Ls of FG (0.5%) had some damage at the injection site but had n o large regions of tissue loss (Figure 4 2A). In contrast, cats that received 3 Ls of FG of the same concentration (0.5%) had several large regions of tissue loss (Figure 4 2C,E). Surprisingly, t he addition of triton to the FG injections (0.5%, 3 L, thre e or seven day survival period; Figure 4 2D,F) which enhances cel l membrane permeability, did not visibly increase the amount of tissue damage at the injection site s in cats with a similar survival period, FG concentration, and volume (Figure 4 2C,E) F luorogold Detection Techniques Depending on the tracing parameters, autofluorescence also may not be as bright and easily detectable prior to any exposure. In the present stud y, FG autofluorescence in the motor cortex was extremely light in the cat following an injection of 3Ls of 0.5% FG, and a survival period of three day s (Figure 4 3A). The addition of triton to the FG injections of a similar volume, concentration, and sur vival period did not enhance autofluorescence (Figure 4 3B). Autofluorescent FG detection also was limited in the red nucleus of the same cats despite being closer to the injection site (Figure 4 3C). Comparisons of FG detection at the injection site using autofluorescent detection versus anti FG IHC detection showed a surprising amount of disparity across these two detection methods Autofluorescent detection of FG at the injection site, the most FG concentrated region of t he nervous system appeared to be strong (Figure 4 3E). However, when compared to an adjacent section with anti F G IHC processing (Figure 4 3F), it was apparent that there wa s a substantial portion of FG not detected with

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112 autofluorescence a lone The IHC processed tissue has substantially more detectable FG Additional benefits to using anti FG IHC processing are that the DAB reaction products, and therefore FG detection, remain strong for years following the initial processing. Images were taken of sections immediately after processing (Figure 4 4A), one (Figure 4 4B), and nine y ears (Figure 4 4C) after processing. There is minimal to no fading at either of these time points. Triton Increases Tracer Travel Time Our past st udies in the cat determined that a 13 day survival period for FG tracing from T12 to the brainstem, specifically red nucleus (Jefferson et al., 2011) as well as the motor cortex (refer to chapter 3) resulted in adequate ne uronal cell body labeling. However, depending on the study being performed, a 13 day survival period may be an unrealistic or inconvenient period of time. For example, the attempt to assess the nervous system immediately following a specific event would be skewed by this 13 day survival period Here, we assess the use of triton as a method for decreasing survival time. The amount of FG labeled neurons in the red nucleus and motor cor tex as detected using anti FG IHC, were compared across cats that received FG only or FG with triton after a three or seven day survival period. As used in our previous studies (Jefferson et al., 2011; Doperalski et al., in prepar ation) a 13 day survival period following injections of FG only led to substantial FG labeling in the red nucleus (Figure 4 5A) and motor cortex (Figure 4 5 F ). Labeling was typically darker in the red nucleus compared to the motor cort ex due to the grea ter proximity to the injection site A survival period of either three (Figure 4 5D) or seven day s (Figure 4 5B) without triton led to some labeling of the red nucleus though was insuffic ient. However, those th at

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113 received FG injections mixed with t riton and a survival period of three or seven days (Figure 4 5C) had more labeled neurons Specifically, the FG only cat (Figure 4 5B) had multiple dark FG labeled neurons but were m issing the more lightly labeled neurons as seen in the FG triton animals (Figure 4 5C). A similar effect was seen in the three day FG triton injected tissue w hich also had additional neurons labeled that were typically of a lighter shade (Figure 4 5E). Group differences between FG neuronal labeling were more extreme in the motor cortex, which is ~2 centimeters further from the injection site compared to the red nucleus. While both the seven (Figure 4 5G) and three day (Figure 4 5I) FG only animals had minimal labeling of just a f ew neurons in this region, the seven (Figure 4 5H) and three day FG triton animals (Figure 4 5J) ha d robust labeling of many neurons A seven day survival period (Figure 4 5H) led to labeling very similar to what was seen in the FG only, 13 day survival period cats (Figure 4 5F), where as a three day survival period (FG triton ; Figure 4 5J ) led to labeling that w as not as dark. However, this three day labeling was readily detectable for neuronal counting. O verall, the results from this study show that FG is necrotic at the injection site and should be used sparingly to prevent extensive damage. Although a minimized FG concentration or volume may lead to underwhelming autofluorescence that is insufficient f or precise FG detection, anti FG IHC is capable of amplifying the FG signal to permit easy detection Furthermore, the mixing of triton with FG injections can enhance tracer speed thus decreasing the necessary survival time for successful tracing. This shortened tracing time has the potential to benefit studies that are time depe ndent and/ or have other rationales that require shorter tracing periods

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114 Discussion Consistent with a few previous studies this study shows extensive tissue damage at the injection site following FG injections (Schmued and Fallon, 1986; Divac and Mogensen, 1990) This study also showed that a slight change in c oncentration or volume greatly a f fected the amount of tissue damage at the injection site. A similar finding was found by Schmued and Fallon, 1986 who compared tissue damage following a larger range of concentrations (~10% versus ~3%) in the rat. In addition to tissue damage at the inje ction site, other studies have reported cytotoxic damage to the targeted cell bodies after FG tracing (Garrett et al., 1991; Naumann et al., 2000) It i s possible that these small alterations in FG concentration an d volume also may result in less damage to the cell bodies Although using small concentrations and volumes of FG injections minimizes tissue damage at the injection site and possibly the targeted cell bodi es, we also showed that it is accom panied by decreased FG autofluorescence that is insufficient for neuronal counts or other quantitative assessments. While this lack of autofluorescence suggests that an insufficient amount of FG was present at the region of interest, we demonstrated that through anti FG IHC amplification there is in fact abundant FG labeling. Similar results have been reported in Akhaven et al., 2006 who showed that anti FG IHC detection led to the detection of FG in cells that no longer displayed FG bas ed autofluorescence Furth ermore, by comparing labeling of an adjacent section that received anti FG IHC processing we demonstrated that in regions displaying bright autofluorescence like the injection site itself, some FG was not being detected by autofluorescence These findings suggest that IHC leads to optimal detection of FG compared to autofluoresce nc e

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115 In large animal models like the cat, tracing often requires a relatively long survival period due to the longer distances being traced We have shown that this survival period can be significantly decreased by mixing triton with the FG in jections. This is most likely due to the increase in cell permeability as it has been reported that tracers taken up by pH trapping, like FG, are done so with greater efficiency if the cell membrane is more permeable (Martin W, 1991) Surprisingly, despite causing increased cell membrane permeability, the addition of triton to FG injections did not visibly enhance damage at the injection site. The re are multiple benefits to shortening FG tra cer survival period. For example, d ecreasing the length of time FG is present in the nervous system will in turn decrease the amount of biochemical changes that are occurring as a response to FG neurotoxic properties Thus, the regions of the nervous system being investigated will be more similar to how they were prior to tracer injection Fewer restrictions on the length of time needed for tracing also will benefit those studies that have certain time l imitations or restrictions. Collectively, the c urrent study shows that by decreasing the concentration and volume of FG injections, as well as enhancing cell membrane permeability with triton, the amount of tissue damage at the injection site can be minimized and the tracing speed optimized. Furthermo re, although these decreases in FG concentration, volume, and survival time s often lead to insufficient autofluorescence, the use of anti FG IHC processing can be used to achieve robust FG detecti on.

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116 Figure 4 1 F luorogold i njection s chematic. At T11 four injection sites were staggered over the span of approximately 2mm. At each injection site, thre e fluorogold deposits of 0.25 L were made for a total of 3 L

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117 Figure 4 2. E ffects of different f luorogold concentrations, volumes, and survival periods on tissue damage at th e injection site. Representative sections from the injection site with the greatest amount of damage were compared across the different f luorogold injection conditions: A) 13 day survi val time, 2L s of 0.5% FG, B) 13 day survival time, 2L of 2.5% f luo rogold C) three day survival time with 3Ls of 0.5% FG injections, E) seven day survival time with 3L of 0.5% fluorogold injections. The effects of triton mi xed with fluorogold on tissue damage also were compared in an imals that received 3 L of 0.5% triton and either a three (D) or seven day survival period (F) Scale bar is 1mm.

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118 Fig ure 4 3 Anti f luorogold immunohistochemical processing leads to greater fluorogold detection compared to native autofluorescent detection. Autof luorescent fluorogold (FG) detection in the motor cortex was weak following 3 L injections of 0.5% FG and a three day survival period (A). The amount of fluorescence was the same in an animal receiving the same injection parameters but with the addition of triton (B), 200m. Autofluorescent detection was similar in the red nucleus of this same animal (C) but anti FG IHC processing using anti FG and DAB of an adjacent section enhanced FG detection (D), 100m. This same effect was seen in regards to detec ting the aerial spread of FG at the injection site with autofluorescence (E), and anti FG IHC (F), 1mm.

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119 Figure 4 4 Long term fluorogold detection using anti fluorogold immunohistochemical processing. A newly stained section of red nucle us containing immunoreactive f luorogold labeled neurons (A) is compared to simil ar sections that were processed one (B), and nine years ago (C) 100m.

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120 Figure 4 5. Triton enhances fluorogold tracing speed. Fluorogold labeled neurons were comp ared in cats that received fluorogold without triton and had a survival time of 13, seven, or three days in the red nucleus (A,B,D) and motor cortex (F,G,I). Comparisons also were made in those that had triton mixed with their flu orogold and a su rvival time of seven or three days in the red nucleus (C,E) and motor cortex (H,J) 100 m.

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121 CHAPTER 5 SUMMARY Defining Recovery There is great debate amongst scientists and clinicians regarding the definition of relative to functional changes associated with central nervous system ( CNS ) injury or disease original functional dynamics are completely restored. Other opinions view any form of (Levin et al., 2009) Thus, it is critical for investigators to clearly define the definition of recovery for their specific study, as was done in Chapters 2 and 3. However, the definition employed in these studies remains controversial as they allowed for compensatory strategies supporting successful movement patterns that differed from pre injury. The decision to use a definition of recovery that allows for compensatory strategies as opposed to the strict definition of recovery descri bed in Levin and colleagues, is primarily due to the extensive changes in axonal circuitry that are known to occur by way of injury induced sprouting and regeneration (Bareyre et al., 2004; Courtine et al., 2009; Blu m, 2010) This novel circuitry, which underlies locomotor performance, will most likely be accompanied by novel movement patterns that differ from pre injury. A more strict definition of recovery may be difficult to establish and not particularly applica ble to individuals with neurological injuries. use of new motor patterns resulting from the adaptation of remaining motor elements or substitution (Levin et al., 2009) may be synonym most likely will strongly incorporate and require enhanced use of spared fibers.

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122 Advancing Chondroitinase ABC to the Clinic definitions for recovery in subsequent studies, as well as the functions being assessed need to be well defined and deliberately chosen. As reported in Chapter 3, models of partial spinal cord injury ( SCI ) similar in many respects to Brown Sequard Syndrome (BSS) m ediated functional effects on adaptive tasks like the peg walkway and were not as readily detected on more simple tasks like the mediated effects may be more noticeable on simple tasks in larger injury models, such as c ontusions. Furthermore, animals frequently employed a variety of compensatory strategies for successful completion of (Jefferson et a l., 2011) Thus, not only is it important to carefully choose the functional assay used for the model of injury being assessed but, it also is critical to assess performance of a variety of different gait features in order to properly understand the effe functional improvements after SCI. For future clinical assessments, the development of a test battery for adaptive locomotor features for the human SCI population would be beneficial and allow for quick assessment of adaptive functions as well as their (Tester and Howland, 2008; Jefferson et al., 2011) Another major translational issue is whether the magnitude of therapeutic efficac y seen in an animal study will translate to humans. Therapeutic effects seen in small rodent models of SCI indicate promising treatment potential, but do not necessarily mean the treatment will be as effective in humans. Moving these treatments to larger animal models provides greater insight into the therapeutic potential of treatments.

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123 Results from Chapter 3 demonstrate this issue. Despite there being multiple reports of ry in rodent SCI models, this treatment duration was not effective in our larger cat model of SCI, but four weeks administration resulted in significant functional benefits. It is t be sufficient for humans, but that six to eight weeks of treatment may be more optimal. It affect functional recovery and if greater recovery can be achieved with this longer are important to assess. Although several studies have begun to look at tre atment windows, the majority have used a single injection paradigm. This single dose approach, as determined in Chapter 3, is most likely not an optimal treatment duration (Yick et al., 2003; Massey et al., 2006; Is eda et al., 2008) Furthermore, determining delivery is via intrathecal administration. However this mode has been reported to cause increased scarring and compression to the spinal cord (Jones and Tuszynski, 2001) and is not particularly feasibl e in humans with acute SCI. Several studies have begun to (Lee et al., 2009) release fibrin gel (Hyatt et al., 2010) (Jin et al., 2011) However, assessments of these delivery modes are limited, and it is unclear how effective they will be. The need to continue to explore clinically applicable

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124 delivery approaches is one of the more criti cal variables for the future translation of this therapeutic. Conclusions As research continues to allow for a better understand ing of the physiological responses and plastic potential of the central nervous system af ter injury, the d evelopment of a treatment paradigm capable of significant ly enhancing functional recovery after inj ury appea r s more plausible than p reviously thought to be A n effective treatment paradigm will most likely require a multi faceted approach and as depicted in Chapters 2 and 3, the therapeutic agent ch abc a nd an increased focus on o ptimizing train ing /rehabilitat ion for simple a s well as adaptive locomoto r functions w ill be importa nt components of this effective treatment paradigm. T he optimization of tract tracing in both small and large pre clinical anima l model s will aid in this treatment s d evelopment by allowing for an understand ing of the treatment mediated circuit ry changes that underl ie the functional c hanges

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149 BIOGRAPHICAL SKETCH Sarah Elizabeth Mondello was born the 2 nd of three daughters during the summer of 1983, in Austin, Texas. Two years later she moved with her family to Niskayuna NY Niskayuna High School where she grew particularly interested in clarinet performance. After graduating from high school in 2002 she attended McGill University as a music performance major and studied with the clarinetist Alain Desgagne of the Montreal Symphony Orchestra. In addition to the multiple music courses she took during her first year of study, Sarah also took several psychology courses and became increasingly mor e interested in that field of study. She switched her major to psychology just prior to beginning her second year of undergraduate studies. For her remaining time at McGill, Sarah became particularly interested in the neuroscience components of her psycho logy studies. She spent two summers as an intern at the Biosciences division of General Electric where she was first introduced to a career path towards neuroscience r esearch. Following graduation from McGill in 2006, Sarah immediately began working on a neuroscience Ph.D. at the University of Florida, Interdisciplinary Program in Biomedical Sciences. She joined the lab of Dena Howland in 2007 and began her studies on plasticity and functional recovery following spinal cord injury. In August 2012 Sarah successfully defended her research and obtained her doctoral degree.