Open Collections

UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Rubrospinal neurons after chronic cervical spinal cord injury Kwon, Brian K. 2004

You don't seem to have a PDF reader installed, try download the pdf

Item Metadata

Download

Media
ubc_2005-99497x.pdf [ 17.2MB ]
[if-you-see-this-DO-NOT-CLICK]
Metadata
JSON: 1.0092332.json
JSON-LD: 1.0092332+ld.json
RDF/XML (Pretty): 1.0092332.xml
RDF/JSON: 1.0092332+rdf.json
Turtle: 1.0092332+rdf-turtle.txt
N-Triples: 1.0092332+rdf-ntriples.txt
Original Record: 1.0092332 +original-record.json
Full Text
1.0092332.txt
Citation
1.0092332.ris

Full Text

RUBROSPINAL NEURONS AFTER CHRONIC C E R V I C A L SPINAL CORD INJURY By Brian K . K w o n B . S c , Queen's University, 1992 M D , Queen's University, 1995  A THESIS S U B M I T T E D IN P A R T I A L F U L F I L L M E N T O F T H E R E Q U I R E M E N T S  FORTHE DEGREE  OF DOCTOR OF PHILOSOPHY IN T H E F A C U L T Y OF G R A D U A T E STUDIES G R A D U A T E P R O G R A M IN N E U R O S C I E N C E  We accept this thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH C O L U M B I A S E P T E M B E R , 2004 © Brian K . K w o n , 2004  II  ABSTRACT Many experimental therapies have been developed that appear to have encouraging therapeutic potential i n animal models o f acute spinal cord injury. It has become increasingly evident however, that their effectiveness is reduced when applied chronically after the injury. This loss o f effectiveness over time is an issue o f obvious and critical relevance for the many individuals with chronic spinal cord injury.  Thus, the overall objective of this thesis was to  evaluate some of the challenges that impede axonal regeneration in a chronic spinal cord injury setting, and develop therapeutic strategies for this condition.  My hypothesis was that that  axonal regeneration can be achieved by chronically injured CNS neurons with the appropriate administration of neurotrophic factors The findings can be summarized as follows:  Two months after cervical axotomy,  rubrospinal neurons undergo significant atrophy and exhibit limited expression o f GAP-43 and Ted tubulin, genes thought to be important for axonal regeneration.  They appear to maintain  full length T r k B receptors on their cell bodies, and while their uninjured axons within the cervical spinal cord also contain T r k B receptors, the injured axons at the level o f the spinal cord axotomy do not.  Consistent with this, B D N F applied to the spinal cord injury site at three  exponentially increasing concentrations  d i d not reverse rubrospinal cell atrophy, did not  stimulate GAP-43 and Ted tubulin expression, and did not promote axonal regeneration o f rubrospinal axons into the permissive environment o f a peripheral nerve transplant. A t 12 months after cervical axotomy, a stereologic evaluation o f rubrospinal neurons demonstrates that rubrospinal neurons are in fact alive, but very atrophic.  Similar to the findings  at 2 months post-injury, the rubrospinal neurons 12 months post-injury display limited expression o f GAP-43 and Tod tubulin but dp maintain full length T r k B receptors on their cell  Ill  bodies.  A t this chronic time point, the administration o f B D N F to the injured cell bodies  reversed neuronal atrophy, stimulated GAP-43 and Tocl tubuin expression, and promoted axonal regeneration into peripheral nerve transplants. These findings suggest that axonal regeneration is possible in the chronic spinal cord injury setting, but that the administration o f neurotrophic factors to promote this growth response must be targetted appropriately. It is hoped that further study in the obstacles that impede axonal regeneration after chronic spinal cord injury w i l l give rise to therapies for this devastating condition.  TABLE OF CONTENTS Abstract  ii  Table o f Contents  iv  List o f Figures  viii  List o f Abbreviations  xi  Statement o f O r i g i n a l Contributions  xii  Acknowledgements Dedication  CHAPTER 1-B A C K G R O U N D  xiii xv  1  1.2. S U M M A R Y O F H Y P O T H E S E S A N D O B J E C T I V E S 3 1.3. P R O M O T I N G A X O N A L R E G E N E R A T I O N I N T H E C N S 5 1.3.1. Obstacles to Axonal Regeneration and Therapies to Overcome Them 5 1.4. N E U R O N A L S U R V I V A L , A T R O P H Y , A N D D E A T H A F T E R A X O T O M Y 8 1.4.1. Introduction 8 1.4.2. Neuronal Survival and C e l l Size are Influenced B y Neurotrophic Factors 8 1.4.3. Measuring Neuronal Death After Axotomy 10 1.5. I N T R I N S I C D E T E R M I N A N T S O F A X O N A L G R O W T H A F T E R C N S I N J U R Y 14 1.5.1. Introduction 14 1.5.2. The C e l l Body Response to Axotomy 14 1.5.3. Regeneration Associated Gene Expression 16 1.5.4. G A P - 4 3 17 1.5.5. Ted Tubulin 19 1.6. N E U R O T R O P H I C F A C T O R S 21 1.6.1. Introduction 21 1.6.2. Brain Derived Neurotrophic Factor ( B D N F ) 22 1.7. T R K N E U R O T R O P H I N R E C E P T O R S 24 1.7.1. Introduction 24 1.7.2. T r k B Receptors 25 1.8. A N I M A L M O D E L I N G O F S P I N A L C O R D I N J U R Y 27 1.8.1. Introduction 27 1.8.2. Anatomical Assessment o f Axonal Growth 27 1.8.3. Sharp Versus Blunt Spinal Cord Injury Paradigms 29 1.9. T H E R U B R O S P I N A L S Y S T E M ...31 1.9.1. Introduction 31 1.9.2. Anatomy o f the Red Nucleus and Rubrospinal Tract 31 1.9.3. Function o f the Rubrospinal System 33 1.10. R A T I O N A L E F O R E X P E R I M E N T A L M O D E L S 35 1.10.1. Cervically Axotomized Rubrospinal Neurons A s A Model o f Chronic C N S Injury 35 1.10.2. Rat Models 36  CHAPTER 2 - MATERIALS AND METHODS  2.1 SURGICAL TECHNIQUES 2.1.1. Anaesthetic Technique 2.1.2. Cervical Axotomy of the Rubrospinal Tract 2.1.3. Reaxotomy of Chronically Injured Rubrospinal Axons ("Refreshment Injury") 2.1.4. BDNF Application to the Spinal Cord Via Gelfoam® 2.1.5. BDNF Application to the Red Nucleus via Osmotic Minipump 2.1.6. Peripheral Nerve Transplantation 2.1.7. Anterograde Tracing of Rubrospinal Axons 2.1.8. Retrograde and Anterograde Labeling of Rubrospinal Neurons and Axons 2.2. HISTOLOGIC TECHNIQUES 2.2.1. Tissue Collection 2.2.2. Cryostat Cutting 2.2.3. NeuN Immunohistochemistry 2.2.4. TrkB Immunohistochemistry 2.3. ANALYSIS OF TISSUES 2.3.1. Disector Counting Technique of Rubrospinal Neurons 2.3.2. Measurement of Cross Sectional Area 2.3.3. In Situ Hybridization (ISH) CHAPTER 3 - RUBROSPINAL NEURONAL ATROPHY AND SURVIVAL AFTER C E R V I C A L A X O T O M Y AND T H E RESPONSE TO BDNF APPLICATION  37  37 37 38 39 41 41 43 44 44 47 47 47 48 51 53 53 54 54  60  3.1. SUMMARY 60 3.2. INTRODUCTION 62 3.2.1. Atrophy and Death of Rubrospinal Neurons After Axotomy and the Administration of Neurotrophic Factors 62 3.2.2. The Targets for Therapeutic Intervention - Cell Body Versus Axon 63 3.2.3. The Timing of Therapeutic Intervention 65 3.3. OVERVIEW OF EXPERIMENTAL QUESTIONS AND HYPOTHESES 67 3.4. RESULTS 69 3.4.1. Neuronal Atrophy 2 Months Post-Axotomy and the Response to Spinal Cord Application of BDNF 69 3.4.2. Neuronal Atrophy 12 Months Post-Axotomy and the Response to Cell Body Application of BDNF 73 3.4.3. Rubrospinal Neuronal Counts 12 Months Post-Axotomy With Cell Body Application of BDNF 78 3.4.4. Neuronal Counts Following a Re-Axotomy 6 Months After Original Injury 81 3.5 DISCUSSION 85 3.5.1. Chapter Summary 85 3.5.2. Rubrospinal Neuronal Survival Versus Death After Axotomy 85 3.5.3. Counting Techniques for Evaluating Neuronal Numbers 91  vi  3.5.4. Rubrospinal Neuronal Atrophy After Axotomy 3.5.6. Administration o f B D N F to the C e l l Body and to the Injured Spinal Cord CHAPTER 4 - REGENERATION ASSOCIATED GENE EXPRESSION IN T H E C H R O N I C A L L Y INJURED RUBROSPINAL SYSTEM  93 98 100  4.1. S U M M A R Y 100 4.2. I N T R O D U C T I O N 101 4.3. O V E R V I E W O F E X P E R I M E N T A L Q U E S T I O N S A N D H Y P O T H E S E S 103 4.4 R E S U L T S 105 4.4.1. R A G Expression Two Months Post Axotomy with B D N F Applied to Cord 105 4.4.2. R A G Expression Two Months Post Axotomy with B D N F Applied to Brainstem.. 108 4.5 D I S C U S S I O N Ill 4.5.1. Chapter Summary 111 4.5.2. R A G Expression In Response to Refreshment Injury and Spinal Cord Application o f BDNF 112 4.5.3. R A G Expression In Response to C e l l Body Application o f B D N F 114 4.5.4. R A G Expression and the Promotion o f Axonal Regeneration 115 CHAPTER 5 - AXONAL REGENERATION OF CHRONICALLY INJURED RUBROSPINAL NEURONS  118  5.1. S U M M A R Y 118 5.2. I N T R O D U C T I O N 119 5.2.1. Neurotrophic Factors and Axonal Regeneration 120 5.2.2. Modifying the Inhibitory Environment o f the Injured C N S 121 5.3. O V E R V I E W O F E X P E R I M E N T A L Q U E S T I O N S A N D H Y P O T H E S E S 122 5.4. R E S U L T S 124 5.4.1. Axonal Regeneration T w o Months Post-Axotomy with B D N F Applied to Cord... 124 5.4.1 Axonal Regeneration 12 Months Post-Axotomy with B D N F Applied to Brainstem 129 5.5. D I S C U S S I O N 134 5.5.1. Chapter Summary 134 5.5.2. Axonal Tracing for the Evaluation o f Axonal Regeneration After Chronic Injury 134 5.5.3. B D N F and the Promotion o f Axonal Regeneration 138 5.5.4. Peripheral Nerve Transplants and Rubrospinal Axonal Regeneration 141 CHAPTER 6 - TRKB RECEPTORS WITHIN CHRONICALLY INJURED RUBROSPINAL NEURONS AND AXONS  146  6.1. S U M M A R Y 6.2. I N T R O D U C T I O N 6.2.1. Expression o f T r k B Receptors in the Uninjured C N S 6.2.2. Expression o f T r k B Receptors in the Injured C N S 6.3. O V E R V I E W O F E X P E R I M E N T A L Q U E S T I O N S A N D H Y P O T H E S E S 6.4. R E S U L T S 6.5 D I S C U S S I O N 6.5.1. Chapter Summary  146 147 147 148 151 152 161 161  vii  6.5.2. Anterograde Labeling of Rubrospinal Tract 6.5.3. T r k B Receptors on the Axons and C e l l Bodies of Rubrospinal Neurons  C H A P T E R 7 - G E N E R A L DISCUSSION 7.1. S U M M A R Y 7.2. M O D E L I N G O F C H R O N I C I T Y I N S P I N A L C O R D P N J U R Y 7.3. A D M I N I S T R A T I O N O F B D N F A S A T H E R A P E U T I C S T R A T E G Y F O R S P I N A L CORD INJURY 7.4. F U N C T I O N O F T R K B R E C E P T O R S I N T H E C H R O N I C A L L Y I N J U R E D RUBROSPINAL SYSTEM 7.5. F U T U R E D I R E C T I O N S 7.6. C O N C L U S I O N S  BIBLIOGRAPHY  162 163  168 168 170 174 176 181 185  186  Vlll  LIST OF FIGURES Figure 1.1. Schematic o f the obstacles to axonal regeneration after spinal cord injury and potential therapeutic strategies  7  Figure 1.2. Schematic illustration of the difference between standard counting and stereologic counting (eg. disector method) 12 Figure 1.3. Schematic o f retrograde tracing paradigm after partial cord transection Figure 2.1. Two months after cervical axotomy, the scar from previous spinal cord injury is readily visible on the dorsal surface of the cord  30  , 40  Figure 2.2. N e u N immunostaining specifically labels neurons and does not label astrocytes or microglia 50 Figure 2.3. In Situ Hybridization Probes  57  Figure 2.4. G A P - 4 3 anti-sense and sense probes on rubrospinal neurons 7 days after cervical axotomy  58  Figure 2.5. T a l tubulin anti-sense and sense probes on rubrospinal neurons 7 days after cervical axotomy 59 Figure 3.1. Rubrospinal neuronal atrophy is not reversed with any o f the three doses o f B D N F applied to the spinal cord injury site 71 Figure 3.2. B D N F applied to the spinal cord injury site 12 months after cervical axotomy does not reverse atrophy o f injured rubrospinal neurons 75 Figure 3.3. Atrophy o f rubrospinal neurons can be reversed by B D N F twelve months after injury  76  Figure 3.4. B D N F administration normalizes the distribution of cell sizes in the chronically injured red nucleus  77  Figure 3.5. Stereologic counting of the injured and uninjured red nuclei demonstrates that chronically injured rubrospinal neurons remain alive long after cervical axotomy  80  Figure 3.6. FluoroGold retrograde labeling helps to identify the boundaries o f the injured red nucleus, and N e u N immunohistochemistry better identifies atrophic neurons than cresyl violet staining 83 Figure 4.1. G A P - 4 3 expression in the injured red nucleus is increased compared to uninjured after spinal cord application of B D N F and P B S two months after axotomy 106  IX  Figure 4.2. T e d tubulin expression in the injured red nucleus is increased compared to uninjured after spinal cord application o f B D N F and P B S two months after axotomy 107 Figure 4.3. B D N F infusion to the red nucleus 12 and even 18 months after cervical axotomy promotes an increase in G A P - 4 3 expression 109 Figure 4.4. B D N F infusion to the red nucleus 12 months after cervical axotomy promotes an increase in Tod tubulin expression 110 Figure 5.1. Double labeling paradigm (FluoroGold and B D A ) to evaluate regeneration into peripheral nerve transplants two months after cervical injury 127 Figure 5.2. Axonal regeneration data from animals treated two months after axotomy at the spinal cord injury site with P B S or B D N F at three different doses ( L O W , M E D I U M , HIGH)  128  Figure 5.3. Double labeling paradigm (FastBlue and D i l ) to evaluate regeneration into peripheral nerve transplants twelve months after cervical injury  130  Figure 5.4. Double labeling paradigm (FastBlue and B D A ) to evaluate regeneration into peripheral nerve transplants twelve months after cervical injury  131  Figure 5.5. Axonal regeneration data from animals treated with cell body administration o f B D N F or vehicle 12 months after axotomy  132  Figure 5.6. Sagittal section o f spinal cord at the interface between the cord and peripheral nerve graft in a 12 month chronically injured animal 133 Figure 6.1. Full length T r k B receptor immunohistochemistry is maintained on rubrospinal neuronal cell bodies 12 months after axotomy  154  Figure 6.2. F u l l length T r k B receptor immunohistochemistry is maintained on rubrospinal neuronal cell bodies 2 months after axotomy  155  Figure 6.3. Anterograde labeling of with B D A provides consistent visualization o f the rubrospinal tract within the dorsolateral funiculus of the spinal cord  156  Figure 6.4. T r k B immunoreactivity is not maintained on the rubrospinal axons at the site o f spinal cord injury, 2 months after the injury  157  Figure 6.5. T r k B immunoreactivity closely colocalizes with B D A labeled rubrospinal axons on cross-sectional images o f the spinal cord at C l , well proximal to the injury site 159 Figure 6.6.  Control T r k B immunohistochemistry slides demonstrate no specific binding  160  Figure 6.7. The current experimental paradigm o f evaluating injured rubrospinal axons on horizontal sections makes it difficult to determine how far proximally the loss o f T r k B receptors occurs  166  Figure 6.8. Immunoreactivity to full length T r k B appears to be less on the injured side o f the spinal cord compared to uninjured well proximal to the injury site (at C I ) 167 Figure 7.1. Experimental paradigm to characterize the loss o f T r k B receptors on rubrospinal axons 182  LIST O F ABBREVIATIONS B D N F - brain derived neurotrophic factor CAP-23 - cortical cytoskeleton-associated protein-23 C G R P - calcitonin gene related peptide C N S - central nervous system GAP-43 - growth associated protein-43 G D N F - glial derived neurotrophid factor cpm - counts per minute I S H - in situ hybridization M A G - myelin associated glycoprotein O M g p - oligodendrocyte-myelin glycoprotein NT-3 - neurotrophin-3 P N S - peripheral nervous system R A G - regeneration associated gene S E M - standard error o f the mean S S C - sodium chloride / sodium citrate T r k B - tropomyosin receptor kinase B  Xll  Statement of Original Contributions This thesis work contains material that has been published in the following:  K w o n B K , L i u J, Messerer C , Kobayashi N R , M c G r a w J, Oschipok L , Tetzlaff W (2002) Survival and regeneration o f rubrospinal neurons 1 year after spinal cord injury. Proceedings o f the National Academy o f Sciences, U S A 99: 3246-3251. K w o n B K , L i u J, Oschipok L , Tetzlaff W (2002) Reaxotomy o f chronically injured rubrospinal neurons results in only modest cell loss. Experimental Neurology, 177: 332-337. K w o n B K , L i u J, Oschipok L , Teh J, L i u Z W , Tetzlaff W (2004) Rubrospinal neurons fail to respond to brain-derived neurotrophic factor applied to the spinal cord injury site 2 months after cervical axotomy. Experimental Neurololgy, epub Jul 3, 2004.  The thesis author, Brian K w o n , was the primary researcher for all results presented in this thesis. Assistance with surgical procedures was provided by J L i u . chronically injured rubrospinal experiments were performed  Pilot studies on the 1 year by N R Kobayashi.  Initial  development and validation o f the physical disector technique for cell counting was performed by C Messerer.  Expertise with in situ hybridization studies and immunohistochemistry  provided by L Oschipok and J M c G r a w , respectively.  was  Technical assistance was provided by J  Teh and Z W L i u .  The above statements and assessment o f work done by the thesis author and collaborators are justified by the senior author (supervisor o f the thesis author), Dr. W Tetzlaff.  W . Tetzlaff, M D , P h D  XIII  Acknowledgements  A s this doctoral work comes to its conclusion, there are many people who I feel enormously indebted to for their help, kindness, and support throughout the last four years. Firstly, to my Professor and supervisor, Dr. Wolfram Tetzlaff, who has steadfastly guided and mentored me through this P h D experience. I cannot thank you enough for your trust in taking me on as an otherwise un-tested laboratory novice in July 2000, and for introducing me to the world o f basic science.  I look forward to many more years o f fruitful collaboration as your  friend and colleague at the International Collaboration on Repair Discoveries. I am deeply grateful to my clinical colleagues and mentors in the Department o f Orthopaedics, with whom I take great pride in working with.  To Dr. Marcel Dvorak, whose  remarkable vision for the Spine Program I bought into some five years ago, and who has remained an unbelievably ardent supporter, advocate, and mentor throughout this time. To Dr. Clive Duncan and Dr. T o m Oxland - how can one not succeed with such role models in the department?  A n d finally, to my clinical colleagues o f the Combined Neurosurgical and  Orthopaedic Spine Program, Dr. Charles Fisher, Dr. Michael Boyd, and Dr. Peter W i n g - one could not ask for a more talented, supportive, and enjoyable group o f partners. One o f the most rewarding aspects o f my P h D experience in the Tetzlaff laboratory has been the friendships and collaborations with my fellow graduate students.  After years o f  orthopaedic residency, I came to appreciate the dedication, industriousness, and brilliance o f this group - students who work evenings and weekends not for money, recognition, or because they are forced to carry a pager, but rather, for the love o f science and a commitment to this important research.  To them all, I take a respectful bow.  In particular, I would like to thank Dr. Jie L i u  xiv  for his undying support and technical assistance, and Loren Oschipok for his help and friendship through the years.  A n d to Dave Stirling, Egidio Spinelli, Carmen Chan, Kourosh Khodarahmi,  Ward Plunet, Clarrie L a m , John M c G r a w , L o w e l l M c P h a i l , Jaimie Borisoff, Chris M c B r i d e and Anthony Choo - thank you for your tremendous support, helpful comments, and many late night conversations in the lab. To Dr. John Steeves, Director o f I C O R D , many thanks for your vision and leadership in creating an institute that I hope to play a large part in. A n d to the support staff at I C O R D , Cheryl Niamath, Jeremy Green, and E m i l y Williamson - thank you for keeping me (and the rest o f us for that matter!) in line, afloat, and running in the right direction. I should also like to thank the other members o f my P h D Committee, Dr. Jane Roskams and D r . T i m O'Connor, for their helpful comments through the years.  Finally, a thank you to the Canadian Institutes for Health  Research, the Neuroscience Canada Foundation, and the generous donation o f Gowan and Michele Guest for their financial support o f my graduate studies. In closing, this would not be at all possible without the love, support, and encouragement of my family - thank you, M o m and Dad, for giving me the tools to do this, and for always being behind me at every step. Y o u give me much to strive for.  To my sister, Janice, whose strength I  derive much inspiration from, and whose friendship I could not be without - I have since gotten over the fact that the brains, athleticism, and musical talent all went your way.  A n d to C o -  thank you for your uncompromising love, support, and for being my #1 fan when I needed it most.  XV  This Thesis is dedicated to Mr. Rick Hansen, Man in Motion, and to the thousands of individuals with chronic spinal cord paralysis whose cause he champions with unimaginable courage, dedication, and commitment.  Over the last four years, he and  my many patients who suffer this unfortunate injury have taught me innumerous lessons that are not found in the books. You were right Rick, the end is just the beginning....  1  - CHAPTER 1 BACKGROUND  1.1.  Each year, over  OVERVIEW  10,000 North Americans sustain  a traumatic spinal cord  injury  (Nobunaga et al., 1999) and are left with one of the most physically and psychologically devastating impairments known to mankind.  The majority o f such paralyzed individuals are  under the age o f 30 and are otherwise healthy at the time of their injury (Sekhon and Fehlings, 2001).  With advances in contemporary medical and surgical care, the survival rate of such  individuals has improved dramatically, resulting in over 250,000 North Americans currently living with chronic spinal cord paralysis.  Beyond the incalculable losses to the individual, the  societal costs o f the medical, surgical, and rehabilitative care for patients with acute and chronic spinal cord injuries are enormous - estimated over a decade ago at four billion dollars per annum (Stripling, 1990). The compelling need for therapies for individuals with spinal cord injuries has sparked great interest in the neuroscience community, where it is widely believed that the cumulative research efforts of the basic science and clinical communities w i l l produce effective therapies in the imminent future. The failure o f paralyzed individuals to regain neurologic function reflects the failure o f disrupted axons to regenerate and re-innervate their distal targets after spinal cord injury.  A  tremendous amount has been learned about what occurs within the spinal cord after injury, the obstacles that are established to impede axonal regeneration and repair, and potential therapeutic interventions to overcome them (reviewed in section 1.3).  Conceptually, the obstacles to  2  regeneration can be divided into the limited intrinsic regenerative response on behalf of CNS neurons, and the extrinsic elements within the injured CNS that inhibit axonal regeneration (reviewed by Kwon and Tetzlaff, 2001 and Steeves and Tetzlaff, 1998).  Following spinal cord  injury, neurons within the brain or brainstem whose axons are disrupted in the spinal cord often undergo severe atrophy and may possibly die (reviewed in section 1.4).  The molecular  mechanisms that are triggered within CNS neurons to promote axonal growth after injury may not be qualitatively and/or quantitatively sufficient to promote long-distance axonal regeneration (reviewed in section 1.5). Many neurotrophic factors exist and have important roles in (amongst many things), neuronal survival and axonal growth, and have thus been utilized as experimental therapies to enhance the regenerative competence of injured CNS neurons. Neurotrophic factors and their receptors are reviewed in sections 1.6 and 1.7. The study of the neurobiology of spinal cord and the development of such strategies to promote recovery are dependent upon animal models of injury (reviewed in section 1.8). In this regard, I have chosen to use the rubrospinal system in adult rats (reviewed in section 1.9). The past two decades have witnessed the emergence of a number of experimental therapeutic strategies for spinal cord injury; some of which have demonstrated modest potential in animal models of acute spinal cord injury.  What has become increasingly evident over the  past decade is that while many experimental therapies appear to be very promising when applied acutely at or near the time of injury, their effectiveness is reduced when applied chronically after the injury. This loss of effectiveness over time is an issue of obvious and critical relevance for the many individuals with chronic spinal cord injury. T h u s , the overall objective of this thesis is to evaluate some of the challenges that impede axonal regeneration i n a chronic spinal c o r d i n j u r y setting, and develop therapeutic strategies for this condition.  3  1.2. SUMMARY OF HYPOTHESES AND OBJECTIVES  The overall hypothesis of this thesis is that axonal regeneration can be achieved by chronically injured CNS neurons with the appropriate administration of neurotrophic factors. The overall objective of these experiments was to investigate the cell body response of chronically injured CNS neurons to determine the obstacles imposed by this condition on axonal regeneration. The specific objectives were as follows. Using a cervical injury model of the rat rubrospinal system I attempted to: 1. Test the hypothesis that providing trophic support in the form of BDNF to chronically injured rubrospinal neurons at the site of spinal cord injury could reverse neuronal atrophy if applied in the correct dose. (Chapter 3) 2. Test the hypothesis that providing trophic support in the form of BDNF to chronically injured rubrospinal neurons directly to their cell bodies could reverse neuronal atrophy. (Chapter 3) 3. Test the hypothesis that chronically injured rubrospinal neurons are not dying after cervical spinal cord injury. (Chapter 3) 4.  Test the hypothesis that regeneration associated gene expression can be stimulated in chronically injured rubrospinal neurons provided with trophic support, (chapter 4)  5.  Test the hypothesis that axonal regeneration of chronically injured rubrospinal neurons can be elicited by providing them with trophic support (chapter 5)  6. Test the hypothesis that responsiveness to of chronically injured rubrospinal neurons to exogenously. provided neurotrophic support is related to the expression of the appropriate receptors (chapter 6)  4  In this Background chapter, I w i l l attempt to briefly review some pertinent considerations to the testing o f these hypotheses.  Although the scope o f these topics is impossible to address  comprehensively within the pages o f this chapter, their relevance to the experiments and their interpretation warrants a brief - albeit somewhat superficial - overview here.  I w i l l begin by  providing a general overview o f the obstacles to axonal regeneration in the injured C N S (Section 1.3).  Then, as a number o f experiments evaluated the response o f rubrospinal neurons to  cervical axotomy, I w i l l discuss issues o f neuronal atrophy and death after axotomy, and methods by which death/survival are measured (Section 1.4).  Further to this discussion o f the  rubrospinal response to axotomy, I w i l l provide an overview o f the "intrinsic determinants" o f axonal growth; specifically, the cell body response to axotomy and some o f the genetic components o f this response, including the expression o f G A P - 4 3 and Tccl tubulin (Section 1.5). The primary intervention that was tested in these experiments was the administration o f the neurotrophic factor, brain-derived neurotrophic factor ( B D N F ) .  I therefore w i l l briefly  discuss neurotrophic factors and provide some background on B D N F specifically (Section 1.6). A s the tropomyosin receptor kinase (Trk) family o f receptors are important for mediating the biologic response o f the classic neurotrophins, I w i l l then discuss Trk receptors and T r k B receptors specifically (Section 1.7). The experiments performed to test the aforementioned hypothesis were largely in vivo experiments, and as such, I w i l l provide an overview o f how spinal cord injury is modeled in the animals, and how axonal regeneration is evaluated in such models (Section 1.8). The neuronal system that I focused on was the rubrospinal system, and therefore I w i l l review some aspects o f rubrospinal anatomy and function (Section 1.9).  Finally, I w i l l summarize the rationale for the  experimental models applied in this thesis (Section 1.10).  5  1.3. PROMOTING A X O N A L REGENERATION IN THE CNS 1.3.1. Obstacles to Axonal Regeneration and Therapies to Overcome Them The fact that surgeons suture together peripheral nerves i n the hand that have been completely transected, but yet offer a poor prognosis to the completely paralyzed individual whose spinal cord has been only bluntly contused, reflects very different expectations about the regenerative capacities o f neurons within the peripheral and central nervous systems. does axonal regeneration fail in the CNS while it occurs fairly readily in the PNS?  So why One could  postulate that this failure is related either to the fact that C N S neurons simply lack the ability to regenerate axons after injury, or alternatively that there exists something i n the spinal cord environment that inhibits C N S neurons from doing so. extent, both o f these are true  (Fawcett, 1998).  It has become apparent that, to some  I w i l l speak more o f the intrinsic growth  propensity o f neurons within the C N S (section 1.4). Suffice to say now, that while the ability o f P N S neurons to regenerate is accompanied by a host o f changes i n gene expression (and i n the extrinsic environment after axotomy), such a gene response i n C N S neurons is transient and generally insufficient for long-term axonal growth (Fernandes and Tetzlaff, 2000).  Enhancing  this limited intrinsic growth propensity o f C N S neurons therefore has a strong rationale for reparative therapies, which would include such strategies as delivering growth factors to the neurons (Kobayashi et al., 1997, K w o n et al., 2002b) and the modulation o f intracellular c A M P (Spencer and Filbin, 2004). Recent years have produced an enormous amount o f insight into some o f the inhibitory molecules within the C N S that make the cord less permissive to axonal regeneration than peripheral nerves (Reviewed by K w o n et al., 2002a).  The two major impediments to axonal  regeneration in the injured spinal cord are C N S myelin and the glial scar/cyst that forms at the  6  injury site.  Within C N S myelin, a number o f molecules that inhibit axonal growth have been  identified and characterized, including Nogo (Chen et al., 2000, GrandPre et al., 2000, Prinjha et al.,  2000),  myelin  associated  glycoprotein  ( M A G ) (McKerracher  oligodendrocyte-myelin glycoprotein (OMgp) (Wang et al., 2002).  et  al.,  1994),  and  Recognizing that C N S  myelin may contain many more as-yet unidentified inhibitory molecules provides the rationale for immunological disruption o f myelin as a potential therapeutic strategy (Dyer et al., 1998). Within the glial scar, astrocytes form a physical barrier to axonal growth and may contribute to the expression o f a number o f inhibitory molecules such as chondroitin sulphate proteoglycans (Morgenstern et al., 2002). Interventions directed at attenuating the inhibitory effect o f these environmental impediments appear promising in their potential to allow axonal regeneration to occur.  In order to "bridge" the inhibitory spinal cord injury site, numerous cellular substrates  are emerging as potential candidates to facilitate axonal regeneration (reviewed by Bunge, 2000). These  would include fetal tissue transplants, embryonic  stem  cells, genetically  fibroblasts, Schwann cells, and olfactory ensheathing cells. (Figure 1.1)  altered  F i g u r e 1.1. Schematic o f the obstacles to axonal regeneration after spinal c o r d injury a n d potential therapeutic strategies.  OBSTACLES TO REGENERATION 1. POOR REGENERATIVE R E S P O N S E at the level of the cell body  2. ENVIRONMENTAL INHIBITORS at the level of the injured axon  Failure to sufficiently express regeneration associated genes and trophic factors eg: GAP-43, CAP-23, Ta1-Tubulin BDNF, FGF-2 At  ofneu'ronf  h  / ^  Glial Scar eg: chondroitan sulfate proteoglycans semaphorins, ephrins Cyst / Cavitation - neuronal and glial death  \  Growth Cone Signalling ess^ -Rho/ROCK inhibition cAMP, cGMP ^ / yy ^ Neurotrophic Factors ^ - BDNF, FGF-2, NT3  jfty  57  Myelin Inhibitors eg: NOGO, MAG, OMgp  /(f V // / /  d  N ^ \ ^ \  Myelin Inhibition - Anti-NOGO antibodies Immunologic disruption , „ Glial Scar Degradation - ChondroitinaseABC  -  Cellular Bridges -Fetal tissue -Schwanncells - stem cells - Olfactory ensheathing cells  THERAPEUTIC STRATEGIES TO PROMOTE REGENERATION  8  1.4. N E U R O N A L S U R V I V A L , A T R O P H Y , A N D D E A T H A F T E R A X O T O M Y  1.4.1. Introduction Fundamentally, the neuropathology associated with spinal cord injury is the result o f the mechanical impact (primary injury) and subsequent pathophysiologic processes  (secondary  injury) disrupting ascending and descending axons within the spinal cord, with the additional demise o f neurons and glial cells at and adjacent to the injury site contributing to the neurologic impairment (reviewed by K w o n et al., 2004b). While much work and attention is directed to these pathologic changes that occur within the spinal cord at the site o f injury, it is important to take into account what is happening to the supraspinal neurons whose axons are disrupted within the spinal cord. Herein I w i l l discuss the issue o f neuronal survival and cell size after axotomy. Apart from the issue o f neuronal survival, the gene expression changes relevant to axonal regeneration that occur in response to axotomy w i l l be reviewed elsewhere (section 1.5).  1.4.2. N e u r o n a l S u r v i v a l and C e l l Size are Influenced B y N e u r o t r o p h i c Factors Neuronal survival after axotomy highlights some important differences between neurons of the central and peripheral nervous systems.  C N S neurons appear to receive substantially less  neurotrophic support after axotomy than their P N S counterparts.  Acutely axotomized P N S  neurons enjoy an abundant supply o f neurotrophic factors from Schwann cells and macrophages (ie. non-target tissue) (Funakoshi et al., 1993, Hoke et al., 2002) and may also upregulate their own production o f both neurotrophic factors and neurotrophic factor receptors (Ernfors et al., 1993, Kobayashi et al., 1996, Piehl et al., 1994). In contrast, while neurotrophic factors are in fact produced by oligodendrocytes (Wilkins et al., 2001, Wilkins et al., 2003), it would appear that the extent to which they support neuronal survival within the C N S after axotomy is indeed  9  quite limited. Rubrospinal neurons, for example, undergo significant atrophy (and possibly also death - see further discussion) after cervical axotomy.  This atrophy can be prevented by the  intraparenchymal infusion o f brain derived neurotrophic factor ( B D N F ) directly to the neuronal cell bodies one week after injury (Kobayashi et al., 1997, Fukuoka et al., 1997). This suggests that while sufficient trophic support may not be arriving at the cell bodies v i a their axons, the cell bodies themselves remain responsive to exogenously applied neurotrophins or they respond indirectly through some effect that the neurotrophins are having on surrounding tissue (discussed further in Chapter 7). A l o n g the same lines, the exogenous intraparenchymal infusion o f B D N F to the motor cortex prevents the near 50% loss o f corticospinal neurons after axotomy at the level of the internal capsule (Giehl and Tetzlaff, 1996). It is well recognized that rubrospinal neurons undergo significant atrophy after cervical axotomy (Kobayashi et al., 1997, K w o n et al., 2002b, K w o n et al., 2002c). Such atrophy after axotomy is certainly not unique to the rubrospinal system, and has been reported in other neuronal populations within the central nervous system such as corticospinal neurons (Giehl et al., 1997) and basal forebrain cholinergic neurons (van der Zee and Hagg, 2002).  A s stated  earlier, the exogenous delivery o f specific neurotrophic factors has the potential to prevent or reverse some o f this atrophy, suggesting that, similar to cell survival, the aspects o f cellular physiology that contribute to the maintenance o f cell size are also dependent on neurotrophic factor support. For the rubrospinal system specifically, Kobayashi et al. reported that the acute administration o f B D N F to the cell bodies o f rubrospinal neurons can prevent their atrophy after cervical axotomy (Kobayashi et al., 1997).  Furthermore, in the experiments o f this thesis, I  observed that the similar cell-body application o f B D N F one year after injury could reverse the atrophy o f rubrospinal neurons ( K w o n et al., 2002b).  The exact mechanism by which cell  10  atrophy occurs and is reversed is not entirely understood, but may relate to the expression and synthesis o f cytoskeletal proteins such as tubulins and neurofilament (McKerracher et al., 1993, Bisby and Tetzlaff, 1992, Bregman et al., 1998). In support o f this association, Fernandes et al. observed that rubrospinal neurons that increased their expression o f G A P - 4 3 and T a l tubulin and regenerated through peripheral nerve transplants were also the largest neurons within the injured red nucleus (Fernandes et al., 1999).  1.4.3. Measuring Neuronal Death After Axotomy The measurement o f neuronal death (or conversely, the counting o f surviving cells remaining) after axotomy requires not only careful histologic techniques but also careful counting methods.  This is by no means an exact science.  A s stated by Guillery and Herrup,  "counting objects in histological material is an exercise in estimation" (Guillery and Herrup, 1997).  In this regard, numerous histologic techniques are available to visualize neurons (eg.  immunohistochemistry, retrograde labeling, Nissl staining), and a number o f techniques have been developed to address the very practical problem that the majority o f neuronal systems contain such large numbers o f neurons that an absolute numerical determination is not feasible. Fundamentally, the problem is that when tissue is sectioned, cells within it may appear in more than one section, which w i l l lead to the overestimation o f the total number o f cells in the population, particularly i f the cells are large.  T o accommodate for this error, one can apply a  mathematical correction that incorporates the height o f the cells within the section and the thickness o f the section, as originally proposed by Abercrombie (Abercrombie, 1946). A n alternative "empirical method" employs a formula that incorporates an estimation o f the number of sections a single cell is visualized within i n order to account for the potential overestimation of cell number (Coggeshall et al., 1984, Coggeshall and Chung, 1984).  In theory, the weakness  11  of the former method is that it requires an estimation o f the height o f the cells within the section (and an assumption o f cell sphericity), while the latter requires an estimation o f the number o f sections per cell (and an assumption o f cell shape). Furthermore, one o f the major considerations in the application o f these mathematical corrections in studies o f neuronal populations after axotomy is the well recognized atrophy that occurs after axotomy.  Stereometric methods had been developed that, in theory require no a  priori knowledge o f the size or shape o f the neurons being counted.  In principle, stereologic  counting compares the cells in one section against the cells in an adjacent section, and by avoiding the double-counting o f cells one arrives at a more true account o f the number o f cells, irrespective o f their size or shape (West, 1999).  The problem that is generated by not  performing stereologic counts in the setting o f atrophy is schematically illustrated in Figure 1.2. There has been considerable debate over the past decade about the validity o f the various counting techniques (Guillery and Herrup, 1997, Benes and Lange, 2001, West and Slomanka, 2001). With no definitive resolution o f the controversy surrounding which counting technique is best, it would appear reasonable to chose the counting technique - recognizing both its strengths and limitations - after accumulating as much prior knowledge o f the behavior o f the neuronal system in question and after deciding upon the research question to be answered.  In this thesis,  I applied a stereologic counting technique (the physical disector method, described in more detail in Chapter 2) to count rubrospinal neurons after axotomy.  12  F i g u r e 1.2.  Schematic illustration of the difference between standard counting and  stereologic counting (eg. disector method). This schematic demonstrates the difference between performing standard and stereologic counting and how the atrophy o f neurons after axotomy might be mis-represented as death i f standard counts alone are used to determine the number o f neurons remaining. In this schematic, the dark grey section ( A ) is the index section that is counted.  In the normal, uninjured state  (above), a standard count would produce a cell count o f 8 (all 8 cells would be visualized in the index section). However, a stereologic count o f the same tissue would compare the cells in A against those visualized in B and then exclude from A those cells that were visualized in both sections.  So in this case, cells 5, 6, 7 and 8 would be seen in both sections, and the  "stereologic" count o f section A would be only 4. N o w , after the axotomy, the neurons atrophy.  Again, performing a standard count on  section A would produce a cell count o f only 5 (cells 2, 3, 4, 6, and 8).  So for the same section  o f tissue, the death o f neurons would be estimated to be 3/8, or 37.5%, merely from atrophy alone. However, a stereologic count comparing section A against B would arrive at a cell count of 4, unchanged from the non-atrophic state.  13  Figure 1.2  Normal Sized Cells A B  "©  CD  0—Cr  1  Standard Count  8  Stereologic Count  -*• 8 cells  4 cells  5 cells  4 cells  Axotomy  A B  O  4  ^^^^^^  o Atrophic Cells  14  1.5. I N T R I N S I C D E T E R M I N A N T S O F A X O N A L G R O W T H A F T E R C N S I N J U R Y  1.5.1. Introduction  Rather pivotal to the issue of promoting neurologic recovery after spinal cord injury is the actual capacity of CNS neurons to regenerate their axons after injury. The ability of CNS neurons to regenerate their injured axons was first described at the turn of the century in the laboratory of the Spanish neuroscientist Ramon y Cajal (Ramon y Cajal S, 1928). Experiments in the 1980s by Aguayo, David, and Richardson (David and Aguayo, 1981, Richardson et al., 1984, Richardson et al., 1980) reiterated and extended these findings by demonstrating that when presented with permissive conditions (ie. a peripheral nerve graft), some CNS neurons are indeed able to regenerate their injured axons.  It has been subsequently shown that the even in non-  permissive conditions, it is possible to influence the intrinsic growth state of the neuron in such a manner as to still promote axonal regeneration (Spencer and Filbin, 2004, Fischer et al., 2004). Characterizing and harnessing this intrinsic ability to regenerate axons after injury is thus a key element in strategies to promote functional recovery after spinal cord injury.  1.5.2. T h e C e l l B o d y Response to A x o t o m y  After axotomy, the intrinsic ability of the neuron to effect long-distance axonal regeneration is thought to be closely tied to molecular and biochemical activity within the cell body. A study by Richardson and Issa in 1984 demonstrated that the response of the neuronal cell body to axonal injury plays a pivotal role in the regenerative capacity of the neuron (Richardson and Issa, 1984). In this study, the central spinal projection of a dorsal root ganglion cell was shown to regenerate into a peripheral nerve transplant only after the peripheral projection had been previously transected, demonstrating that the cell body response evoked by  15  transection o f the peripheral projection was in some manner essential for the regeneration o f the central spinal projection.  subsequent  It was subsequently demonstrated that transection  o f the peripheral axon induces changes in gene expression in the parent neuron which are not seen after injury o f the central process (Schreyer and Skene, 1993).  These observations  suggested that these neurons did in fact possess the appropriate regenerative machinery, but required the initial stimulus o f the peripheral transection to activate it and thus become "regeneration-capable".  Significant interest has therefore been generated to delineate these gene  regulatory mechanisms that must be initiated at the cell body level, with hopes that such understanding w i l l allow for strategies to persuade otherwise incompetent neurons to regenerate their injured axons.  We now know that this regenerative competence and the response to  axotomy is unequal amongst different neuronal types (Woolhead et al., 1998, Morrow et al., 1993), and also varies significantly with neuronal age (Chen et al., 1995) and distance from the site o f injury (Fernandes et al., 1999, Richardson et al., 1984, Mason et al., 2003). In summary, while injured axons may intrinsically be capable o f short disorganized terminal sprouting and in some cases even slow axonal elongation (Bisby et al., 1996, Andersen and Schreyer, 1999), the sustained and distant growth o f axons requires the participation o f the cell body, manifested by the expression o f a number o f regeneration associated genes (Smith and Skene, 1997, Fernandes and Tetzlaff, 2000).  16  1.5.3. Regeneration Associated Gene Expression A number o f genes have been shown to be up-regulated or constitutively expressed in association with axonal growth, both during development and during axonal regeneration.  These  have collectively been termed "regeneration associated genes" or R A G s (reviewed by Fernandes and Tetzlaff, 2000).  The increases in R A G expression that occur in response to axotomy o f  C N S neurons are weaker and more transient (or altogether absent) than those that occur in the P N S , and the successful regeneration o f the latter implicate these R A G s as important mediators o f axonal regeneration (reviewed by Plunet et al., 2002).  Regeneration o f the central (spinal)  axons o f the dorsal root ganglion has been demonstrated  after the combined  transgenic  overexpression o f G A P - 4 3 and C A P - 2 3 , but not when each o f these genes was expressed alone (Bomze et al., 2001). Interestingly, the number o f regenerating axons with the dual G A P - 4 3 and C A P - 2 3 overexpression was still only a third o f that observed after a pre-conditioning peripheral nerve axotomy, which presumably initiates the " f u l l " cell body response.  The results o f this  study highlight two important considerations: that R A G expression is a prerequisite rather than an associative phenomenon for axonal elongation, and that a complex, coordinated expression o f a battery o f genes (rather than just one or two) is necessary for the optimal growth response. The pace at which the many components o f this molecular response to axotomy are identified in the future w i l l undoubtedly be facilitated by gene chip technology (Fan et al., 2001, Gris et al., 2003).  The products o f these genes include transcription factors such as c-jun which mediates subsequent gene expression (Jenkins et al., 1993, Herdegen et al., 1997), cell adhesion molecules such as L l and N C A M involved in growth cone guidance (Becker et al., 1998, Jung et al., 1997, Woolhead et al., 1998), cytoskeletal proteins involved in axonal extension such as T a l - t u b u l i n  17  (Fernandes et al., 1999, M i l l e r et al., 1989), and growth cone proteins involved in mediating axonal guidance and synaptic plasticity such as G A P - 4 3 and C A P - 2 3 (Frey et al., 2000, Skene, 1989).  The importance o f these genes in axonal regeneration has generally been extrapolated  from the correlation o f their upregulation with axonal growth and the absence o f their expression with regenerative failure (Anderson and Lieberman, 2000, Fernandes et al., 1999, Tetzlaff et al., 1991, Becker et al., 1998, Anderson and Lieberman, 2000, Schreyer and Skene, 1993).  In this  thesis, I have evaluated the expression of Tod-tubulin and G A P - 4 3 as a manifestation of the regenerative response to axotomy of rubrospinal neurons.  1.5.4. GAP-43 GAP-43  (also  known  as  B50, neuromodulin,  and  FI)  is  a  calcium-regulated  phosphoprotein that is closely related to initial axonal outgrowth during differentiation and successful axonal regeneration in both the C N S and P N S . It is a major constituent o f the developing growth cone, where it appears to be involved in axonal steering and the formation o f new synaptic connections (reviewed by Benowitz and Routtenberg, 1997). B y modulating the assembly o f phosphoinositide lipid PI(4,5)P2 containing rafts on the inner surface o f the cell membrane and its interactions with calmodulin and G proteins, G A P - 4 3 appears to play an important role in the regulation o f the actin cytoskeleton (Caroni, 2001, Frey et al., 2000, Laux et al., 2000).  G A P - 4 3 knockout mice exhibit defects in axonal guidance (Maier et al., 1999), and  growth cones o f primary sensory neurons depleted o f G A P - 4 3 in vitro demonstrate poor adhesion and limited resistance to inhibitory substrates (Aigner and Caroni, 1995).  Conversely,  the transgenic overexpression o f G A P - 4 3 in adult mice induces significant spontaneous axonal sprouting (Aigner et al., 1995).  18  While P N S neurons express high levels o f G A P - 4 3 during development and regeneration (Skene, 1989), the ability o f C N S neurons to increase and sustain G A P - 4 3 expression after axotomy is generally limited (reviewed by Fernandes and Tetzlaff, 2000).  Within the C N S ,  rubrospinal neurons were observed to increase the expression o f G A P - 4 3 after axotomy at the cervical but not at the thoracic level, and correspondingly only regenerated into peripheral nerve transplants inserted into the cervical spinal cord but not the thoracic spinal cord (Fernandes et al., 1999).  Similar observations correlating regeneration into peripheral nerve transplants and G A P -  43 expression have been made in other C N S neuronal systems (Vaudano et al., 1995, Woolhead et al., 1998).  In interpreting these findings, however, one should not forget that the expression  of a number o f other R A G s such as L l and c-jun and a host o f as yet unidentified genes may also be stimulated in response to axotomy (Chaisuksunt et al., 2000).  Furthermore, there are  examples in which the overexpression o f G A P - 4 3 alone was not sufficient to  promote  regeneration (Buffo et al., 1997, Mason et al., 2000, Bomze et al., 2001). Conversely, G A P - 4 3 knockout mice have a grossly normal nervous system with axonal growth characteristics not unlike wild-type mice, but they demonstrate abnormal axonal pathfinding (Strittmatter et al., 1995).  These findings suggest that while much evidence points to G A P - 4 3 being an indicator  of neuronal growth propensity, it is neither absolutely necessary for axonal growth due to other compensatory mechanisms, nor is it by itself sufficient to promote axonal growth.  19  1.5.5.  Ted Tubulin Microtubules are one o f the most important constituents o f the neuronal cytoskeleton,  playing an essential role in the development and maintenance o f neuronal morphology, neurite outgrowth, and intracellular transport (reviewed by Laferriere et al., 1997).  Microtubules are  formed by the polymerization o f tubulin molecules into protofilaments .which then laterally associate to form a hollow tube.  Most commonly, tubulin molecules are comprised o f an a and  a p subunit, although new members o f the tubulin superfamily have been discovered more recently, including gamma (y), delta (8), epsilon (s), zeta (Q, eta (r|), and iota (i) tubulin (reviewed by Dutcher, 2003). A number o f a-tubulin and P-tubulin isoforms have been identified in neuronal tissue. The expression o f Tod tubulin m R N A was reported to be very high in the embryonic nervous system, particularly in neurons actively undergoing neurite extension (Miller et al., 1987).  This  study also demonstrated that P C 12 cells stimulated in vitro by N G F significantly increased their Tod  tubulin m R N A expression concurrent with their differentiation and their extension o f  neurite processes.  M i l l e r and colleagues subsequently demonstrated a rapid increase in Tod  tubulin m R N A expression in facial and sciatic motor neurons after axotomy, which remained elevated until axonal growth re-established terminal connections (Miller et al., 1989).  Tetzlaff  and colleagues reported that rubrospinal neurons also upregulate T o d tubulin expression after cervical axotomy (Tetzlaff et al., 1991), findings that were extended by Fernandes  and  colleagues who demonstrated that while T o d tubulin expression was increased after cervical axotomy o f the rubrospinal tract, it was not significantly increased by thoracic axotomy (Fernandes et al., 1999).  The expression (and lack thereof) o f T o d tubulin in the latter study  corresponded with the ability to regenerate into peripheral nerve transplants inserted into the  20  cervical spinal cord but not the thoracic cord.  Combined, these studies suggest that T a l tubulin  is an aspect o f the regenerative response o f axotomized neurons, with an increase in its expression correlating with a growth propensity.  21  1.6. NEUROTROPHIC FACTORS 1.6.1. Introduction Neurotrophic factors are proteins that exert considerable influence on a wide spectrum o f processes within the developing and mature nervous system, including neuronal survival, axonal growth, synaptic plasticity, and neurotransmission (reviewed by Tuszynski, 1999).  Since the  identification o f the first neurotrophic factor, nerve growth factor ( N G F ) by Levi-Montalcini and Hamburger over 5 decades ago (Lev-Montalcini and Hamburger, 1951, Lev-Montalcini and Hamburger, 1953), dozens o f such factors have been uncovered.  The classical description o f  neurotrophic factor action on neurons is that o f a target-derived, retrogradely transported signal delivered back to the neuronal soma by the axon. This concept has been expanded somewhat, as it has become apparent that neurons receive trophic support not only from their distal targets in a retrograde fashion, but also from afferent neurons in an anterograde manner, from adjacent or ensheathing glial cells in a paracrine manner, and even from themselves in an autocrine manner (reviewed by Korsching, 1993).  While the term "growth factors" is used loosely to collectively  describe these proteins, they are structurally very diverse, and as such, have distinct targets within the nervous system, and exert their activity through different receptors and signalling pathways.  The responsiveness o f a particular cell population within the nervous system to a  specific neurotrophic factor is therefore partially dependent on the expression o f the appropriate receptors. Because o f their wide-ranging influence on many aspects o f neural biology, neurotrophic factors have been extensively studied as a potential therapeutic strategy for promoting neural repair after spinal cord injury (Jones et al., 2001).  Within this context, it is important to  recognize the extremely diverse nature o f neurotrophic factors and their functions. However, the  22  exogenous delivery o f a single trophic factor - while demonstrating some potential as a therapeutic strategy for spinal cord injury - is unlikely by itself to elicit a comprehensive regenerative response in all relevant neuronal and glial cell populations. The "classic" family o f neurotrophic  factors,  otherwise  referred  to  as  neurotrophins,  includes  NGF, BDNF,  Neurotrophin-3 (NT-3), NT-4/5, N T - 6 , and N T - 7 .  1.6.2. Brain Derived Neurotrophic Factor (BDNF) Brain derived neurotrophic factor ( B D N F ) was identified by Barde and colleagues in 1982 as a 12.3 k D a protein purified from pig brain that promoted both the survival and neurite outgrowth o f embryonic chick sensory neurons in an additive fashion to N G F (Barde et al., 1982).  The B D N F gene was subsequently cloned and sequenced by Barde's laboratory in 1989,  where it was also found that the brain and spinal cord contained much larger amounts o f B D N F m R N A than N G F (Leibrock et al., 1989).  The temporal pattern o f B D N F m R N A expression  demonstrates a higher level o f expression during adulthood than during development (exactly in contrast to N T - 3 expression), suggesting that B D N F may be more involved as a maturation and maintenance  factor later in development, while N T - 3 plays an earlier role in neuronal  development (Maisonpierre et al., 1990).  The exact role that B D N F (and other neurotrophic  factors for that matter), plays in the developing nervous system is difficult to elucidate in knockout mice lacking the B D N F gene given the compensation and redundancy that undoubtedly exists with many other neurotrophic factors.  Such B D N F null mice demonstrate substantial loss  o f neurons in a number o f sensory ganglia, including the D R G , trigeminal, vestibular, and nodose ganglia, but no loss o f neurons in the cortical and hippocampal regions o f the brain and motor neurons in the spinal cord (Ernfors et al., 1994, Jones et al., 1994).  23  Neuronal populations known to be responsive to B D N F include sensory neurons o f the D R G , nodose ganglion and geniculate ganglion, dopaminergic neurons o f the substantia nigra, basal forebrain cholinergic neurons, hippocampal neurons, cerebellar granule cells, and retinal ganglion cells (Korsching, 1993, Barde et al., 1987).  The widespread expression o f B D N F and  its many potential targets has made it an appealing neurotrophic factor to evaluate as a potential therapeutic agent for a number o f neurological disorders, including stroke (Schabitz et al., 2004), amyotrophic lateral sclerosis (for which it has already undergone human clinical trials) (Ochs et al., 2000, B D N F Study Group, 1999), Alzheimer's and Parkinson's disease (reviewed by Murer et al., 2001), head injury (Blaha et al., 2000), peripheral nerve injury (Ho et al., 1998) and, as w i l l be discussed in more detail later, spinal cord injury.  24  1.7. T R K NEUROTROPHIN RECEPTORS 1.7.1. Introduction A s stated earlier, the biological activity o f neurotrophic factors depends on the presence o f the appropriate receptors on target tissue.  The neurotrophins (eg. N G F , B D N F , N T - 3 ) act  primarily through high-affinity binding to the Trk (tropomyosin receptor kinase) family o f protein tyrosine kinases, but also bind to the low-affinity p75 receptor (reviewed by Barbacid, 1995).  The trk oncogene, present in a human colon carcinoma, was originally described as a  transforming gene containing sequences o f both a non-muscle tropomyosin and a protein tyrosine kinase (Martin-Zanca et al., 1986).  Activity o f the tyrosine kinase was found to be  stimulated by N G F , which identified the product o f the trk oncogene to be a putative receptor for this neurotrophic factor (Kaplan et al., 1991a, Hempstead et al., 1991, Kaplan et al., 1991b, K l e i n et al., 1991). Additional studies revealed other highly related Trk receptors, including T r k B (Klein et al., 1989) and T r k C , (Lamballe et al., 1991) which also serve to bind to and effect the signal transduction o f members o f the neurotrophin family. While highly related, the Trk receptors demonstrate specificity in their binding, with N G F binding to Trk (TrkA), B D N F and NT-4/5 acting through T r k B , and N T - 3 acting primarily on T r k C .  A l l neurotrophins bind with low affinity to the p75 receptor, a cell  surface  glycoprotein belonging to the tumor necrosis factor receptor superfamily (Chao, 1994). Structurally, the Trk receptors are characterized by an extracellular ligand binding domain consisting o f cysteine clusters, a leucine-rich motif with Ig-like domains, and an intracellular domain consisting o f the tyrosine kinase catalytic domain (Reviewed by Barbacid, 1994). Extracellular ligand binding facilitates a dimerization o f the Trk receptor, autophosphorylation o f the intracellular tyrosine residues (Jing et al., 1992).  allowing for These serve as  25  docking sites for adaptor proteins that mediate their interaction with the residues v i a S H 2 domains (Schlessinger and U l l r i c h , 1992) and intracellular signaling cascades.  phosphotyrosine  then initiate downstream  Intracellular signalling pathways initiated in this fashion  influence cell survival and neurite outgrowth through P L C y , A k t 1/2, and M A P kinase (reviewed by Kaplan and M i l l e r , 2000).  1.7.2. TrkB Receptors T r k B receptor m R N A expression is widely found in the brain, spinal cord, and peripheral nervous system (Klein et al., 1989, 1990, and 1993).  Immunoreactivity o f the T r k B  extracellular domain is also widespread but is most intensely seen within the olfactory bulb, pyramidal neurons o f the hippocampus, granular cells o f the dentage gyrus, striatal neurons, Purkinje cells o f the cerebellum, substantia nigra pars compacta, locus coeruleus, brainstem and spinal motoneurons, and rubrospinal neurons (Yan et al., 1997). T r k B knockout mice appear phenotypically normal at birth, but die typically within days from starvation which is thought to be related to sensory and motor deficiencies in systems related to feeding and gastrointestinal functions (Klein et al., 1993).  Neuronal loss in the trigeminal ganglia, facial nucleus, and D R G  in particular was noted in these animals. Interestingly, while T r k B knockout mice demonstrate significant loss o f motorneurons (Klein et al., 1993),  B D N F knockout mice have normal  numbers o f spinal motorneurons (Jones et al., 1994), suggesting that during the development o f the latter, other neurotrophic factors can compensate for the absence o f B D N F . The T r k B locus encodes not only a full length receptor, g p l 4 5 t r k B (or T r k B T K + ) , with both the extracellular and intracellular domains described above, but also two truncated versions, T r k B . T l and T r k B . T 2 that lack the catalytic intracellular kinase domain (Middlemas et al., 1991).  In the rat forebrain, the expression o f the full length T r k B predominates in early  26  development, while the truncated forms predominate in late postnatal and adult life (Fryer et al., 1996).  The function o f these truncated forms o f the receptor is not clear.  It has been proposed  that they act in a dominant-negative fashion to sequester B D N F and thereby restricts the diffusion and binding o f this neurotrophin to full length receptors (Haapasalo et al., 2001, Biffo et al., 1995, Offenhauser et al., 2002).  Alternatively, their truncated intracellular domains may  in fact have some signal transduction function after B D N F binding (Baxter et al., 1997), possibly by activating a G protein that leads to inositol-1,4,5-trisphosphate-dependent calcium release (Rose et al., 2003) or by interacting with other cytoplasmic or membrane proteins one o f which has been identified and named T r k B - T l Interacting Protein, or TTIP ( K r y l and Barker, 2000). While the physiologic function o f these truncated forms o f T r k B is still in question, it is interesting to note that they are significantly upregulated at the site o f spinal cord injury (Frisen et al., 1993, K i n g et al., 2000).  In this regard, it is proposed that they may restrict the local  bioavailability o f exogenously administered or endogenously produced B D N F . this upregulation o f truncated T r k B receptors at the injury site is unknown.  The purpose o f  27  1.8. ANIMAL MODELING OF SPINAL CORD INJURY 1.8.1. Introduction The delineation o f the pathology and pathophysiology o f spinal cord injury and the development o f strategies to overcome the paralysis associated with them depends heavily upon animal models o f spinal cord injury.  The interest in this line o f research has led to the  establishment and refinement of a number of animal models which employ a variety of animal species and a spectrum o f injury paradigms, ranging from sharp transection to blunt contusion (reviewed by K w o n et al., 2002d and Rosenzweig and McDonald, 2004).  Recently, to more  closely reproduce the clinical situation in which the spinal cord is injured by the mechanical failure o f the surrounding spinal column, an animal model o f spinal cord injury via spinal dislocation rather than by impaction has been reported (Fiford et al., 2004).  Currently, the rat  and mouse are the most popular animals utilized in spinal cord injury research, both for cost and accessibility reasons, as well as for the latter's transgenic potential (Jakeman et al., 2000). Experimental interventions in animal spinal cord injury models are most commonly evaluated anatomically, biochemically, neurophysiologically, and/or behaviorally.  1.8.2. Anatomical Assessment of Axonal Growth Anatomic assessment of axonal growth in animal models is highly dependent upon immunohistochemistry  and on axonal tract tracers.  Immunohistochemical techniques utilize  antibodies targeted against proteins uniquely found i n certain axonal populations, allowing for the visualization of these axons in histologic sections o f the spinal cord.  Examples of such  proteins include calcitonin gene related peptide ( C G R P ) , which is a marker o f small diameter primary sensory axons, serotonin (5-HT), a marker of raphe-spinal axons, tyrosine hydroxylase  28  (TH), a marker o f coerulospinal axons and sympathetic axons, and choline acetyltransferase ( C h A T ) , a marker o f cholinergic motor axons (Jones et al., 2001). A x o n a l tracers are molecules that can be picked up by neurons or axons and transported in either an anterograde or retrograde fashion.  Anterograde tracers are applied within the  vicinity o f the cell bodies o f neurons and are then transported along the axons where they can be visualized.  Such tracers are therefore useful for visualizing axons that are injured and/or  regenerating at the injury site. Examples o f such anterograde tracers include biotinylated dextran amine ( B D A ) , the enzyme horseradish peroxidase conjugated to wheat germ agglutinin ( H R P W G A ) , and the cholera toxin B subunit ( C T B ) ( A l i s k y and Tolbert, 1994).  A tracer that is used  retrogradely is applied in the vicinity o f the axons and, depending on the characteristics o f the tracer, is taken up by cut and/or intact axons, or by their terminal endings and transported back to the cell body.  The previously mentioned anterograde tracers ( H R P - W G A , C T B , and B D A ) can  also serve as retrograde tracers. A variety o f fluorochromes such as FluoroGold, Fast-Blue, and Nuclear Y e l l o w are commonly used retrograde tracers (Cowan, 1998).  The differential  absorption and emission characteristics o f these fluorochromes allows them to be distinguished on histologic sections with various filters and wavelengths o f light. It is a common practice in C N S regeneration studies to apply a tracer distal to the injury, and then evaluate its presence in the neuronal bodies proximally. For example, i f the corticospinal tract is cut completely at C 3 , and a retrograde tracer is applied at T l , the presence o f tracerlabeled corticospinal neurons in the motor cortex implies that their axons successfully regenerated across the lesion at C 3 , picked up the tracer at T l , and transported it back (Figure 1.3). This tracing paradigm can be misleading i f the tract is incompletely cut and axons have been spared, or i f excessive tracer diffuses proximally beyond the lesion.  29  1.8.3. S h a r p Versus Blunt Spinal C o r d Injury Paradigms  The vast majority o f spinal cord injuries that occur i n humans are the result o f blunt trauma which rarely causes a complete transection o f the spinal cord. To this effect, a number o f devices have been developed to cause reproducible blunt contusive or compressive injuries to the spinal cord ( K w o n et al., 2002d). Similar to human injuries, these injury models typically create a central area o f damage within the spinal cord, leaving variable amounts o f spared tissue along the periphery ( H i l l et al., 2001).  Alternatively, sharp injury models either partially or  completely transect the spinal cord.  It is important to recognize that while the blunt injury  paradigm may be more representative o f clinical reality, both sharp and blunt injury paradigms are important for spinal cord injury research.  Models in which the spinal cord is fully or  partially transected are useful for examining axonal regeneration because they more dependably disrupt the axons o f a tract, while the sparing that results from contusion injuries is much more difficult to control and account for. Hence, the anatomical study o f axonal regeneration and the cell body response to axotomy is best performed in sharp spinal cord injury models, where one can be reasonably confident that the axons were actually severed in the first place. O n the other hand, models i n which the spinal cord is bluntly injured are useful for examining the acute pathophysiologic responses to injury and are the setting for the development o f neuroprotective agents that act to minimize secondary damage.  A s this thesis is focused on the study of the  cell body response to axotomy and methods f o r promoting axonal regeneration, the injury model I employ is that of a sharp, partial transection of the spinal cord which unilaterally disrupts the laterally placed rubrospinal tract.  30  Figure 1.3. Schematic of retrograde tracing paradigm after partial cord transection After injury/axotomy o f the corticospinal tract (A), the distal axons degenerate.  Axons  that regenerate across the lesion site w i l l pick up the tracer which is injected distally (B). The tracer is then retrogradely transported back to the cell body (C). Misinterpretation within this tracing paradigm can occur i f the tracer diffuses proximal to the lesion site, which w i l l occur i f too much tracer is injected or i f it is injected too close to the lesion site (D). A l s o , i f the partial injury is incomplete and misses some o f the axons o f the tract (E), spared axons w i l l pick up the label.  In both scenarios (D and E), the neuronal cell bodies w i l l be labeled and may be mistaken  for regenerating neurons. B Cortical Motor Neuron ; | | t  Labeled Neuron  Tracer retrogradely transported to cell body  Axotomy Regenerating Axon Degenerating Corticospinal Tract  Tracer^**™ Injection  Tracer picked Excessive I ^ » u p by injured ortraoer | ^ ~ ( b u t not regenerated) injected too close to axotomy  NP  AXON  Incomplete.  axotomy  Tracer AK Injection  Spared axon • picks up tracer  31  1.9. T H E R U B R O S P I N A L S Y S T E M  1.9.1. Introduction  There are many neuronal systems o f interest to those who study repair strategies for spinal cord injury.  In general, the study o f a C N S neuronal system in this context is facilitated  by access to both the neuronal cell bodies (to evaluate morphologic and molecular changes that occur at the level o f the soma) and to the axonal tract in the spinal cord (to both mimic the spinal cord injury and to evaluate the axonal response to injury and its capacity to regenerate).  The  rubrospinal system in rats possesses both o f these characteristics and as such is a useful neuronal system to study with regards to identifying regeneration obstacles within the C N S and potential therapeutic strategies.  1.9.2. A n a t o m y of the Red Nucleus and R u b r o s p i n a l T r a c t  The rubrospinal system consists o f rubrospinal neurons concentrated within the red nucleus and their descending axons which almost exclusively cross midline and travel as the rubrospinal tract. In rats, the red nucleus is identified as a ovoid collection o f neurons beginning approximately 2.5 m m rostral to the interaural line and extending rostrally for approximately 1 mm to a less well defined cephalad border (Huigrok and Cella, 1995).  The relatively well  defined boundaries o f the nucleus allow for its identification histologically and facilitates stereotactic access to the rubrospinal neurons and their exiting axons in vivo (Murray and Gurule, 1979, Whishaw et al., 1990, Jeffery and Fitzgerald, 2001, Houle and Jin, 2001).  Two  distinct populations o f rubrospinal neurons are thought to exist within the red nucleus, referred to generally as the magnocellular neurons located predominantly ventrolaterally in the caudal pole of the nucleus and the smaller, parvicellular neurons located dorsomedially in the more rostral  32  aspects o f the nucleus (Kennedy et al., 1986).  The caudal, ventromedial magnocellular neurons  contribute the majority o f axons to the descending rubrospinal tract, particularly to the distal lumbar spinal cord, while the smaller dorsomedial parvocellular neurons project axons to the cervical spinal cord (although the distinction between these neuronal subtypes and their projections is not absolute) (Murray and Gurule, 1979, Daniel et al., 1987, Strominger et al., 1987).  It is interesting to note that while the magnocellular component o f the red nucleus  appears to play a predominant role in the rubrospinal tract o f lower mammals such as rodents, it diminishes in importance as one moves up the evolutionary ladder and becomes rudimentary in man (ten Donkelaar, 1988, Nathan and Smith, 1982).  almost  Rubrospinal neurons  receive their afferent input primarily from the cerebellum, but also are targets for cortical, posterior thalamic, dorsal raphe, and locus coeruleus afferents (Huigrok and Cella, 1995). Rubrospinal axons cross the ventral tegmental decussation and descend within the dorsal part o f the lateral funiculus as the rubrospinal tract (Brown, 1974). In this position, it is possible to reliably transect the rubrospinal tract unilaterally, leaving the  contralateral  side for  comparison. The rubrospinal axons terminate mainly within laminae 5 to 7 o f the dorsal horn where they synapse with excitatory and inhibitory interneurons (Antal et al., 1992), but they have also been shown to extend into the ventral horn where they directly innervate pools o f motoneurons supplying the distal and intermediate forelimb muscles (Kuchler et al., 2002).  The  rubrospinal tract extends along the entire length o f the rat spinal cord, although for the most part it appears to end within the cervical spinal cord, with only 20% extending to the lumbosacral enlargement (Huisman et al., 1982).  33  1.9.3. Function of the Rubrospinal System The actual function o f the rubrospinal system is subject to some uncertainty.  A s its  neuronal anatomy appears to change as one ascends through phylogeny (ten Donkelaar, 1988, Nathan and Smith, 1982), it is quite likely that its function changes.  The actual presence o f the  system appears to be related to the presence o f limbs or limb-like structures, suggesting a role in motor control o f the extremities.  A rubrospinal tract has not been identified in some primitive  vertebrates such as the shark and boid snakes, while rays which use their enlarged pectoral fins for locomotion do possess a rubrospinal tract (ten Donkelaar, 1988).  While this association  implicates the rubrospinal system i n extremity motor control, in Nathan and Smith's classic report on the rubrospinal tract in humans - obviously, a species with high demands for extremity control - the rubrospinal tract was reported to be virtually non-existent below the mid-cervical spinal cord (Nathan and Smith, 1982). These findings, however, were based on degeneration studies rather than more sophisticated axonal tracing techniques. From studies in lower mammals, Kennedy has proposed that the rubrospinal tract mediates the automation o f motor skills that are first learned and established through activity o f the corticospinal tract (Kennedy, 1990).  While such a theory has some appeal, it is difficult to  reconcile with the lack o f an evident corticospinal tract in avian species such as prehensile parrots, ducks, and geese, who clearly have automated motor skills in their extremities (Webster et al., 1990, Webster and Steeves, 1988). Whishaw et al. ascribed subtle deficiencies in forelimb accuracy and grasp to lesions o f the red nucleus in animals having combined rubrospinal and corticospinal lesions, although animals with isolated red nucleus lesions generally did not demonstrate forelimb impairments, possibly due to corticospinal tract compensation (Whishaw et al., 1990).  However, red nucleus ablation was found to cause measurable and sustained  34  alterations in overground locomotion (Muir and Whishaw, 2000).  Electrophysiologic studies in  monkeys have suggested that the rubrospinal system preferentially activates extensor muscles in both proximal and distal joints o f the extremities (Belhaj-Saif et al., 1998). Determining the exact function o f the rubrospinal tract (and many other tracts for that matter) in rodents is made difficult by neuronal plasticity and their ability to compensate over time (Raineteau et al., 2001).  Increasing sophistication in the behavioral testing o f animals may  eventually provide a more clear picture o f the relationship between the rubrospinal system and neurologic function, which w i l l obviously be o f pragmatic importance to the interpretation o f therapeutic interventions in animal models o f spinal cord injury.  I should note that behavioral  testing was not a component o f this thesis work, although it is recognized that functional improvement is ultimately the goal o f regeneration strategies.  35  1.10. R A T I O N A L E F O R E X P E R I M E N T A L  MODELS  The general approach taken in this thesis is to investigate the response the rubrospinal system in the state o f chronic injury due to a sharp partial transection o f the spinal cord at the mid-cervical level.  1.10.1. Cervically Axotomized Rubrospinal Neurons A s A M o d e l of C h r o n i c C N S Injury  A s discussed earlier, the rubrospinal neurons are contained within the red nucleus, a relatively discrete nucleus within the midbrain, allowing the neurons to be accessed in vivo using stereotactic techniques (eg. for the administration o f trophic factors or the anterograde tracing o f axons), and facilitating histologic (eg. immunohistochemistry) and molecular (eg. in situ hybridization) evaluation.  The axons that emerge from the rubrospinal neurons cross the  midbrain tegmentum and descend within the spinal cord as the rubrospinal tract in the dorsal part of the lateral funiculus.  In this lateral position, the tract can be completely transected on one  side o f the spinal cord, leaving the contralateral side intact for comparison o f both the axons within the spinal cord and the neuronal cell bodies in the red nucleus. The acute cell body response o f rubrospinal neurons to cervical and thoracic axotomy is fairly  well  characterized  within the  rubrospinal system, which provides some  information for the evaluation o f chronic changes.  baseline  For example, transection o f the rubrospinal  tract in the cervical spinal cord results in severe neuronal atrophy and the transient expression o f the R A G s G A P - 4 3 and T a l tubulin (Fernandes et al., 1999, Tetzlaff et al., 1991). The inability to sustain this elevation in R A G gene expression correlates with regenerative failure (Kobayashi et al., 1997, Fernandes et al., 1999), and in this regard, the rubrospinal system is representative o f the "intrinsic" regenerative incompetence that impedes axonal regeneration within the C N S in  36  general. The rubrospinal axons within the spinal cord are subjected to the "extrinsic" inhibitors of regeneration such as CNS myelin and glial scarring after axotomy.  Thus, the rubrospinal  system allows for the evaluation of both the intrinsic and extrinsic obstacles to axonal regeneration. 1.10.2. Rat M o d e l s  In this thesis, I have exclusively used the Sprague-Dawley rat model. The animals are readily available, and the surgical protocols for such procedures as the spinal cord axotomy and stereotactic injections into the brainstem have been established.  The protocols for a number of  histologic and molecular techniques have been established within our lab for this species. The rats can be subjected to a contusion type injury as well, allowing for the extension of the findings from an axotomy injury paradigm to be taken forward into a more clinically relevant injury model. Finally, as stated, the rubrospinal system in rats exemplifies both the intrinsic and extrinsic obstacles to axonal regeneration;  as such, the fact that the rubrospinal system's  importance in humans is uncertain does not negate the valuable lessons that can be learned about these obstacles and methods for overcoming them.  37  - CHAPTER 2 MATERIALS AND METHODS  2.1 SURGICAL TECHNIQUES  2.1.1.  Anaesthetic Technique The animals used in this study were adult male Sprague-Dawley rats, weighing between  275-550 grams (the variation related to the chronicity o f some o f the animals). The animals were obtained from Charles River Laboratory (Quebec, Canada) or bred within the University o f British Columbia animal facility. Throughout the course o f experiments, they were housed in an alternating 12 hour light-dark cycle with free access to a standard diet and water.  A l l animal  experiments were performed in accordance with the guidelines o f the Canadian Council for A n i m a l Care and were approved by the University o f British Columbia Committee on Animal Care. The anesthetics used for surgical procedures were xylazine (Rompun®, Bayer, Toronto, O N ) , and ketamine (Ketalean®, B i m e d a - M T C , Cambridge, O N ) . The standard anesthetic mixture was 2 m l o f xylazine (20 mg/ml) and 3 m l o f ketamine (100 mg/ml) in 45 m l o f distilled water (to make a solution o f 0.8 mg/ml xylazine, 6 mg/ml ketamine).  Each animal received  approximately 1 m l o f solution per 100 gm body weight v i a intraperitoneal injection, and then was checked regularly during the procedures to ensure adequate anesthesia and analgesia.  With  the length o f some o f the surgical procedures being quite long (over an hour), it was frequently  38  necessary to provide additional anesthesia with 0.5 to 1.0 m l bolus injections o f anesthetic mixture.  2.1.2.  C e r v i c a l A x o t o m y of the R u b r o s p i n a l T r a c t After inducing deep anesthesia, the dorsal cervical region o f the animal was shaved and  the head was gently secured within a stereotactic frame, with light traction (approximately 100 gm) applied to the tail to spread the lamina and allow easier access to the spinal cord.  The  exposed skin was sterilized with Betadine® solution (Purdue Pharmaceuticals, Wilson, N C ) , a 10% providone-iodine solution that acts as a wide spectrum antimicrobial.  Using a surgical  operating microscope with 4.5x to 6.5x magnification, a midline longitudinal incision was made over the mid-cervical spine and the overlaying muscles split to expose the posterior elements o f C3 and C 4 . A small laminectomy was performed with a bone rongeur and the left side o f the spinal cord was visualized.  A t the lateral border o f the dorsal horn, a 26 gauge needle was  inserted to create a small hole just medial to the rubrospinal tract, and the left dorsolateral funiculus was then cut sharply by inserting one blade o f a pair o f fine iris scissors into this hole to a depth o f approximately two thirds that o f the spinal cord, and then closing the scissors. A l l spinal cord injuries were performed on the left side. After making the initial cut in the spinal cord, a defect o f approximately half a millimeter in length along the rostro-caudal axis was created by aspiration through a tapered glass pipette. N o attempt was made to suture the dura. The muscle and subcutaneous tissue were allowed to fall back towards midline and surgical clips were used to close the skin. The animal was taken out o f the stereotactic frame and placed back into a cage with an electric heating blanket underneath convalescence.  to prevent hypothermia during  39  2.1.3. Reaxotomy of Chronically Injured Rubrospinal Axons ("Refreshment Injury") A number o f time periods, from 2 months to 18 months, were utilized during these experiments to induce a state o f "chronicity" in the rubrospinal system.  After the period o f time  to establish chronicity, the rats were anesthetized and their cervical region prepared and exposed in the same fashion as described earlier.  O n occasion, the area o f prior laminectomy  occasionally fills with bone which needs to be re-resected to gain access to the spinal cord; most times, however, the previous laminectomy defect is easy to identify and the prior spinal cord injury site can be accessed by enlarging the defect slightly with a rongeur. within the  spinal cord by aspiration is typically  filled  The defect created  with scar tissue  which, under  magnification, is readily distinguished from the normal spinal cord tissue on either side o f it. (Figure 2.1) Reaxotomy o f the rubrospinal tract (ie. "refreshment"  injury) is performed by again  puncturing the cord at the lateral border o f the dorsal column with a 26 gauge needle and cutting it with a pair o f fine iris scissors, approximately 1 mm proximal to the rostral border o f the previous defect.  Houle has demonstrated that retraction or "die back" o f rubrospinal axons  following cervical axotomy is quite modest, averaging approximately 500 p m with terminal end bulbs "rarely" visualized more than 1 m m rostral to the injury (Houle and Jin, 2001). this second axotomy should re-injure the majority o f rubrospinal axons in this area.  Therefore,  40  Figure 2.1. Two months after cervical axotomy, the scar from previous spinal cord injury is readily visible on the dorsal surface of the cord. The incision through the dorsolateral funiculus is readily visible on the dorsal surface of the spinal cord; the original injury is performed by making an incision with a pair o f fine iris scissors, and then aspirating the cord for a length o f approximately 0.5 mm.  The refreshment  injury extends the injury site proximally about 1 mm. (rostral is to the left, caudal to the right)  41  2.1.4. B D N F Application to the Spinal C o r d V i a Gelfoam®  After the refreshment injury was performed, small pledgets o f Gelfoam® (Pharmacia & Upjohn, Peapack, N J ) were soaked with B D N F and then placed into the spinal cord defect. The concentrations o f B D N F that we employed for local application to the spinal cord included a low concentration o f 50 ng/pl, a medium concentration o f 1,000 ng/pl, and a high concentration o f 20,000 ng/pl. The BDNF-soaked Gelfoam was left in place for 5 minutes, then replaced by another pledget o f fresh, BDNF-soaked Gelfoam. This procedure was repeated three more times for a total o f 4 applications o f freshly soaked Gelfoam over one hour.  Control animals received  Gelfoam soaked in sterile phosphate-buffered saline (PBS, p H 7.5). This method o f applying the BDNF  in Gelfoam and then replacing the  BDNF-soaked  Gelfoam sponge  with  fresh  neurotrophic factor every 15 minutes over one hour, was reported to attenuate rubrospinal death after second axotomy performed 4 weeks after initial injury (Houle and Y e , 1999), and so we chose to employ this same regimen in our studies. The B D N F used for the experiments in which direct spinal cord application o f the neurotrophic factor was performed was kindly provided by Regeneron Pharmaceuticals Inc., Tarrytown, N Y .  2.1.5. B D N F Application to the Red Nucleus via Osmotic M i n i p u m p  To established.  apply B D N F  directly, to the rubrospinal cell bodies, an infusion system was  A n Alzet osmotic minipump (Alzet no 2001, 1 ml/hr, D U R E C T Corp., Cupertino,  C A ) was filled with B D N F (approximate total volume o f 250 pl) at a concentration o f 500 ng/ml within a vehicle solution o f 20 m M sterile P B S , 100 U Penicillin/Streptomycin, and 0.5% rat serum albumin (Sigma-Aldrich Canada, Oakville, O N , #A-6272).  Minipumps were filled with  vehicle solution alone for control animals. The pumps were connected to a 28 gauge, 8 m m long  42  cannula (Plastic One Inc., Roanoke, V A ) v i a a 6 to 8 cm silastic tubing (no 508-003, V W R Canlab, Mississauga, O N ) . This whole assembly (minipump, tubing, cannula) was preincubated for 4 to 12 hours in sterile 20 m M P B S at 37°C to initiate an even flow rate before implantation. W i t h the anesthetized animal held within the stereotactic frame, the Betadine-sterilized skin over the dorsal aspect o f the cranium was incised in a longitudinal midline fashion.  With  the cranium exposed, an electric high speed burr was used to make a small hole (approximately 1 m m in diameter) in the skull 6.3. m m posterior to Bregma and 1.7 m m to the right o f midline. Great care was taken to ensure that the burr did not plunge through the inner table o f the cranium and injure the epidural or subdural structures.  The cannula for the B D N F infusion pump was  then positioned on the stereotactic frame 6.3 m m posterior to Bregma and 1.7 m m to the right o f midline, in line with the burr hole.  The cannula was carefully lowered through the burr hole  until its tip rested on the dural surface.  From this point, it was lowered 6.5 m m into the brain  parenchyma, with the intention o f leaving the tip o f the cannula just lateral to the red nucleus. Two watchmaker screws and acrylic cement were used to secure the cannula rigidly in place and seal the burr hole. The skin was closed with skin clips and the animal placed back into a cage with an electric heating blanket underneath to prevent hypothermia during convalescence. The B D N F used for application at the level o f the rubrospinal cell bodies was kindly provided by Regeneron Pharmaceuticals Inc., Tarrytown, N Y .  A pegylated version o f B D N F  was also generously provided by Dr. Qiao Y a n o f A M G E N ( A M G E N , Thousand Oaks, C A ) under a Materials Transfer Agreement.  The non-pegylated B D N F  (from  Regeneron  Pharmaceuticals) was used for studies o f the rubrospinal cell body response (ie. atrophy, cell number, and in situ hybridization) while the pegylated B D N F was used for studies o f regeneration into peripheral nerve transplants.  43  2.1.6. Peripheral Nerve Transplantation Because o f the many inhibitory elements within myelin and the glial scar that prevent axonal regeneration within the C N S , the transplantation o f peripheral nerve grafts into the spinal cord  is intended to provide C N S axons with  a permissive environment for growth.  Approximately ten days prior to anticipated transplantation, the anesthetized animal underwent a transection o f the right sciatic nerve at the level o f the obturator tendon. After being cut, the nerve was left in situ to allow Wallerian degeneration o f the distal stump, thus creating the optimal trophic environment for axonal regeneration.  Ten days, later, the animal was  anesthetized again and secured within the stereotactic frame. The cervical spinal cord was then re-exposed and re-axotomized as described in Section 2.1.3.  The right sciatic nerve was re-  exposed and a 30 to 35 m m segment was harvested. The proximal end o f this pre-degenerated nerve graft was then inserted into the spinal cord defect and held in place with two 10-0 Prolene sutures (Ethicon, Somerville, N J ) . The distal end o f the graft was brought out to the subcutaneous tissue and marked with a suture for easy identification later.  The wounds were  closed in the standard fashion with skin clips. T w o months later, the animal was re-anesthetized, the distal end o f the graft was exposed, the distal 2 m m o f the graft resected (to provide a fresh nerve ending) and a carbocyanine dye, D i l (Molecular Probes, Eugene, O R ) or Biotin Dextran A m i n e ( B D A ) (Molecular Probes, Eugene, O R ) was applied to this tip to retrogradely label neurons whose axons regenerated to the end o f the graft.  44  2.1.7. Anterograde T r a c i n g of R u b r o s p i n a l A x o n s In order to evaluate T r k B receptor expression in rubrospinal axons within the spinal cord, the axons were anterogradely labeled via a stereotactic injection o f B D A .  The anesthetized  animals were gently held within the stereotactic frame and the dorsal surface o f the cranium was exposed.  A n electric high-speed burr was then used to create a 1 m m hole in the skull centered  6.2 m m caudal to Bregma and 0.7 m m lateral to midline. A 25% solution o f B D A (10,000 M W , Molecular Probes, Eugene, O R ) i n 0.5% D M S O was drawn up into a glass micropipette with a Hamilton syringe. The tip o f the micropipette was positioned on the dural surface 6.2 m m caudal to Bregma and 0.7 m m lateral to midline, then advanced into the brainstem to a depth o f 7.2 mm. B D A was injected at a rate o f 0.04 pl/min to a total volume o f 0.6 p l (15 minutes), and the micropipette was left in place for an additional 5 minutes to allow diffusion o f the tracer.  The  injection was performed on each side o f midline to label both the injured and uninjured rubrospinal axons.  The coordinates o f this stereotactic injection were those described by Houle  and J i n i n their analysis o f anterogradely labeled rubrospinal axons and their dieback from a cervical injury site (Houle and Jin, 2001), with a slight modification to accommodate for the larger size o f our animals.  2.1.8. Retrograde a n d Anterograde L a b e l i n g o f R u b r o s p i n a l Neurons a n d A x o n s A number o f retrograde tracers were used i n these experiments, including 1% Fast Blue (Sigma-Aldrich Canada, Oakville, O N ) , 5% FluoroGold (Fluorochrome Inc. Eaglewood, C A ) , D i l (Molecular Probes, Eugene, O R ) and Biotin Dextran Amine ( B D A ) (Molecular Probes, Eugene, O R ) .  The Fast Blue and FluoroGold tracers were micro-injected with a Hamilton  syringe into the left side o f the spinal cord at the T l level to label the lumbar-projecting population o f rubrospinal neurons (predominantly magnocellular neurons, as discussed in the  45  Background Section 1.8.2).  These tracers have the ability to be taken up by intact axons en  passage, although the ability o f FluoroGold to do so appeared to be better than Fast Blue, the latter being more readily picked up by intact nerve terminals (Kobbert et al., 2000).  A 0.2 p l  injection o f 5% FluoroGold directly into the spinal cord was therefore used for the majority o f experiments in which retrograde labeling o f the rubrospinal tract was required. known to be retrogradely transported  FluoroGold is  in a robust fashion, even by chronically transected  rubrospinal axons (McBride et al., 1990), and we found it to be present within the rubrospinal cell bodies even 6 months post-injection ( K w o n et al., 2002c) (see Figure 3.6), although we recognize that others have not found it to be maintained within the cell bodies for quite so long (Novikova et al., 1997). The FluoroGold retrograde labeling o f rubrospinal neurons was performed prior to cervical axotomy in these experiments to identify the lumbar projecting neurons for one o f two reasons:  (1) so that the boundaries o f the injured aspects o f the red  nucleus could be identified, or (2) so that rubrospinal neurons could be identified as being chronically injured in the evaluation o f axonal regeneration into peripheral nerve transplants (see further description o f the double-labeling paradigm).  Alternatively, i n earlier stages o f the  animal model development, the FluoroGold was used after the cervical axotomy to ensure that no sparing o f fibers had occurred.  After some experience with the technique for unilaterally  transecting the rubrospinal tract, we felt confident that our cervical axotomies were not leaving any rubrospinal axons spared (based on the absence o f FluoroGold retrograde labeling o f rubrospinal neurons) and thus this step was abandoned. D i l is a lipophilic carbocyanine dye that actively can be retrogradely transported or passively diffuses laterally within the membrane as phospholipid molecules, and can thus label both live and fixed cells. In vivo, D i l can diffuse rapidly (up to 6 mm/day) (Holmqvist et al.,  46  1992).  D i l was employed in axonal regeneration experiments to evaluate regeneration through  the peripheral nerve transplant.  Using the tip a needle, we applied a small crystal o f D i l directly  to the distal end o f the peripheral nerve transplant to retrogradely label axons that grew to the tip of the graft. Dextrans are hydrophilic polysaccharides that can be. used as axonal tracers when biotinylated or conjugated to fluorescent molecules (Vercelli et al., 2000). Biotin dextran amine ( B D A ) requires an immunohistochemical reaction for detection, but this provides a much more stable end-product for visualization than the fluorescent dextrans. anterograde or retrograde tracer, and in our studies was used as both.  It can be used as an Anterogradely, it was  injected medially to the red nucleus (coordinates described above) to label the descending axons. Retrogradely, it was injected into the distal tip o f the peripheral nerve graft to label axons that had successfully regenerated through the graft.  47  2.2. HISTOLOGIC TECHNIQUES 2.2.1. Tissue Collection Animals were given a lethal dose o f chloral hydrate (approximately 1 g/kg). The animals were transcardially perfused with 350 m l o f P B S followed immediately by 350 m l o f ice cold, freshly hydrolyzed 4% paraformaldehyde (pH 7.4).  Following exposure o f the cranium and  spinal column, the brainstem and spinal cord around the injury site was carefully extracted and placed in 4% paraformaldehyde overnight. The tissues were then sequentially cryoprotected in 12%, 16%, and 22% sucrose, before being rapidly frozen in isopentane cooled on dry ice.  2.2.2. Cryostat Cutting The brainstem was mounted with the rostral side down for axial cryostat cutting in a caudal to rostral direction. The spinal cord was mounted either with the rostral side down for axial cutting or with the dorsal surface down for longitudinal, coronal cryostat cutting. sections were cut at 20 p m thickness at temperatures ranging from -16 to -19°C.  Brain  Sections were  mounted onto Superfrost Plus slides (Fisher Scientific, Pittsburgh, P A ) and stored at -80°C until use.  While cutting the brainstem tissue in a caudal to rostral direction, the tissue block was  adjusted when the facial nucleus came into view i n order to achieve the correct left-right balance before reaching the red nucleus.  A s the number and size o f rubrospinal neurons was to be  compared between injured and uninjured red nuclei, it was felt that left-right balance should be achieved so that each section that was cut represented equivalent levels o f the injured and uninjured nuclei. Because the rubrospinal neurons in general become smaller as one progresses from the caudal (more magnocellular) to rostral (more parvocellular) end o f the nucleus, a leftright imbalance in the cutting might produce a section with a higher percentage o f smaller,  48  parvocellular neurons in one red nucleus compared to the other. Hence, great attention was paid to ensuring this balancing prior to reaching the red nucleus. Brain sections used for the counting o f rubrospinal neurons using the physical dissector method (described in more detail in the Background) were mounted adjacent to each other on the same slide so that they would be subjected to identical histological conditions. For the spinal cord, the sections were taken in a ventral to dorsal fashion so that the tissue could be balanced in a left-right fashion using the ventral grey matter before arriving at the more dorsally placed rubrospinal tracts.  This right-left balancing was performed so that when  evaluating T r k B expression within the injured spinal cord, both the injured and uninjured rubrospinal tracts would be visualized on the same section.  2.2.3. NeuN Immunohistochemistry N e u N immunohistochemistry was performed to enhance the identification o f rubrospinal neurons, particularly after axotomy and the resultant atrophy.  The N e u N monoclonal antibody  was initially described by Mullen et al. (Mullen et al., 1992) who observed that the antigen recognized by this antibody was neuron-specific. specific marker o f neuronal populations. characterized.  It has subsequently been popularized as a  The antigen to which it binds has yet to be fully  Recent work by M c P h a i l et al. has demonstrated that after cervical axotomy,  rubrospinal neurons maintain their expression o f N e u N for at least 28 days post-injury, while axotomized facial neurons do not (McPhail et al., 2004). Slides containing the perfusion-fixed sections o f the caudal 500 p m o f the red nucleus were taken from the -80°C freezer and thawed for 10 minutes at room temperature. Following several washes in 0 . 0 I M P B S containing 0.1% Tween-20, sections were incubated overnight at 4°C in the primary antibody solution (NeuN mouse monoclonal, 1:100; Chemicon International  49  Inc. Temecula, C A ) .  Following this, the slides were washed in 0.01 M P B S , blocked with 5%  normal goat serum, and incubated overnight at 4 ° C in a solution containing a secondary antibody raised in goat, conjugated to A l e x a 488 or Cy3 (goat anti-mouse, 1:100; Molecular Probes Incorporated, Eugene, Oregon). The slides were then washed in 0.01 M P B S and coverslipped with a glycerol / 0.% sodium azide mounting medium (Sigma Diagnostics Inc, St. Louis, M O ) . Although the N e u N antibody has been widely used as a neuronal-specific marker, crossreactivity o f N e u N  with astrocytes  and microglial cells was ruled out by  performing  immunohistochemistry for both N e u N as well as an additional primary antibody either to G F A P (Dakopatts Corp, Carpinteria, C A , Z334) or to isolectin B 4 (Sigma-Aldrich Canada, Oakville, O N , L2140) respectively. A 1:200 secondary antibody conjugated with E x t r A v i d i n Cy3 (SigmaAldrich Canada, Oakville, O N , E-4142) was used for the G F A P and isolectin B 4 , and the sections evaluated for cross-reactivity with N e u N (using a secondary antibody conjugated to A l e x a 488).  These "control" slides demonstrated no cross-reactivity between N e u N and  astrocytes or microglia. (Figure 2.2)  50  Figure 2.2 NeuN immunostaining specifically labels neurons and does not label astrocytes or microglia. N e u N (green) and G F A P  (red) immunohistochemistry for neurons and  respectively shows no overlap o f labeling (top row).  astrocytes  N e u N (green) and isolectin B 4 (red)  immunohistochemistry for neurons and microglia respectively shows no overlap o f labeling (bottom row). Scale bar, 50 pm.  51  2.2.4.  TrkB Immunohistochemistry T r k B immunohistochemistry was performed on both the rubrospinal cell bodies and on  the rubrospinal tracts within the spinal cord.  For sections o f the red nucleus, the slides were  taken from the -80°C freezer and thawed for 10 minutes. After initial washes with 0.01 M P B S with 0.1% Tween-20, the slides were incubated overnight at 4°C with rabbit polyclonal T r k B antibody directed at the intracellular carboxy terminus o f the full length T r k B receptor (1:100; Santa Cruz Biotechnology Inc., Santa Cruz, C A , sc-12).  Following blocking in 5% normal  donkey serum, slides were incubated at room temperature for 2 hours i n a solution containing a biotinylated  donkey  anti-rabbit  secondary  antibody  (1:200;  Jackson  ImmunoResearch  Laboratories, West Grove, P A ) , and subsequent development with the A B C kit (Elite A B C K i t , Vector Laboratories, Burlinghame, C A ) .  After several brief washes in distilled water, the slides  were dehydrated using a graded series o f ethanol, being immersed briefly in 100% isopropanol, and cleared in toluene before being mounted in Entellan® (Electron Microscopy Sciences, Hatfield, P A ) and coverslipped. To visualize T r k B receptor expression in the spinal cord, slides were incubated overnight at 4°C with T r k B primary antibody (1:100; Santa Cruz Biotechnology Inc., Santa Cruz, C A , sc12). Slides were then blocked using 5% donkey normal serum, and incubation overnight with both an A l e x a 488 conjugated anti-rabbit secondary antibody raised in donkey (1:200; Molecular Probes  Incorporated,  Eugene, Oregon) to visualize T r k B  immunoreactivity, as well  as  streptavidin conjugated to Cy3 (1:200; Jackson ImmunoResearch Laboratories, West Grove, P A ) to visualize the anterograde B D A labeling o f the rubrospinal tract.  Finally, slides were washed  in 0.01 M P B S and coverslipped with an anti-fade media to reduce  fluorescent  bleaching  52  (SlowFade®, Molecular Probes, Eugene, OR), with the anticipation that bleaching o f the fluorescence would be a problem with confocal imaging o f this tissue.  53  2.3. ANALYSIS OF TISSUES 2.3.1. Disector Counting Technique of Rubrospinal Neurons The principles o f the disector method o f stereologic counting have been described in the background chapter. In brief, the method involves comparing the cells identified on one section with the cells identified on the adjacent section, and counting only those that are not visualized on both i n order to avoid double-counting the same cell. The caudal 500 p m o f the red nucleus was sectioned into 25 sections o f 20 p m thickness.  After N e u N immunohistochemistry to  identify the rubrospinal neurons, the image o f each red nucleus was captured with a Zeiss A x i o s k o p microscope equipped with a S P O T digital colour camera (Diagnostic Instruments Inc, Sterling Heights, M I ) and Northern Eclipse image analysis software (Empix, Mississauga, O N ) . The application o f FluoroGold into the spinal cord allowed for the retrograde labeling o f the rubrospinal neurons and outlined the boundaries o f the injured aspect o f the red nucleus. NeuN-positive rubrospinal neurons were then outlined on each section (the "sampling" section) and compared to neurons on the rostral adjacent section (the "lookup" section).  Neurons were  counted on the sampling section only if they were not present on the lookup section.  NeuN-  positive neurons were outlined i f their nucleus or nucleolus could be detected, or i f they possessed a characteristic stellate morphology. Neuronal counts were performed on both the injured and uninjured red nuclei.  The  evaluation o f neuronal survival was performed i n a comparative fashion, with the number o f neurons i n the injured red nucleus compared to the number o f neurons in the contralateral uninjured red nucleus.  54  2.3.2. Measurement of Cross Sectional A r e a Six sections spaced evenly throughout the caudal 500 p m o f the red nucleus (each approximately 60-80 p m apart from one another) were used for the measurement o f crosssectional area.  Again, NeuN-positive rubrospinal neurons were outlined i f their nucleus or  nucleolus could be detected, or i f they possessed a characteristic stellate morphology.  A n image  analysis application for Adobe PhotoShop was used to measure the cross sectional area o f each outlined neuron (Image Processing Took K i t , Reindeer Graphics, Asheville, N C ) . The evaluation o f neuronal atrophy was performed in a comparative fashion, with the cross sectional area o f neurons in the injured red nucleus compared to the cross sectional area o f neurons in the contralateral uninjured red nucleus.  2.3.3. In Situ H y b r i d i z a t i o n (ISH) To evaluate the expression o f the regeneration associated genes G A P - 4 3 and T a l tubulin, we performed in situ hybridization (ISH) on sections o f the red nucleus.  This technique allows  for the cellular localization o f specific m R N A sequences, from which a semi-quantitative analysis o f gene expression changes can be performed.  I S H was performed with radioactively  labeled oligonucleotide probes complementary to G A P - 4 3 and T a l tubulin. The oligonucleotide sequence for the G A P - 4 3 probe and T a l tubulin probes are listed i n Figure 2.3. oligonucleotides were end-labeled with  3 5  The  S - d A T P using deoxynucleotide terminal transferase  according to standard molecular protocols (Ausubel et al., 1987). This adds a radioactive p o l y A tail to the 3' end o f the oligonucleotide probe.  The specific activity o f the probes was at least  600,000 cpm/pl. Control slides were performed to confirm the sensitivity and specificity o f the sense and antisense G A P - 4 3 and T a l tubulin probes on animals that were acutely injured. (Figures 2.4 and 2.5)  55  Slides with 20 pm thick sections o f the red nucleus were taken from storage in the -80°C freezer, dried for 5 minutes at room temperature, and then post-fixed for 30 minutes in 4% paraformaldehyde at 4°C before being rinsed in two washes o f l x P B S with D E P C .  Sections  were then permeabilized in a solution o f proteinase K (20 pg/ml in 50 m M Tris and 5 m M E D T A ) to improve the access o f the oligonucleotide probes to their complementary m R N A s within the cells.  The slides were rinsed in P B S and fixed in 4% paraformaldehyde again for 5  minutes to stabilize the proteins after permeabilization. The slides were washed twice in P B S and dehydrated in a graded series o f ethanol. The slides were placed in chloroform for 5 minutes to delipidize the sections before being placed back into 100 and 95% E t O H washes for one minute each.  The sections were air-dried at room temperature for 10 minutes.  lOOpl o f  hybridization cocktail containing 1.2 x 10 cpm o f labeled oligonucleotide was applied to each 6  slide, which was coverslipped and incubated at 43°C for 16-18 hours. Each m l o f hybidization cocktail contains 1.2 x 10 cpm o f labeled oligonucleotide, 200 mg o f salmon sperm D N A , and 7  200 mg t R N A , in a solution o f 50% deionized formamide, 10% dextran sulfate, 5x S S C , 5x Denhardt's solution, and 200 m M dithiothreitol. After hybridization, the slides were washed in decreasing concentrations sodium chloride / saline citrate (SSC) solutions in order to remove unbound and non-specifically bound oligonucleotide probe. A l l S S C wash solutions contained 2-mercaptoethanol (200ml/ml) except for the final two washes in 0.25x and O.lx S S C .  After removing the coverslips in 4x S S C at  room temperature, the slides were washed for 20 minutes each at 48-50°C in 2x S S C , l x S S C , and 0.5x S S C . The two final washes were at 55°C in 0.25x and O.lx S S C . Remaining salts were rinsed from the slides using distilled water at room temperature, and the sections were dehydrated in 60 and 95% E T O H .  56 For autoradiographic development, the slides were dipped in Kodak N T B 2 photographic emulsion (Kodak Canada, Toronto, O N ) diluted 1:1 with distilled water.  For G A P - 4 3 , the slides  were incubated for 5 days prior to development, while for T a l tubulin, slides were incubated for 2 days prior to development.  Slides were developed using Kodak D-18 developer and fixed  with Kodak Fixer. To visualize the rubrospinal neurons, the slides were immersed in 0.01% ethidium bromide in 1:100 P B S , washed for one hour in tap water, then coverslipped. A semi-quantitative analysis o f G A P - 4 3 and T a l tubulin I S H signal was performed in order to compare R A G expression in the injured red nucleus with that o f neurons in the uninjured red nucleus.  Darkfield images o f the I S H silver grains and fluorescent images o f the ethidium  bromide stained rubrospinal neurons were captured with the Zeiss Axioskop, and stored as TIFF files.  Using SigmaScan Pro ImageAnalysis 5.0 Software (Systat Software, Inc, Point Richmond,  C A ) , the ethidium bromide stained rubrospinal neurons were then outlined, using the same criteria as described above for the counting o f rubrospinal neurons and measurement o f neuronal cross sectional area. The darkfield image o f I S H signal was then overlaid on top o f the neuronal profiles and the area fraction occupied by the grains (ie. grain density) was calculated per neuron.  A n region o f the section devoid o f neuronal or glial cells was outlined (with an area  approximately equal to that o f a rubrospinal neuron) and the I S H signal from this area was measured as the "background" autoradiographic signal. The I S H signal per cell was then calculated as the difference between the I S H signal within the cell profile and the background signal.  57  Figure 2.3. In Situ Hybridization Probes  GAP-43 Probe (52mer): 5' - G C A T C G G T A G T A G C A G A G C C A T C T C C C T C C T T C T T C T C C A C A C C A T C A G C A A - 3 ' 100% complementary to bases 273-324 o f rat G A P - 4 3 m R N A {Basi, Jacobson, et al. 1987 310 /id} (accession # J02809).  T a l Tubulin Probe (47mer): 5' - A A A C C C A T C A G T G A A G T G G A C G G C T C G G G T C T C T G A C A A A T C A T T C A - 3 ' 100 % complementary to bases 1548-1594 o f rat T a l a-tubulin m R N A {Lemischka, Farmer, et al. 1981 952 /id} (accession# V01227).  58  Figure 2.4. GAP-43 anti-sense and sense probes on rubrospinal neurons 7 days after cervical axotomy G A P - 4 3 expression is low in adult rubrospinal neurons, but increases rapidly after cervical axotomy (unfortunately, this upregulation is not sustained, correlating with the failure o f axonal regeneration).  We therefore performed control I S H on the axotomized red nucleus, 7  days after injury. A s expected, while the G A P - 4 3 anti-sense probe demonstrated robust binding, there was no specific binding o f the sense probe.  GAP-43 anti-sense probe o n rubrospinal neurons 7 d a y s after cervical axotomy. (Note the expected rise of GAP-43 expression at this early time point after axotomy - this expression eventually falls back down to a low, baseline level of expression.)  G A P - 4 3 s e n s e probe o n rubrospinal neurons 7 days after cervical axotomy. (Note the absence of specific binding.)  59  Figure 2.5. T a l tubulin anti-sense and sense probes on rubrospinal neurons 7 days after cervical axotomy Baseline T a l  tubulin expression  is maintained  at relatively high levels in adult  rubrospinal neurons (and tends to decrease over time after axotomy). We therefore evaluated the binding o f our sense and antisense probes on the uninjured red nucleus. A s expected, while the T a l tubulin anti-sense probe demonstrated robust binding, there was no specific binding of the sense probe.  T o r t tubulin anti-sense probe on uninjured rubrospinal neurons  (This is the expected high baseline expression in the adult)  T(/1 tubulin s e n s e probe on uninjured rubrospinal neurons  (Note the absence of specific binding)  60  - CHAPTER 3 RUBROSPINAL NEURONAL ATROPHY AND SURVIVAL AFTER CERVICAL AXOTOMY AND THE RESPONSE TO BDNF APPLICATION  3.1. S U M M A R Y Following cervical axotomy, rubrospinal neurons undergo a well-documented atrophy and have also been described as undergoing extensive cell death within 4 to 8 weeks. prevention, attenuation, or reversal o f this atrophy has been associated with a regenerative propensity o f rubrospinal neurons.  The  greater  Obviously, the prevention o f cell death is o f  paramount importance to the possibility o f promoting regeneration o f this neuronal system after injury.  The retrograde death o f supraspinal neurons after spinal cord injury has obvious  implications for the therapeutic potential o f interventions to promote axonal regeneration or plasticity, as the more neurons that die after spinal cord injury, the less likely recovery w i l l be achieved with such treatments.  In this chapter, I summarize a body o f work that examines  rubrospinal atrophy and survival at various time points after cervical axotomy and the effect that B D N F applied either at the spinal cord injury site or to the red nucleus has on this. I found that two months after cervical axotomy, injured rubrospinal neurons were significantly smaller than their contralateral uninjured counterparts.  The application o f B D N F  within gelfoam pledgets directly to the spinal cord injury site, over a wide spectrum o f  61  concentrations, did not reverse this atrophy ( K w o n et al., 2004a).  Compared to control  axotomized rubrospinal neurons with no interventions, a slight increase i n neuronal cross sectional area was collectively observed with all animals that received a repeat excision o f the spinal cord injury site (a "refreshment" injury) and application o f both B D N F or P B S , suggesting an effect o f the  refreshment injury itself.  In contrast, the application o f B D N F v i a osmotic  mini-pumps directly to the injured red nucleus did reverse rubrospinal atrophy, even 12 months after cervical axotomy. The measurement o f the number o f surviving rubrospinal neurons using stereologic counting techniques and N e u N immunohistochemistry (a neuron specific marker), demonstrated that rubrospinal neurons were still present 12 months after cervical axotomy, albeit in a severely atrophic state ( K w o n et al., 2002b). Even with conventional histologic techniques (cresyl violet staining), equal numbers o f injured and uninjured rubrospinal neurons were observed in animals treated with cell body administration o f B D N F . This indicated the reversal o f neuronal atrophy and the ability to detect the larger neurons within the injured red nucleus.  Using the same  counting and histologic techniques, I found that a second cervical injury (refreshment) caused only a modest amount o f cell loss ( K w o n et al., 2002c).  62 3.2.  INTRODUCTION  3.2.1. A t r o p h y and Death of Rubrospinal Neurons After Axotomy and the Administration of Neurotrophic Factors  Rubrospinal neurons that are axotomized in the cervical spinal cord undergo significant atrophy within weeks o f injury (Tetzlaff et al., 1991, Kobayashi et al., 1997) and reportedly undergo substantial cell death within months o f injury (Goshgarian et al., 1983, Houle and Y e , 1999, M o r i et al., 1997).  The cause o f this atrophy and cell loss is likely closely related to the  interruption o f target-derived neurotrophic factor support (reviewed by Goldberg and Barres, 2000).  Support for this concept is derived from both developmental and adult studies o f the  rubrospinal system.  For example, axotomy o f the rubrospinal tract in developing animals prior  to the establishment o f axon collaterals causes severe retrograde atrophy and cell loss (Bregman and Reier, 1986). It was postulated by these authors that axon collaterals would have provided the means for attaining neurotrophic support from other targets that might have prevented such death.  Such was supported by further work which demonstrated  survival and axonal  regeneration o f rubrospinal neurons in neonatal rats that maintain axon collaterals (BernsteinGoral and Bregman, 1997), and that the exogenous application o f B D N F to the spinal cord o f newborn rats acutely after hemisection prevents the loss o f rubrospinal neurons (Diener and Bregman, 1994). O f particular relevance to spinal cord injury in the mature nervous system, the exogenous administration o f B D N F and N T - 3 into the intrathecal space o f adult rats acutely after cervical axotomy promoted neuronal survival and lead to reversal o f rubrospinal atrophy (Novikova et al., 2000). These authors noted that the combined infusion o f both B D N F and N T - 3 effected better neuronal survival and hypertrophy than the neurotrophic factors alone.  The atrophy o f  63  adult rubrospinal neurons after thoracic axotomy was shown to be prevented by the acute transplantation o f fetal tissue with or without B D N F and NT-3 into the lesion site, again with the most striking results seen in animals receiving the full combination o f transplants, B D N F , and NT-3 (Bregman et al., 1998). In  summary therefore,  the relationship between the interruption o f target-derived  neurotrophic support and the atrophy and loss o f rubrospinal neurons after cervical axotomy provides a compelling rationale for the exogenous application o f neurotrophic factors to the rubrospinal system after injury.  3.2.2. The Targets for Therapeutic Intervention - Cell Body Versus Axon For patients with spinal cord injuries, the obvious site for therapeutic intervention would be their injured spinal cord, where the disrupted axons and their hostile C N S environment can be directly accessed.  Indeed, most experimental therapies (including neurotrophic factors) in  animal models o f spinal cord injury have been initially applied to the spinal cord injury site where their potential effectiveness has been first established.  Studies in which B D N F has been  applied exogenously to the spinal cord in the acute injury setting have demonstrated both a neuroprotective and regenerative effects (Novikova et al., 2002, Sayer et al., 2002, Sharma et al., 2000, Jakeman et al., 1998, L u et al., 2001, Ankeny et al., 2001, Bamber et al., 2001, Blits et al., 2003).  Specifically relevant to my work in the rubrospinal system, the acute administration o f  B D N F to the spinal cord injury site (either by direct infusion or by genetically modified cell lines) has been reported to reduce retrograde atrophy ( L i u et al., 2002) and loss (Diener and Bregman, 1994) o f axotomized rubrospinal neurons, reduce oligodendrocyte apoptosis (Koda et al., 2002), promote rubrospinal axonal regeneration ( L i u et al., 1999), and promote functional  64  recovery ( L i u et al., 1999, N a m i k i et al., 2000, Jakeman et al., 1998, L i et a l , 2003, Ikeda et al., 2002). Alternatively, the target o f intervention after spinal cord injury can be the actual cell body of the axotomized neuron. Kobayashi et al. demonstrated that the direct infusion o f B D N F to the red nucleus prevented rubrospinal atrophy at cervical axotomy, promoted the expression o f regeneration  associated  genes, and facilitated axonal regeneration  transplants (Kobayashi et al., 1997).  into peripheral  nerve  Fukuoka et al. also observed the complete prevention o f  rubrospinal atrophy after cervical axotomy with the direct infusion o f B D N F into the vicinity o f the red nucleus (Fukuoka et al., 1997).  Transfection o f rubrospinal neurons by adeno-  associated virus vector mediated B D N F gene therapy also reversed rubrospinal atrophy after cervical axotomy (Ruitenberg et al., 2004).  The cell body as a potential target o f intervention  extends outside the rubrospinal system as well. Berry et al. demonstrated increased regeneration o f retinal ganglion cells through an optic nerve injury after the application o f a peripheral nerve segment into the vitreous body o f the eye, where the many neurotrophic factors being expressed by reactive Schwann cells o f the peripheral nerve would be in the vicinity o f the retinal ganglion cell bodies (Berry et al., 1996). Also, Toma et al. demonstrated on sympathetic neurons using compartmentalized cultures that N G F exposure to the cell bodies elicited a much higher gene expression response than N G F exposure to the distal axons (Toma et al., 1997).  While the  application o f such interventions aimed at the cell bodies is moderately invasive (and would therefore limit their clinical translation), these studies highlight the importance o f the cell body as a potential target for intervention.  65  3.2.3. The Timing of Therapeutic Intervention While substantial excitement and hope has been generated over experimental therapies such as neurotrophic factors that appear to be beneficial in animal models o f spinal cord injury, these models typically employ an acute injury paradigm where the intervention occurs at or just after the actual injury.  The applicability o f such therapies for individuals whose spinal cord  injury occurred long ago and who are considered "chronic" is less clearly defined (reviewed by Houle and Tessler, 2003).  Unfortunately, axonal regeneration therapies in chronic models o f  spinal cord injury have frequently been met with a reduced efficacy compared to that seen with acute application (discussed further in Chapter 5). O f course, such animal studies in which a delay in therapeutic intervention is included raise the question o f how to define the term "chronic" as it relates to the neurobiology o f spinal cord injury.  From a clinical perspective, the improvement in neurologic function that patients  who sustain a complete or incomplete spinal cord injury achieve w i l l usually plateau around 1 year post-injury (Marino et al., 1999). H o w this pattern o f recovery is best reproduced i n a small rodent model is unclear, and thus it is difficult to know how long o f a delay needs to be instated in order to simulate a chronic state.  A large amount o f work was done on animals 12 months  post-axotomy, and while this period o f convalescence represents a fairly substantial part o f the animals' total life span, it is a somewhat impractical length o f time to propose for a model o f injury chronicity.  Keeping the animals alive for this period o f time is expensive, and in an  elderly state their ability to tolerate anesthesia and invasive surgical interventions is reduced. One subsequent experiment was carried out with a 6 month delay in intervention, but the remainder were performed two months post-axotomy, justified in part by the reports o f other authors o f gross morphologic changes within the spinal cord after injury ( H i l l et al., 2001, Houle  66  and Jin, 2001) and of molecular changes within the red nucleus after injury (Tetzlaff et al., 1991, Kobayashi et al., 1997, Fernandes et al., 1999). in Chapter 7.  This rationalization is discussed in more detail  67  3.3. OVERVIEW OF E X P E R I M E N T A L QUESTIONS AND HYPOTHESES In this chapter, I evaluated the cross-sectional area and the survival o f chronically injured rubrospinal neurons after cervical axotomy to test the following hypotheses: 1.  Investigators who have applied B D N F directly to the injured spinal cord have done so in  various doses/concentrations (ie. a standard dose/concentration does not currently exist).  Given  that rubrospinal neurons undergo significant atrophy after cervical axotomy, and that such atrophy was not reversed by the chronic administration o f B D N F to the spinal cord injury site (Storer et al., 2003), / hypothesized that the loss of BDNF effectiveness in the ''chronic" might be related to providing the appropriate dose/concentration  of the neurotrophic  setting  factor.  To test this hypothesis, animals underwent a cervical axotomy, then two months later, B D N F in 3 exponentially increasing concentrations (50, 1000, and 20,000 ng/ul) was applied within gelfoam to the refreshed injury site (in addition to PBS-treated controls).  Cross-sectional  area of N e u N immunolabeled injured and uninjured neurons was measured and compared.  2. Given that the infusion o f B D N F into the vicinity o f the rubrospinal cell bodies was shown to prevent their atrophy when applied acutely after cervical axotomy (Kobayashi et al., 1997), / hypothesized that the cell-body administration  of BDNF could reverse the atrophy of rubrospinal  neurons when applied chronically after cervical axotomy. To test this hypothesis, animals underwent a cervical axotomy, then twelve months later, B D N F was infused through an osmotic minipump cannula stereotactically placed just lateral to the red nucleus. Cross-sectional area o f N e u N immunolabeled injured and uninjured rubrospinal neurons was measured and compared.  68  3.  Given that the reported rates o f retrograde rubrospinal death after spinal cord axotomy have  been quite variable and have employed different techniques for counting neurons (eg. different histologic techniques and counting techniques - see Table 3.1), / hypothesized that substantial cell death was not occurring in chronically axotomized rubrospinal  neurons.  To test this hypothesis, animal underwent a cervical axotomy, then twelve months later, B D N F was infused into the vicinity o f the red nucleus (as above), and the physical dissector method o f stereologic counting was employed to count injured and uninjured rubrospinal neurons after both N e u N immunohistochemistry and cresyl violet staining.  4.  While performing an intervention on the chronically injured spinal cord (eg. a cell  transplantation), a second axotomy o f already injured axons may be unavoidable (or even necessary).  Given that second axotomy o f chronically injured rubrospinal neurons has been  reported to result in a significant acceleration o f cell death using non-stereologic counting techniques (Houle and Y e , 1999), / hypothesized that this cell death after second axotomy might be over-estimated. To test this hypothesis, animal underwent a cervical axotomy, then six months later, the injury site was refreshed to re-axotomize these chronically injured neurons.  The physical  dissector method o f stereologic counting was employed to count injured and uninjured rubrospinal neurons after N e u N immunohistochemistry.  69  3.4. RESULTS 3.4.1. Neuronal Atrophy 2 Months Post-Axotomy and the Response to Spinal Cord Application of BDNF Two months post-axotomy, I observed significant atrophy o f the injured rubrospinal neurons compared to uninjured. In six animals sacrificed two months after injury with no other interventions, the mean cross sectional area o f the axotomized rubrospinal neurons was 224.7± 15.9 p m while the mean cross sectional area o f the contralateral uninjured rubrospinal 2  neurons was 379.6±50.0 p m (p<0.001, paired t test). 2  Compared to the contralateral uninjured  neurons, the mean cross sectional area o f the injured rubrospinal neurons was 59.7±2.3%. (Figure 3.1) I tested the hypothesis that the application o f various concentrations o f B D N F to the spinal cord could reverse this atrophy.  T w o months post-axotomy, the spinal cord was re-  exposed, the chronic lesion was "refreshed" by extending it rostrally by 1 mm, and either P B S or BDNF-soaked gelfoam was inserted.  The mean cross sectional areas o f injured rubrospinal  neurons treated with BDNF-soaked gelfoam in concentrations o f 50 ng/pl (low), 1000 ng/pl (medium), and 20,000 ng/pl (high) were 231.2±6.8, 232.0±4.7, and 219.9±12.1 p m respectively 2  (n=5 animals per group).  Expressed as a percent o f the contralateral uninjured neurons, the  mean cross sectional areas were 69.0±4.5%, 67.1±1.2%, and 6 6 . 7 ± 4 . 8 % for the low, medium, and high dose B D N F groups.  In animals treated with PBS-soaked gelfoam, the injured  rubrospinal neurons had a cross sectional area o f 234.7±24.2 p m (n=5 animals per group), and were 6 3 . 9 ± 3 . 3 % o f the contralateral uninjured neurons. (Figure 3.1)  70  There was no significant difference in the cross sectional area percentages between the P B S treated animals (63.9±3.3%) and the three groups o f B D N F treated animals (69.0±4.5%, 6 7 . 1 ± 1 . 2 % , and 66.7±4.8%) (p=0.81, one way A N O V A ) .  Given that the cross sectional area  percentages were each higher for the B D N F treated groups than the P B S treated group, I pooled the B D N F treated groups to look for a neurotrophic factor effect.  However, when comparing  the percent cross-sectional area o f the pooled BDNF-treated animals ( 6 7 . 6 ± 2 . 1 % ; n=15) against the P B S treated animals (63.9±3.3%; n=5), there was again no statistically significant difference (p=0.38, unpaired t test).  This suggests that B D N F at any o f the three doses did not promote  the reversal o f neuronal atrophy when applied to the refreshed spinal cord injury site, 2 months post-axotomy. I then compared the cross sectional area percentages o f the injured rubrospinal neurons without a refreshment injury against those o f the P B S and B D N F treated animals. Amongst all 5 groups (No refreshment injury, P B S , low, medium, and high dose B D N F ) there was no significant difference (p=0.34, one way A N O V A ) .  Given  that the cross sectional area  percentages were each higher for the groups o f animals undergoing a second intervention (the P B S and B D N F treated groups) than the P B S treated group, I pooled the animals undergoing the second intervention to look for an effect o f the refreshment injury.  When comparing the percent  cross-sectional area o f the animals that did not have a refreshment injury (59.7±2.3%., n=6) against the combined P B S and B D N F treated animals (66.7±1.8%; n=20), there was a trend towards a significant difference (p=0.057, unpaired t test). The biological relevance o f a mean difference o f approximately 7% in cross sectional area is uncertain, but does suggest that the neurons may have reacted to the refreshment injury with a slight increase in cell size.  71  Figure 3.1. Rubrospinal neuronal atrophy is not reversed with any of the three doses of B D N F applied to the spinal cord injury site.  The  atrophy o f the rubrospinal neurons and the manner in which the FluoroGold  retrograde tracer was utilized to outline the borders o f the injured red nucleus is illustrated. With the neuronal atrophy ( A ) , the borders o f the red nucleus become less distinct. Therefore, the border is outlined in the FluoroGold image (B) and this is overlaid (C) on top o f the N e u N image (A).  Cross sectional area is then measured.  Note the significant atrophy o f the injured  rubrospinal neurons compared to the uninjured contralateral neurons. There was no significant difference between the cross sectional areas o f the groups on one way A N O V A (E). However, when pooling the P B S , L o w , Medium, and H i g h B D N F dose groups (ie. all animals that received a refreshment injury) and comparing them to the N o Refreshment group, there was an increase in mean cross sectional area o f approximately 7%, which approached statistical significance (p=0.057).  73  3.4.2.  Neuronal A t r o p h y 12 Months Post-Axotomy and the Response to C e l l Body  Application of B D N F  Twelve months after cervical axotomy, I evaluated rubrospinal neuronal atrophy with cresyl violet staining and tested the hypothesis that the application o f B D N F directly to the cell bodies rather than to the cut axons in the spinal cord could reverse this atrophy.  Pilot studies o f  3 animals 12 months post-axotomy were performed with the application o f B D N F 500 ng/pl within osmotic minipumps directly to the spinal cord injury site. I observed no apparent reversal of neuronal atrophy (Figure 3.2) which prompted the attempts to apply the B D N F to the cell bodies. In control animals treated 12 months post-axotomy with osmotic minipumps filled with vehicle solution alone (n=8), I observed severe atrophy o f injured rubrospinal neurons.  The  cross sectional area o f these injured neurons was 178.6±5.8 p m , which was significantly smaller 2  than the contralateral uninjured neurons with a mean cross-sectional area o f 277.0±23.2 p m ( p O . O O l , paired t test).  (Figure 3.3)  2  Represented as a percentage o f the uninjured neurons, the  cross sectional area o f the injured rubrospinal neurons in these control animals was 6 6 . 7 ± 4 . 1 % . When evaluating the distribution o f neuronal sizes across the injured and uninjured red nuclei, I observed a preponderance o f smaller, atrophic neurons in the injured red nucleus. (Figure 3.4). In animals treated 12 months post-axotomy with osmotic minipumps filled with B D N F (n=5), the mean cross-sectional area o f the injured and contralateral uninjured rubrospinal 2  2  neurons was 240.0±26.8 pm and 266.6±30.7 p m respectively. (Figure 3.3)  Represented as a  percentage o f the uninjured neurons, the cross sectional area o f the injured rubrospinal neurons in these B D N F treated animals was 90.9±6.2%.  While still slightly smaller, the difference  between the injured and uninjured cross sectional areas was not statistically significant (p=0.074, paired t test).  Comparing the injured rubrospinal neurons between those animals treated with  74  vehicle solution alone against those treated with B D N F , the increase in cross sectional area observed with B D N F treated animals was statistically significant (240.0±26.8 u m 178.6+5.8 p m , p=0.009, 2  unpaired t test).  2  versus  While the control animals had a preponderance o f  smaller, atrophic neurons in the injured red nucleus, the distribution o f neuronal sizes in the animals treated with cell body application o f B D N F was very similar to that seen in the uninjured red nucleus. (Figure 3.4) O f note, there was no significant difference in size between uninjured neurons o f the control and B D N F treated animals (p=0.395, unpaired t test).  75  Figure 3.2. BDNF applied to the spinal cord injury site 12 months after cervical axotomy does not reverse atrophy of injured rubrospinal neurons. In pilot experiments leading to the decision to try administering B D N F directly to the cell bodies o f chronically injured rubrospinal neurons, B D N F (500 ng/pl) was applied via osmotic minipumps to the spinal cord injury site, 12 months post-axotomy.  This unfortunately was not  associated with any noticeable reversal o f atrophy in these chronically injured rubrospinal neurons, thus stimulating the interest in providing the neurotrophic factor via infusion to the brainstem.  Note the significant atrophy o f the injured rubrospinal neurons.  76  Figure 3.3. Atrophy of rubrospinal neurons can be reversed by BDNF twelve months after injury. N e u N immunohistochemistry demonstrates numerous atrophic neurons on the injured side treated with vehicle alone.  Note the recovery in cell size o f the B D N F treated injured  neurons to almost normal size as seen on the contralateral side.  A l l sections are taken from  comparable areas o f the rubrospinal nucleus, approximately 240 pm from the caudal pole. Scale bar. 50 pm.  Neu N Immunohistochemistry Injured  Contralateral Uninjured  77  Figure 3.4. BDNF administration normalizes the distribution of cell sizes in the chronically injured red nucleus. Histogram o f cross-sectional area plotting neurons in 100 p m increments demonstrates a 2  normalization o f the distribution o f cell sizes with the B D N F treatment. Note the predominance of small neurons in the vehicle treated group.  Neuronal Cross Sectional Area (jam ) Vehicle BDNF 2  L-  <100  100 -199  200 -299  300 -399  400 500 >600 -499-599  fc  <100  • Injured Uninjured  100 200 300 -199 -299 -399  400 500 >600 -499-599  78  3.4.3.  Rubrospinal Neuronal Counts 12 Months Post-Axotomy With Cell Body Application  of BDNF The disector method o f counting was used to determine the number o f rubrospinal neurons in the injured and uninjured red nucleus, to test the hypothesis that significant cell death was not occurring after cervical axotomy.  A s discussed earlier, this technique compares each  section against its adjacent section so that neurons are counted only once. For each animal, the number o f neurons on the side o f the injured red nucleus was counted and compared to the number o f neurons found on the contralateral uninjured red nucleus.  The number o f injured  neurons is therefore represented as a percentage o f the presumably normal number o f neurons counted on the uninjured side.  (Figure 3.5)  In the B D N F treated animals, using N e u N  immunohistochemistry I counted similar numbers o f neurons ( 1 0 1 . 8 ± 1 . 3 % , p—0.11) in the injured red nucleus compared to uninjured.  Similarly, in the vehicle treated animals, equal  numbers o f neurons were counted between injured and uninjured red nuclei (99.5±4.2%, p=0.47) using the N e u N marker.  With cresyl violet staining, in the B D N F treated animals I again  counted similar numbers o f neurons  in the injured red nucleus compared to uninjured  (106.8±18.5%, p=0.42). For the vehicle treated animals I counted 8 9 . 0 ± 7 . 6 % as many neurons in the injured red nucleus compared to uninjured (p=0.10).  These results using stereologic  counting techniques suggest that twelve months after injury, while significantly atrophic, cervically axotomized neurons remain alive, and that using a neuronal specific marker (NeuN) aids in the visualization o f atrophic neurons.  That I counted on the cresyl violet sections a  normal number o f injured rubrospinal neurons with B D N F treatment suggests that the reversal o f atrophy induced by the neurotrophic factor made it easier to visualize these neurons and thus count them as being present.  Conversely, neurons that remain atrophic with the vehicle  79  treatment may not be visualized on cresyl violet staining, but may be better identified with N e u N immunohistochemistry.  This was confirmed in further experiments at a 6 month post-axotomy  time point (see below and Figure 3.6).  80  Figure 3 . 5 . Stereologic counting of the injured and uninjured red nuclei demonstrates that chronically injured rubrospinal neurons remain alive long after cervical axotomy. The number o f injured neurons is represented as a percentage o f the number o f contralateral uninjured neurons. Note that with B D N F treatment, the number o f injured neurons counted is approximately 100% o f the uninjured, in both N e u N and cresyl violet staining. With cresyl violet staining only 89% o f the number o f uninjured neurons was detected in the vehicle treatment group.  This difference is likely the result o f the N e u N immunohistochemistry being a  more effective method o f identifying atrophic neurons than cresyl violet staining (see Figure  3.6).  Neuronal Cell Counts 125i  1  BDNF Treated Vehicle Treated  NeuN  Cresyl Violet  81  3 . 4 . 4 . Neuronal Counts Following a Re-Axotomy 6 Months After O r i g i n a l Injury  Houle and Y e reported that a second axotomy o f chronically injured rubrospinal axons (injured one month previously) caused a pronounced acceleration o f cell death when compared to single-axotomized rubrospinal neurons at eight weeks after the initial lesion (Houle and Y e , 1999). In that study, the authors used retrograde labeling o f rubrospinal neurons and observed approximately 70% cell loss in double-axotomized rubrospinal neurons compared to 25% cell loss in those with a single-axotomy (ie. a massive acceleration o f cell death after second axotomy).  In followup to my above findings o f neuronal survival long after cervical axotomy  with the use o f a stereologic counting technique and a neuronal specific marker, I re-evaluated this issue o f cell loss after second axotomy in rubrospinal neurons, six months after their original cervical injury. FluoroGold retrograde labeling o f the injured rubrospinal neurons was performed to help delineate the boundaries o f the injured red nucleus.  (Figure 3.6, panels A - D )  One month after the second axotomy (ie. 7 months after the original axotomy), using N e u N and the disector method o f counting, I counted 8 7 . 3 ± 2 . 3 % o f neurons in the injured red nucleus compared to the uninjured (n=4 animals).  This cell loss o f 13% represented a  statistically significant decrease in the number o f injured rubrospinal neurons (p<0.05, paired t test). Measurement o f cross sectional area again demonstrated that axotomized rubrospinal neurons undergo marked atrophy. Injured rubrospinal neurons measured 182.±5.4 p m versus 2  290±6.0 p m for the contralateral uninjured neurons. This reduction in size o f the injured 2  rubrospinal neurons to 6 2 . 9 ± 2 . 1 % o f the cross sectional area o f uninjured contralateral neurons was statistically significant (p< 0.01, paired t test). Analysis o f sections on which N e u N immunohistochemistry was performed initially, the digital images captured, and then cresyl violet staining was performed demonstrated a number o f  82  atrophic N e u N positive neurons in the injured red nucleus that did not stain effectively with cresyl-violet.  (Figure 3.6, panels E-J)  This finding is consistent with the differences in the  cell counts between N e u N and cresyl violet staining that I recorded in my evaluation o f rubrospinal neurons 12 months post-axotomy.  Note that in the counts o f the atrophic, vehicle-  treated neurons, with N e u N immunohistochemistry I detected 9 9 . 5 ± 4 . 2 % o f the number of neurons in the uninjured red nucleus, while with cresyl violet staining I detected 89.0±7.6%. The reversal o f atrophy with cell body application o f B D N F facilitated the identification o f the injured rubrospinal neurons and/or their distinction from surrounding glial cells with the standard cresyl violet staining.  83  F i g u r e 3.6. F l u o r o G o l d retrograde labeling helps to identify the boundaries o f the injured red nucleus, and N e u N immunohistochemistry better identifies atrophic neurons than cresyl violet staining. N e u N immunohistochemistry and FluoroGold (FG) retrograde labeling o f rubrospinal neurons approximately 160 pm from the caudal pole o f the red nucleus (magnocellular portion). N e u N immunohistochemistry is shown in the green channel ( A and D ) while FluoroGold labeling o f the injured rubrospinal neurons is represented in false colour in the red channel (B). For the injured red nucleus, the N e u N (A) and FluoroGold (B) images o f are combined (C) so that the FluoroGold labeling facilitates identification o f the rubrospinal neurons projecting down to the lumbar spinal cord (ie. those that are axotomized at the C3/4 level).  Note the atrophic  neurons in the ventrolateral portion o f the injured red nucleus that stain poorly with FluoroGold but are seen well with the N e u N staining (arrow).  The FluoroGold staining is, however,  particularly useful for identifying the borders o f the axotomized population o f neurons in the red nucleus.  In the N e u N immunostaining o f the injured red nucleus ( A ) , note the large neurons in  the dorsomedial part o f the nucleus (arrowhead) that are located i n an area devoid o f FluoroGold staining (B). Such neurons would be considered to be outside the area o f the axotomized population o f rubrospinal neurons and would therefore not be included in the evaluation o f neuronal counts or cross sectional area.  In E and F, the sections were stained with cresyl  violet. Note on the injured side (E), the difficulty in identifying neurons and the borders o f the red nucleus after the significant atrophy.  Higher magnification o f injured rubrospinal neurons  ( G versus H and I versus J) the demonstrates the poor visualization o f these atrophic neurons with cresyl violet staining compared to N e u N immunohistochemistry. Scale bar: 50 pm.  85  3.5 DISCUSSION  3.5.1. Chapter Summary This chapter reviews rubrospinal atrophy and survival at a number o f time points after cervical axotomy and in a number o f experimental paradigms (spinal cord versus cell body treatment). In summary, using stereologic counting techniques and a specific neuronal marker, I observed that rubrospinal neurons do not undergo significant cell loss after cervical axotomy, but rather, undergo severe atrophy which makes them hard to detect using conventional histologic techniques.  I found that this atrophy can be reversed with B D N F applied directly to the cell  bodies 12 months after injury, but was not reversed with B D N F applied directly to the spinal cord injury site 2 months after injury, despite using different doses o f B D N F .  I also determined  that following a second axotomy, rubrospinal neurons undergo a modest but statistically significant amount o f cell death.  3.5.2. Rubrospinal Neuronal Survival Versus Death After Axotomy The retrograde death o f rubrospinal neurons in rat species following cervical axotomy has been reported as approximately 25% to 35% by 2 months after injury ( M o r i et al., 1997, Goshgarian et al., 1983, Houle and Y e , 1999).  Clearly, the significant loss o f supraspinal  neurons in response to spinal cord injury could have tremendous implications on the prognosis for recovery.  M y observation that 12 months after cervical axotomy, rubrospinal neurons are  atrophic but otherwise present in normal numbers is therefore somewhat at odds with other authors who have reported the death o f a significant proportion o f adult rubrospinal neurons after axotomy.  The response o f rubrospinal neurons to axotomy is an issue that has been examined  86  extensively in the literature, in rats predominantly (Goshgarian et al., 1983, Houle and Y e , 1999, N o v i k o v a et al., 2000, Feringa et al., 1988, M o r i et al., 1997, L i u et al., 2002) but also in mice (Zhou et al., 1999), and opposums ( X u and Martin, 1990, Wang et al., 1999).  While neuronal  atrophy appears to occur universally after axotomy, these studies report different rates o f cell loss between and amongst species (See table 3.1).  Determining whether these represent true  intra and inter-species differences is made difficult by variations in histologic technique (eg. retrograde tracing o f neurons versus cresyl violet staining) and counting methods (eg. stereologic versus non-stereologic counts).  Influence of Histologic Technique - NeuN vs Cresyl Violet vs Retrograde Tracing The influence o f histologic technique on the reporting o f cell death was demonstrated by Feringa et al. (Feringa et al., 1988).  In this study, the authors used both horseradish peroxidase  ( H R P ) retrograde tracing and hematoxylin and eosin ( H & E ) staining to evaluate rubrospinal cell death in Wistar rats undergoing thoracic axotomy.  Using H R P , they found no statistically  significant difference in the number o f injured rubrospinal neurons compared to uninjured at 15 and 25 weeks post-axotomy. However, using H & E staining, they found a significant reduction in rubrospinal neurons in the injured compared to the uninjured red nucleus (42% loss at 52 weeks post-axotomy).  X u and Martin in their study o f rubrospinal cell death in adult opposums  also commented that the use o f cresyl violet staining likely "underestimated the proportion o f neurons that survived axotomy" ( X u and Martin, 1990).  87  T a b l e 3.1. Reported rates of retrograde cell death of rubrospinal neurons after spinal cord injury vary widely in the literature  Note that these studies employ many different animal species, counting techniques, and histologic techniques to arrive at quite varied rates o f cell loss after injury. Authors  Animal  Lesion  Counting Technique  Egan et al. Acta Neuropathologica, 1977  Rat Male Wistar  C4 orT13 hemisections  Goshgarian et al. J Comp Neurol, 1983  Female OsbornMendel rats  McBride et al. i Neuropath Exp Neurol, 1989  Female Wistar rats  Tl hemisection and C2 hemisection T9 transection  Xu and Martin, Exp Neurol, 1990  Female opposums  T5 hemisection  Standard - every 10 section - 30 and 60 days post  Theriault and Tator, J Comp Neurol 1994  Female Wistar rats  Standard - every 2 section  Mori et al, Exp Neurol, 1997  Female SpragueDawley rats  C7-T2 clip compresion or T2-T3 transection C3/4 hemisection  Feringa et al. Exp Neurol 1998  Rat Female wistar  T9 transection  Standard - counts of caudal and total RSNat5, 10, 15, 25, 52 weeks  Zhou et al, NeuroReport, 1999 Houle and Ye, Neurosci, 1999  C57BL/6J mice and Bcl-2 overexpressing Female SpragueDawley rats  C4-5 hemisection  Liu P et al., Exp Neurol 2003  Female Wistar rats  C2 lateral funiculus incision or brainstem  Liu et al, Exp Neurol 2002  Female SpragueDawley rats  C3 hemisection  Optical disector method 1, 2, or 3 months post axotomy Standard - counts on alternating sections (25 microns apart) at 1,4, and 8 weeks post axotomy Standard - counts of every 3 section (60 microns apart) at 2, 4, and 10 weeks post axotomy Optical disector method - counts of caudal half - 1 or 2 months post axotomy  C3 Hemisection  Standard - counts of the caudal 200 microns at 1,3,4,7,14,21 days Standard - counts from 9-219 days  Standard - counts of 910 microns - 10 and 20 weeks post  th  nd  Standard - every 3 section - corrected for cell size - 2 and 4 months post rd  Histology Cresyl violet  HRP retrograde labeling and neutral red counters tain Fluorogold retrograde prelabeling  Fast blue retrograde prelabeling and cresyl violet Fluorogold or HRP retrograde labeling after injury Fluorogold retrograde prelabeling or Nissl-Myelin staining HRP retrograde tracing at Tl H & E staining  Cresyl violet staining  Cell loss Equal numbers of neurons counted at days 1 and 21 in both groups (cervical or thoracic) With HRP, 63% loss of RSNs; In total (labeled and non-labeled) 35% loss. No significant difference in surviving rubrospinal neurons at 10 and 20 weeks post axotomy. With Fastblue, 25% cell loss With cresyl violet, 20% cell loss with cresyl violet With Fluorogold, 27% cell loss at 8 weeks  35-38% cell loss at 2 and 4 months postaxotomy  With HRP, no significant difference in caudal or total cells at 15 or 25 weeks. With H&E staining, cell death of 22-41% 45% cell loss at 1, 2, and 3 months  True blue retrograde labeling at injury site  25% cell loss at 8 weeks post axotomy.  Cresyl violet  15% cell loss at 4 and 10 weeks post axotomy with C2 lesion; 75% cell loss at 10 weeks post axotomy with brainstem lesion 45% cell loss at 2 months post axotomy  rd  Fluorogold retrograde prelabeling or cresyl violet  88 In my own series o f animals at both 6 or 12 months post-axotomy, I also observed the influence o f histologic technique in the number o f rubrospinal neurons counted. With the use o f N e u N immunohistochemistry, equal numbers o f neurons were present in the injured and uninjured red nuclei, indicating the absence o f cell death after axotomy. W i t h the use o f cresyl violet staining, however, there were fewer neurons in the injured red nucleus o f animals treated with vehicle solution. This decrease in cell numbers would otherwise imply the retrograde death o f rubrospinal neurons, i f not for the observation that in animals treated with cell body infusions of B D N F , equal numbers o f cresyl violet stained neurons were present in the injured and uninjured red nuclei. With the reversal o f neuronal atrophy seen with the application o f B D N F , the discrepancy in cell counts between vehicle treated and B D N F treated animals on cresyl violet stained sections suggests that severely atrophic neurons are not detected (and thus deemed as dead) using this histologic technique. The reversal o f atrophy with B D N F makes it possible to see these neurons again and thus count them.  I indeed observed in slides stained first with  N e u N and then with cresyl violet that the visualization o f small neurons is much enhanced with N e u N (see Figure 3.6).  Interestingly, a recent report by Ruitenberg et al. (Ruitenberg et al.,  2004) used cresyl violet staining to evaluate rubrospinal neurons 1 and 6 months after cervical axotomy and treatment with adeno-associated  virus vector-mediated B D N F gene transfer.  While the cell counting data was not made available in the manuscript, the authors report that the B D N F gene transfer effected a reversal o f neuronal atrophy and that similar numbers o f rubrospinal neurons were counted on cresyl violet stained sections in both injured and uninjured red nuclei, "indicating the absence of massive lesion-induced  death of RSNs".  This to some  extent provides independent substantiation o f my findings o f rubrospinal neuronal survival one year after cervical axotomy, as I reported in 2002 ( K w o n et al., 2002b).  89  Retrograde tracers have also been used to quantify cell loss after axotomy, with the tracer injected caudally into the spinal cord to label the neuronal population prior to axotomy, and then the loss o f retrogradely labeled neurons representative o f cell death.  Such was the technique  used by M o r i et al. (FluoroGold) (Mori et al., 1997), M c B r i d e et al. (FluoroGold) (McBride et al., 1989), Houle and Y e (True Blue) (Houle and Y e , 1999), and Feringa et al. (horseradish peroxidase)(Feringa et al., 1988).  While the strategy o f pre-labeling with a retrograde tracer  would seemingly have the advantage o f distinctively identifying rubrospinal neurons, the interpretation o f subsequent cell counts requires one to make a number o f assumptions: 1. that the tracer was reliably taken up and retrogradely transported by most i f not all the neurons/axons; 2. that the axotomized neurons w i l l actually maintain the tracer for long periods o f time, despite all the metabolic changes that occur after axotomy that culminate in their severe atrophy; and 3. that the retrograde tracer does not leach out and get picked up by glial cells which could be mistaken for atrophic neurons.  A s for the first assumption, Theriault and Tator demonstrated  significantly better retrograde prelabeling o f rubrospinal neurons with FluoroGold than with H R P , demonstrating that the efficacy o f labeling differs amongst the tracers (Theriault and Tator, 1994).  Pilot data from our lab suggests that the uptake by rubrospinal neurons o f retrograde  tracers applied to the spinal cord can indeed suffer from some variability.  W i t h regards to the  second assumption, the ability o f axotomized rubrospinal neurons to maintain each o f the varying retrograde tracers over time is unknown.  Novikova et al. compared the retrograde  labeling o f axotomized spinal motoneurons using five different retrograde tracers, including Fast Blue, FluoroGold, FluoroRuby, M i n i R u b y , and FluoroEmerald (Novikova et al., 1997).  They  found that the number o f Fast Blue-labeled motoneurons remained constant over time (up to 24 weeks), while a time-dependent decrease in the number o f neurons labeled with the other tracers  90  was observed, related to the degradation and leakage o f the tracer.  Finally, M c B r i d e et al.  demonstrated that between 10 and 20 weeks post axotomy, there was increased leakage o f FluoroGold from rubrospinal neurons and subsequent uptake by surrounding glial cells (which they assumed to be oligodendrocytes) (McBride et al., 1989). These results all point to the limitations o f using retrograde tracers to determine rubrospinal cell death after axotomy.  I felt, however, that even i f the rubrospinal axons picked  up and retrogradely transported FluoroGold i n a somewhat inconsistent manner and then maintained the tracer over time in a similarly inconsistent fashion, its presence could at least be used to outline the general area o f the red nucleus where the injured neurons resided.  Egan et al.  pointed out in cresyl violet stained sections that following axotomy, the atrophy o f rubrospinal neurons makes it difficult to identify the boundaries o f the red nucleus and thus distinguish this neuronal population from surrounding reticular neurons (Egan et al., 1977).  Indeed, I found this  to be somewhat problematic in my evaluation o f rubrospinal neurons 12 months post-axotomy. Therefore, in my studies o f rubrospinal neuronal atrophy and cell number at 2 and 6 months post-axotomy, the prelabeling with FluoroGold greatly assisted in outlining the region o f the red nucleus within which to count the descending population o f injured, atrophic rubrospinal neurons.  Indeed,  many  atrophic  rubrospinal  neurons  identified  on  NeuN  immunohistochemistry in the injured red nucleus had very faint or absent FluoroGold labeling (see Figures 3.6).  91  3.5.3. Counting Techniques for Evaluating Neuronal Numbers M y reported findings o f rubrospinal survival 12 months after cervical axotomy ( K w o n et al., 2002b) and o f modest cell death (approximately 13%) in response to a second axotomy 6 months after the original cervical axotomy ( K w o n et al., 2002c) are based on the application o f stereologic counting methods.  In summary, the physical disector technique involves outlining  the neurons on one section (the "index" section) and comparing them to neurons on the next adjacent section (the "lookup" section) and excluding those neurons seen i n both sections. A s stated in the Background chapter, it is well recognized that significant controversy surrounds the subject o f optimal counting techniques (Benes and Lange, 2001).  It is important to note,  however, that while rubrospinal atrophy is almost universally observed after axotomy, and cellular atrophy is known to influence the results o f standard counting techniques, almost all o f the studies that describe the death o f rubrospinal neurons after axotomy do not employ either a stereologic counting method or even a mathematical correction to account for the change in cell size (such as the Abercrombie correction). The loss o f approximately 4 5 % o f rubrospinal neurons after cervical axotomy in both mice (Zhou et al., 1999) and rats ( L i u et al., 2002) was reported by authors that did apply stereologic counting techniques to the injured red nucleus. In both o f these studies, cresyl violet staining was used to identify the rubrospinal neurons, which, based on the compative analysis performed  in my  experiments,  appears  to  be  a  immunohistochemistry for identifying atrophy neurons.  less  reliable technique  than  NeuN  The stereologic counting techniques  also differed slightly, in that I have used the physical disector method i n my studies, while these studies employed the optical disector (also called the optical fractionator) method.  In the optical  disector technique, one evaluates a section o f tissue through its vertical height by sequentially  92  moving the plane o f focus in small increments through the vertical axis, and evaluating the cells as they come into focus.  In essence, the optical disector technique counts the cells that come  into focus as one moves vertically through the section in very thin slices (planes o f focus), then excludes those cells that are in focus in the last plane o f focus o f the section, as these would presumably be in focus also in the next adjacent section.  The technique is applied typically to  small portions o f the overall population o f interest, and in the papers o f Zhuo et al. and L i u et al., for each section the red nucleus was divided into quarters and one quarter randomly examined (Zhou et al., 1999, L i u et al., 2002).  The optical disector method is more dependent than the  physical disector on mathematical calculations that involve the estimated thickness o f the section and the area o f the sampling compared to the total area o f interest, and to some extent, assumes that the cells are evenly distributed throughout that total area o f interest (in this case, the red nucleus).  M y technique o f using the physical disector counts is done at a magnification that  allows one to count all o f the neurons in the red nucleus on a given section. Given the fact that the neurons are not evenly distributed throughout the nucleus, with the dorsomedial "quadrant" housing the most neurons (particularly as one moves rostrally in the nucleus), this would seemingly provide less error. O n the other hand, the optical disector technique is not affected by the small extent o f tissue distortion that occurs between two physically separate sections.  En  balance, I think that the differences in these two stereologic counting techniques account for less o f the discrepancy between my results and those o f Zhou et al. and L i u et al. than the histologic techniques that I employed, with the N e u N immunohistochemistry facilitating the identification o f atrophic, injured rubrospinal neurons.  93  3.5.4. Rubrospinal Neuronal Atrophy After Axotomy Regardless o f histologic technique (NeuN or cresyl violet staining), I observed statistically significant atrophy o f axotomized rubrospinal neurons at 2 months, 6 months, and 12 months post-axotomy.  Injured rubrospinal neurons measured approximately 65 to 70% o f the  cross sectional area o f their uninjured counterparts.  Such atrophy is consistent with numerous  other reports o f rubrospinal atrophy in response to axotomy in rats, mice, and opposums ( L i u et al., 2002, Egan et al., 1977, Kobayashi et al., 1997, Storer et al., 2003, Wang et al., 1999, X u and Martin, 1990, Zhou et al., 1999).  The most rapid extent o f rubrospinal neuronal atrophy begins  between 7 and 14 days after cervical axotomy (Tetzlaff et al., 1991, Barron et al., 1989).  The  application o f B D N F to either the spinal cord ( L i u et al., 2002, L i u et al., 1999) or to the rubrospinal cell bodies themselves (Fukuoka et al., 1997, Kobayashi et al., 1997, Ruitenberg et al., 2004) acutely after injury has been shown to prevent or reverse this atrophy. In contrast to the effectiveness o f acute B D N F application, I found that rubrospinal atrophy was not reversed by the application o f B D N F to the injured cervical spinal cord 2 months post-axotomy.  The ineffectiveness o f B D N F applied to the spinal cord 2 months post-  axotomy is consistent with the recently reported findings o f Storer et al, who found that B D N F applied to the spinal cord 1 month post axotomy also did not reverse rubrospinal atrophy (Storer et al., 2003).  M y experiments at two months post-axotomy used exactly the same dose o f  B D N F (50 ng/pl) and the same technique o f gelfoam application (replacing the gelfoam with freshly soaked gelfoam every 15 minutes) as Storer and colleagues. Additionally, I used two substantially higher concentrations o f B D N F (1000 ng/pl and 20,000 ng/pl) to evaluate for a dose dependent effect, and again found no reversal o f rubrospinal neuronal atrophy.  Tobias et  al. applied grafts o f B D N F and N T - 3 secreting fibroblasts into the cervical spinal cord 6 weeks  94  after hemisection (Tobias et al., 2003).  When comparing the cross sectional area o f injured  against contralateral uninjured neurons, they reported only a slight reversal o f rubrospinal atrophy, from 5 0 . 8 ± 3 % in control animals to 5 8 . 7 ± 2 % in neurotrophic factor treated animals. Because the grafts contained a combination o f B D N F and N T 3 , it is difficult to delineate from this study which neurotrophic factor this small increase in cell size should be attributed to. Clearly, my results and those o f others indicate that the application o f B D N F (even in a range o f concentrations) to the injured spinal cord at a "chronic" time point after injury has limited to no appreciable effect on rubrospinal cell size. While the application o f B D N F to the injured spinal cord had limited effect on rubrospinal neuronal size 2 months post-axotomy, application to the rubrospinal cell bodies 12 months after cervical axotomy was associated with a near-complete reversal o f neuronal atrophy. M y reporting o f these observations represented the first description o f B D N F administration directly to the cell bodies o f rubrospinal neurons in such a chronic injury state ( K w o n et al., 2002b). These findings, however, were corroborated recently by Ruitenberg and colleagues with the in vivo gene transfer o f B D N F into rubrospinal neurons axotomized 18 months prior (Ruitenberg et al., 2004). These authors found that 3 months after virus injection (21 months after the original axotomy), the rubrospinal neurons o f animals injected with the BDNF-encoding adeno-associated viral vector had a mean cross sectional area o f 2 8 4 ± 1 7 um  compared to  uninjured neurons measuring 338±9 p.m (representing atrophy o f approximately 80 to 85% the size o f normal rubrospinal neurons).  While the reversal o f rubrospinal atrophy was not  complete, the size o f the chronically injured rubrospinal neurons after B D N F treatment was significantly greater than those o f animals that received the control virus (p<0.006).  95  A s neuronal atrophy and its reversal or prevention after axotomy is a common outcome measure for studies o f experimental therapeutic interventions, it is worth considering the potential physiologic implications o f neuronal atrophy (and its reversal).  While neuronal body  size in metazoan organisms is determined largely by D N A content and transcriptional activity (Cavalier-Smith, 1978), vertebrate neurons possess generally constant amounts o f nuclear D N A ( M c l l w a i n , 1991, Pearson et al., 1984), and thus transcriptional activity appears to be the most important determinant o f cell size (Pena et al., 2001).  In support o f this, Sato et al.  demonstrated in frog motoneurons that the largest cells contained the largest nuclei, which also possessed the highest transcriptional activity (Sato et al., 1994).  The size o f non-neuronal cells  also appears to correlate with transcriptional activity. Schmidt and Schibler found in cells from the liver, lung, kidney, spleen, and thymus o f rats and mice that large cells synthesize more R N A than small cells, and that the ratio between R N A (variable) and D N A (fixed) per cell correlated with cell size (Schmidt and Schibler, 1995). Given this relationship between transcriptional activity and cell size, it would be reasonable to postulate that the atrophy o f rubrospinal neurons after axotomy represents a reduction in their transcriptional activity, while the reversal o f neuronal atrophy likely represents an increase i n transcriptional activity.  Exactly which o f the genes whose expression changes  after axotomy or after neurotrophic factor application are most influential in determining rubrospinal neuronal cell size is a matter o f speculation, although genes for structural cytoskeletal proteins such as neurofilament (Verge et al., 1990) are likely candidates.  In  support o f this proposed relationship in axotomized rubrospinal neurons, Fernandes et al. demonstrated that within the injured red nucleus, the largest rubrospinal neurons also possessed the highest level o f G A P - 4 3 and T a l tubulin m R N A expression, and were typically the neurons  96  to have regenerated through a peripheral nerve transplant, thus also receiving trophic support from the transplant itself (Fernandes et al., 1999).  In studies that evaluate experimental  therapies to promote axonal regeneration after spinal cord injuries, it would therefore seem reasonable to view the prevention o f neuronal atrophy or its reversal in the chronic setting as a potentially beneficial outcome.  3.5.5.  Dosage Considerations for BDNF A s stated in the introductory section o f this chapter, the direct application o f neurotrophic  factors such as B D N F to the spinal cord injury site is a frequently employed experimental treatment strategy.  In general, the exogenous administration o f B D N F to the spinal cord injury  site has been performed in one o f three methods:  single application (injection or within  gelfoam), via continuous infusion, and by cellular expression through ex vivo or in vivo genetic transfer techniques (Novikova et al., 2002, Sayer et al., 2002, Sharma et al., 2000, Jakeman et al., 1998, L u et al., 2001, Ankeny et al., 2001, Bamber et al., 2001, Blits et al., 2003).  Clearly,  there are substantial differences in the actual quantity and mechanism o f B D N F administration to the injured spinal cord in these studies.  A n optimal dose/concentration for exogenously applied  B D N F has not yet been established, and in keeping with this, a variety o f doses and treatment modalities have in fact been employed.  These range from as low as 50 ng/pl in gelfoam soaked  sponges as described by Houle and colleagues (Houle and Y e , 1999, Y e and Houle, 1997) to as high as 12,500 ng/pl infused over 28 days through an osmotic minipump as described by Jakeman et al. (Jakeman et al., 1998).  Fibroblasts genetically altered to produce B D N F have  been measured to produce between 12.8 to 94 ng o f B D N F / 1 0 cells/24 hours in vitro, ( L i u et al., 6  1999, L i u et al., 2002, Tobias et al., 2003) but the actual in vivo delivery to the cord over time as the transplant incorporates into host tissue is difficult to determine.  The study by Jakeman et al.  97  employed three different concentrations o f B D N F in a 28 day osmotic minipump infusion (4150, 8300, and 12,500 ng/pl, providing a total dose o f 50, 100, 150 pg/day). The authors reported increased functional recovery in the two higher concentrations o f B D N F , including improved plantar stepping, increased early locomotor recovery, and more consistent forelimb and hindlimb coordination.  The anatomic correlate behind such functional recovery is difficult to ascertain,  but this study points to an influence o f B D N F concentration, despite the fact that even the lowest of these concentrations is likely orders o f magnitude higher than what is expressed within the native cord after injury (Ikeda et al., 2001). From a therapeutic standpoint, the issue o f dose is obviously one o f particular relevance, and for this reason I chose to evaluate three B D N F concentrations concentration separated by an order o f magnitude.  in my study, each  Conveniently, a 1/20 dilution o f my stock  B D N F solution (20,000 ng/pl) provided the 1,000 ng/pl concentration o f B D N F that Bregman and colleagues had used to supplement a fetal tissue transplant in a over-hemisection model o f acute spinal cord injury (Bregman et al., 1997, Bregman et al., 1998)  This concentration o f  B D N F prevented the atrophy o f acutely axotomized rubrospinal neurons (at the thoracic level) and promoted growth o f serotonergic, noradrenergic, and corticospinal axons within the fetal tissue transplants.  A 1/20 dilution o f this concentration provided the 50 ng/pl that Houle and  colleagues have employed in numerous studies o f the rubrospinal system after cervical spinal cord injury (Houle and Y e , 1999, Y e and Houle, 1997). The method o f applying the B D N F in gelfoam and then replacing the BDNF-soaked gelfoam sponge with fresh neurotrophic factor every 15 minutes over the first hour was reported to attenuate rubrospinal death after second axotomy performed 4 weeks after initial injury (Houle and Y e , 1999), and so I chose to employ this same regimen and B D N F concentration in my study.  I decided against using an osmotic  98  minipump infusion of BDNF into the spinal cord injury site two months after axotomy as an additional treatment arm for a number of reasons.  For one, I felt that the lowest BDNF  concentration (50 ng/pl) was likely supra-physiologic in nature.  Secondly, BDNF infused  directly to the cervical spinal cord through osmotic mini-pumps 12 months after cervical axotomy did not reverse rubrospinal neuronal atrophy in the hands of Nao Kobayashi and Wolfram Tetzlaff (n=3 animals, unpublished data) and thus instigated my subsequent investigations into cell-body treatment at this chronic time point, which I later reported on (Kwon et al., 2002b). Finally, the efficiency of such osmotic minipump infusions over time has been shown to be modest, making it difficult to predict exactly how much trophic factor actually gets delivered (Jones and Tuszynski, 2001). 3.5.6. A d m i n i s t r a t i o n o f B D N F to the C e l l B o d y and to the Injured S p i n a l C o r d  Obviously, one of the most striking observations from this chapter is the difference between the spinal cord and the cell body administration of BDNF in terms of the reversal of rubrospinal neuronal atrophy. While the time frame differed between these two experimental paradigms (2 months post-axotomy for the spinal cord application of BDNF versus 12 months post-axotomy for the cell body application of BDNF), it is probably reasonable to postulate that the effective reversal of neuronal atrophy with cell body application of BDNF at 12 months could have been reproduced at 2 months post-axotomy. In their in vitro study of sympathetic neurons, Toma et al. found that the increased effectiveness of cell body administration of NGF compared to axonal administration was not the result of differences in TrkA receptors, but rather to the spatial localization of the NGF receptor-ligand complex (Toma et al., 1997). Nonetheless, in the absence of effectiveness of the spinal cord application of BDNF at two months post-axotomy I considered whether there were in vivo differences in TrkB receptor  99  expression between the chronically injured cell bodies and spinal cord. investigations are described in Chapter 6.  My results of these  100  - CHAPTER 4 REGENERATION ASSOCIATED GENE EXPRESSION IN THE CHRONICALLY INJURED RUBROSPINAL SYSTEM  4.1. SUMMARY The intrinsic ability o f neurons to regenerate axons after injury is thought to be closely linked to the coordinated expression o f a multitude o f genes, collectively termed regeneration associated genes, or R A G s .  The failure o f C N S neurons to regenerate axons after injury is  thought to be i n part related to the inability to mount and sustain a sufficient R A G expression response.  In this chapter I review the expression o f two important R A G s , G A P - 4 3 and T a l  tubulin in rubrospinal neurons two and 12 months after cervical axotomy.  Using in situ  hybridization, I observed that the expression o f G A P - 4 3 and o f T a l tubulin was l o w in injured rubrospinal neurons as compared to the uninjured contralateral control neurons at both the two and 12 month time points. The application o f B D N F to the spinal cord injury site 2 months postaxotomy did not stimulate an increase in G A P - 4 3 or T a l tubulin expression over that which was induced by the refreshment injury performed during B D N F application ( K w o n et al., 2004a). However, the application o f B D N F directly to the rubrospinal cell bodies 12 months postaxotomy did stimulate an increase i n G A P - 4 3 and T a l tubulin expression ( K w o n et al., 2002b). These results suggest that the chronically injured rubrospinal neurons maintain responsiveness to B D N F at the level o f the cell body but not at the injured spinal cord.  101  4.2.  INTRODUCTION  A s discussed in the Background chapter, the cell body response to axotomy is felt to be an element o f the neuron's axonal regenerative competence after injury. That neurons within the C N S possess an ability to extend their axons into the permissive environment o f peripheral nerves after injury was demonstrated first by Tello and Ramon y Cajal almost a century ago (Ramon y Cajal S, 1928), and reiterated with more contemporary methods in work performed by Richardson, Aguayo, and colleagues (Richardson et al., 1980). The somewhat limited nature o f this intrinsic regenerative nature in mature C N S neurons, however, lies i n stark contrast to the fairly robust regenerative abilities o f motor and sensory neurons projecting through the peripheral nervous system. While the injury environments encountered by neurons o f the C N S and P N S are clearly different in their permissiveness to regeneration (the latter being far more conducive), it is also apparent that C N S neurons fail to mount or to maintain some o f the gene expression changes thought to be necessary for a peripheral regenerative response (Fernandes et al., 1999, Tetzlaff and Bisby, 1990).  The genes thought to be related to the regenerative  response encode a wide spectrum o f proteins, including transcription factors, cytoskeletal proteins, growth cone proteins, and cell adhesion molecules (reviewed by Fernandes and Tetzlaff, 2000).  The identification o f these proteins and their enhancement may augment the  regenerative capacity o f the injured neuron and thus represents a potential therapeutic target for spinal cord repair (reviewed by Plunet et al., 2002). While a large number o f genes likely undergo changes after axotomy, those that have been associated with axonal growth propensity have been termed "regeneration associated genes", or R A G s (Plunet et al., 2002).  The failure o f neurons within the C N S to incite a  sufficient and sustained up-regulation o f R A G s is considered to embody their "intrinsic" inability  102  to regenerate after axotomy. The two R A G s that our laboratory have studied most extensively are G A P - 4 3 and T a l tubulin, the descriptions o f which are detailed in the Background chapter, Sections 1.5.4 and 1.5.5. In brief, the importance o f R A G expression in axonal regeneration after C N S injury is implied by the correlation between their upregulation and the regenerative propensity o f neurons both in the C N S and the P N S . upregulate  For example, rubrospinal neurons o f the C N S transiently  G A P - 4 3 and tubulins after cervical axotomy but not thoracic axotomy, and  correspondingly regenerate axons into permissive peripheral nerve transplants engrafted into the cervical but not thoracic spinal cord (Tetzlaff et al., 1991, Tetzlaff et al., 1994). Fernandes et al. observed on a cell-by-cell basis that those rubrospinal neurons that were capable o f regenerating through a peripheral nerve transplant after cervical axotomy were those that upregulated G A P - 4 3 expression (Fernandes et al., 1999).  Peripheral axotomy o f D R G neurons increases G A P - 4 3  expression while central axotomy does not (Chong et al., 1994), and correspondingly the regeneration o f the central axons is promoted only by a preceding peripheral axotomy (the "conditioning lesion") (Richardson and Issa, 1984, Neumann and Woolf, 1999), demonstrating the correlation between this gene and axonal regeneration for neurons o f the P N S .  A  relationship between G A P - 4 3 expression and axonal elongation has been demonstrated across numerous species, such as the optic nerves o f fish, toads, and rabbits (reviewed by Skene, 1989). A s I am interested in evaluating measures that reflect the regenerative capacity o f rubrospinal neurons in the chronic injury state, I examined G A P - 4 3 and T a l tubulin m R N A expression using in situ hybridization.  103  4.3. OVERVIEW OF EXPERIMENTAL QUESTIONS AND HYPOTHESES In this chapter, I evaluated the mRNA expression of GAP-43 and T a l tubulin in rubrospinal neurons to test the following hypotheses: 1.  Investigators who have applied BDNF directly to the injured spinal cord have done so in  various doses/concentrations.  Given that the expression of GAP-43 and T a l tubulin decreases  in rubrospinal neurons after axotomy (Kobayashi et al., 1997, Storer and Houle, 2003), and that BDNF administration to the spinal cord injury site appears to diminish in its ability to increase RAG expression (Storer et al., 2003) and promote a regenerative response after a delay in intervention (Tobias et al., 2003), / hypothesized that the loss of effectiveness in promoting RAG expression in  the  "chronic" setting might be  related to providing  the appropriate  dose/concentration of the neurotrophic factor.  To test this hypothesis, animals underwent a cervical axotomy, then two months later, BDNF in 3 exponentially increasing concentrations (50, 1000, and 20,000 ng/ul) was applied within gelfoam to the refreshed injury site (in addition to PBS-treated controls).  In situ  hybridization for GAP-43 and T a l tubulin mRNA was then performed on injured and uninjured rubrospinal neurons. 2. Given that the infusion of BDNF into the vicinity of the rubrospinal cell bodies was shown to promote RAG expression in acutely injured rubrospinal neurons (Kobayashi et al., 1997), / hypothesized that the cell-body administration of BDNF could promote GAP-43 and Tal tubulin expression when applied chronically after cervical axotomy.  To test this hypothesis, animals underwent a cervical axotomy, then twelve months later, BDNF was infused through an osmotic minipump cannula stereotactically placed just lateral to  104  the red nucleus.  In situ  hybridization for GAP-43 and T a l tubulin mRNA was then performed  on injured and uninjured rubrospinal neurons.  105  4.4 RESULTS 4.4.1. R A G Expression Two Months Post Axotomy with BDNF Applied to Cord Two months after cervical axotomy, the lesion site was re-exposed and extended rostrally approximately 1.0 m m (the "refreshment injury").  Gelfoam soaked with P B S or with one o f  three concentrations o f B D N F (50, 1000, 20,000 ng/pl) was inserted into the lesion.  In situ  hybridization was performed on 20 p m thick sections through the caudal half o f the red nucleus. Counterstaining o f rubrospinal neurons was performed with ethidium bromide. The in situ hybridization signal in the injured neurons was expressed as a ratio o f that in the uninjured neurons.  For G A P - 4 3 m R N A expression (n=7 animals), this ratio was on average  1.6 to 1.8 in the injured neurons.  This upregulation i n G A P - 4 3 expression that was similar  amongst all four experimental groups ( P B S , L o w , Medium, and H i g h Concentration B D N F ) . (Figure 4.1)  For T a l tubulin expression (n=8 animals), this ratio was, on average, close to 1.0  and again was not noticeably different amongst the four groups. (Figure 4.2) Compared to the well-recognized decrease in the expression o f these genes i n the cervically axotomized rubrospinal system (Kobayashi et al., 1997, Storer and Houle, 2003), these values represent an increase in gene expression that is similar in all groups, suggesting that this may be more a result o f the refreshment injury (ie. second axotomy) than the B D N F treatment.  106  Figure 4.1. G A P - 4 3 expression in the injured red nucleus is increased compared to uninjured after spinal cord application of B D N F and P B S two months after axotomy Two months after cervical axotomy and spinal cord application o f P B S or B D N F (in one of three concentrations) G A P 4 3 expression appeared in the injured rubrospinal neurons to increase above contralateral uninjured neurons (approximately 1.7-1.8 times).  N o noticeable  difference between P B S and BDNF-treated animals was observed, suggesting that a B D N F effect was lacking and that the increase in G A P - 4 3 expression was related to the refreshment injury.  The darkfield image o f the I S H silver grains is represented in red, while the sections are  counterstained with ethidium bromide and represented in green (false colours). Scale Bar, 50 pm  w  •a * 2.0 C  TP  15  I I I . Ff PBS  LOW  MED  4 HIGH  107  Figure 4.2. T a l tubulin expression in the injured red nucleus is increased compared to uninjured after spinal cord application of BDNF and PBS two months after axotomy Two months after cervical axotomy and spinal cord application o f P B S or B D N F (in one of three concentrations) T a l tubulin expression appeared to increase to levels similar to the contralateral rubrospinal neurons in all animals tested, with again, no noticeable difference between P B S and B D N F treated animals. This suggests that the gene expression changes are related to the refreshment injury, and not the application o f this neurotrophic factor. darkfield  image o f the I S H silver grains is represented  The  in red, while the sections are  counterstained with ethidium bromide and represented in green (false colours). Scale Bar, 50 pm  PBS  LOW  MED  HIGH  108  4.4.2. R A G Expression Two Months Post Axotomy with BDNF Applied to Brainstem 12 months after cervical axotomy, an osmotic mini-pump cannula was stereotactically inserted into the brainstem to infuse either BDNF or its vehicle solution alone into the vicinity of the rubrospinal neurons. In situ hybridization was performed on 20 um thick sections through the caudal half of the red nucleus. Quantification of thesefindingswas not performed, as these experiments were conducted simply to identify whether rubrospinal neurons this long after injury (one year) were still able to mount some form of a regenerative gene expression response when treated with BDNF. Consistent with the findings at 2 months post-injury, 12 months after cervical axotomy the ISH signal for GAP-43 and T a l tubulin in injured rubrospinal neurons was low in the control animals treated with vehicle solution alone. (Figures 4.3 and 4.4) For GAP-43, the ISH signal in the injured red nucleus was comparable to the low ISH signal of the uninjured neurons, while for Tal tubulin, the ISH in the injured red nucleus appeared lower than that of the uninjured red nucleus. Given what I found at 2 months post-axotomy and what has been described about the acute upregulation of these genes after cervical axotomy, it is reasonable to postulate that these 12 month chronically injured neurons similarly mounted a transient regenerative response after axotomy that abated by 4-8 weeks post-injury and remained quiescent over the next 10 to 11 months.  BDNF infusion at 12 months post-axotomy stimulated an increase in ISH signal for  GAP-43 and for T a l tubulin. For GAP-43, this effect was observed in some animals treated at 18 months post-injury.  109  Figure 4.3. BDNF infusion to the red nucleus 12 and even 18 months after cervical axotomy promotes an increase in GAP-43 expression Twelve months after cervical axotomy, the cell body application o f B D N F increased G A P - 4 3 expression in the injured red nucleus to levels greater than that observed in the uninjured red nucleus. Note that the effect was achieved even in animals that were 18 months post-axotomy (bottom row). The infusion o f the vehicle solution alone did not promote G A P - 4 3 expression.  Scale bar, 50 pm. Injured  Contralateral Uninjured  110  Figure 4.4. BDNF infusion to the red nucleus 12 months after cervical axotomy promotes an increase in T a l tubulin expression Twelve months after cervical axotomy, the cell body application o f B D N F increased T a l tubulin expression in the injured red nucleus, restoring it to levels comparable to that o f the uninjured red nucleus. The infusion o f the vehicle solution alone did not promote T a l tubulin expression.  Scale bar, 50 pm.  Injured  Contralateral Uninjured  4.5 DISCUSSION 4.5.1. Chapter Summary Similar to Chapter 3, this chapter reviews the effects o f B D N F treatment at two different time points and i n two different modalities: spinal cord application two months post-axotomy, and cell body application 12 months post-axotomy.  M y observations that B D N F application to  the injured spinal cord did not stimulate either G A P - 4 3 or T a l tubulin expression over that o f the refreshment injury at two months post-axotomy suggest that the rubrospinal axons were not responsive to spinal cord application o f this neurotrophic factor, and is consistent with the failure to reverse neuronal atrophy with this mode o f application (Chapter 3).  M y observations that  B D N F application to brainstem stimulates G A P - 4 3 and T a l tubulin expression over that o f vehicle application suggest that the rubrospinal neuronal bodies remain responsive to the application o f B D N F 12 months after axotomy. To put the findings o f this chapter into perspective, it is important to review the changes in G A P - 4 3 and T a l tubulin expression that occur over time after cervical axotomy.  It has been  repeatedly shown that G A P - 4 3 and T a l tubulin expression in rubrospinal neurons increases acutely after cervical axotomy but then diminishes over the subsequent weeks (Fernandes et al., 1999, Kobayashi et al., 1997, Tetzlaff et al., 1991).  More recently, using a similar cervical  axotomy model, Storer and Houle demonstrated an acute rise in rubrospinal expression o f G A P 43 within 1 day o f injury, and a further increase o f this gene expression and that o f (311 tubulin by 3 days post-axotomy (Storer and Houle, 2003).  However, by 28 days post-axotomy, G A P - 4 3  expression had returned to original baseline expression levels, and p i l tubulin expression had fallen more than 20% below original levels.  This early increase in R A G expression within  rubrospinal neurons likely represents an initial regenerative attempt by these C N S neurons, while  112  the inability to sustain this response over the ensuing weeks is reflective o f the axonal regeneration failure observed within the C N S after injury.  4.5.2. R A G Expression In Response to Refreshment Injury and Spinal Cord Application of BDNF Two months post-axotomy I observed an increase in G A P - 4 3 and T a l tubulin expression in all animals undergoing a refreshment injury and gelfoam application with P B S or B D N F . With no apparent differences in the G A P - 4 3 and T a l tubulin expression response observed amongst these animals (ie. P B S versus B D N F ) , it would appear that chronically injured rubrospinal neurons do not initiate changes in the expression o f these R A G S specifically in response to B D N F applied to the axons within the injured spinal cord.  Rather, the change in  R A G expression observed in the injured red nucleus is likely the result o f the refreshment injury itself that was performed as part o f the insertion o f the BDNF-soaked and PBS-soaked gelfoam. Indeed, the role o f the refreshment injury is suggested by Storer and Houle in their observations o f the red nucleus following cervical re-axotomy 4 weeks after the initial injury (Storer and Houle, 2003).  They reported that the second injury initiated another rise in G A P - 4 3  and p i l tubulin expression that was more rapid than the increase in their expression after the initial injury. In the case o f G A P - 4 3 , the magnitude o f this increase in m R N A expression after a second axotomy was even greater than the magnitude o f the increase after the first axotomy. The increase I observed in G A P - 4 3 expression amongst all the animals ( P B S and B D N F ) to approximately 1.8 times that o f uninjured rubrospinal neurons is similar to the results o f Houle and colleagues who reported an increase o f at least 2.0 times that o f uninjured (Storer and Houle, 2003). Similarly for T a l tubulin, with a level o f expression falling below that o f uninjured by as early as 14 days post axotomy (Kobayashi et al., 1997), the reported expression in my current  113  study o f approximately 1.0 times that o f uninjured in the two month chronic state likely represents an acute upregulation due to the second axotomy. While the implication from the findings from Storer and Houle at 4 weeks post-axotomy and my observations at 2 months post-axotomy is that chronically injured rubrospinal neurons retain the ability to upregulate the expression o f certain R A G s in response to another injury, what remains somewhat unanswered is how much o f the gene expression changes are attributable to neurons that are axotomized again versus those that terminated just rostral to the initial injury and are axotomized for the first time during the refreshment injury.  M y analysis o f the red  nucleus is performed only on the caudal half which is predominantly the lumbar-projecting region o f the nucleus, so it would be anticipated that most o f the neurons evaluated were injured during the original axotomy and thus re-injured during the refreshment injury. The method by which the quantification o f I S H signal was performed warrants some discussion as it pertains to the interpretation o f the results.  The silver grains from the darkfield  images were overlaid upon the ethidium bromide counterstained rubrospinal neurons which were outlined using SigmaScan Pro ImageAnalysis 5.0 Software (Systat Software, Inc, Point Richmond, C A ) .  The extent to which each neuron is filled with silver grains is then calculated  by the SigmaScan software and converted into an Excel spreadsheet where it is averaged across the neurons that were outlined. This average (minus background I S H signal) from the injured neurons is then compared against the similarly derived average (minus background I S H signal) from the contralateral uninjured neurons.  While the algorithm is conceptually simple, it does  not account for differences in neuronal cross sectional area.  Given that the cross sectional area  of the rubrospinal neurons was quite uniform across all four groups o f animals ( P B S , L o w , M e d i u m , and H i g h Dose B D N F ) , as described in Chapter 3,1 feel that the final interpretation o f  114  this semi-quantitative analysis is valid and meaningful. H a d there been substantial differences between the mean cross sectional areas between treatment groups (for example,  BDNF  significantly reversing neuronal atrophy), then a more sophisticated quantification algorithm that accounted for the size o f each neuron would be required. A l s o consistent with my in situ hybridization data and the ineffectiveness o f B D N F applied to the spinal cord at two months post-axotomy, Storer et al. found B D N F to be ineffective at promoting G A P - 4 3 and (311 tubulin expression within the red nucleus when applying this neurotrophic factor to the spinal cord 4 weeks after cervical hemisection (Storer et al., 2003).  The injury model (unilateral rubrospinal tract disruption), refreshment injury, and  mode o f B D N F application (gelfoam soaked in 50 ng/pl B D N F ) were identical between our studies, although my B D N F intervention occurred at 2 months rather than 4 weeks postaxotomy.  O f note, Storer et al. observed that while B D N F did not stimulate G A P - 4 3 and (311  tubulin expression in rubrospinal neurons injured 4 weeks previously, the application o f 10 ng/pl G D N F did (Storer et al., 2003).  This demonstrates that the failure o f rubrospinal neurons to  initiate R A G expression changes was not solely a function o f the chronically axotomized state, but rather a neurotrophic-specific phenomenon.  The potential role o f T r k B receptor expression  in this lack o f B D N F effect is explored in Chapter 6.  4.5.3.  R A G Expression In Response to Cell Body Application of BDNF I observed an increase in G A P - 4 3 and T a l tubulin m R N A expression in rubrospinal  neurons injured 12 months previously when B D N F was applied directly to the cell bodies. These observations suggest a retained responsiveness o f the injured red nucleus to cell-body application o f this neurotrophic factor.  The small numbers o f animals, however (n=7) make it difficult to  perform a truly quantitative comparison between the experimental groups however.  Kobayashi  115  et al. reported that cell body application o f B D N F after acute injury promoted the expression o f these genes (Kobayashi et al., 1997).  The demonstration o f R A G expression with cell body  application o f B D N F 12 months after injury had not been described prior to my reporting o f this in 2002 ( K w o n et al., 2002b).  The upregulation o f G A P - 4 3 and T a l tubulin expression in these  chronically injured neurons is consistent with the reversal o f their neuronal atrophy (Chapter 3) and the increased regeneration into peripheral nerve transplants (Chapter 5).  4.5.4. R A G Expression and the Promotion of Axonal Regeneration While I specifically examined G A P - 4 3 and T a l tubulin within the rubrospinal system, I recognize that these are only a fraction o f the molecular changes that likely occur in response to axotomy.  Our current appreciation o f the full extent o f this battery o f gene expression changes  and how they interact with each other to ultimately produce axonal outgrowth is fairly rudimentary, although micro-array technology w i l l likely accelerate our understanding o f this in the future. G A P - 4 3 is a growth associated protein localized to the growth cone where, by virtue o f its interactions with calmodulin, PI(4,5)P2, G proteins, and the actin cytoskeleton, it influences axonal growth and synaptic plasticity (reviewed by Benowitz and Routtenberg, 1997 and Caroni, 2001). The correlation between increased G A P - 4 3 expression and axonal growth and sprouting has made G A P - 4 3 a commonly used indicator o f the neuronal growth propensity (reviewd by (Caroni, 1997) and (Skene, 1989)).  One should be aware, however, that there do exist examples  in which G A P - 4 3 was neither sufficient (Buffo et al., 1997) nor necessary (Strittmatter et al., 1995) for axonal growth.  Bisby and colleagues demonstrated that even without a noticeable  increase in G A P - 4 3 expression, mouse motoneurons were capable o f initiating collateral sprouting in response to muscle inactivity or partial muscle denervation (Bisby et al., 1996).  116  Andersen and Schreyer demonstrated that axonal regeneration not only occurred from DRG neurons expressing GAP-43 but also from DRG neurons devoid of GAP-43 immunoreactivity (Andersen and Schreyer, 1999). Axonal regeneration, however, did not occur uniformly, with rapid growth observed only from those neurons constituitively expressing GAP-43.  These  results imply that while GAP-43 may not be an absolute requirement for axonal regeneration within this neuronal system, its expression is correlated with the ability to promote rapid axonal growth. Bomze at al. demonstrated in vivo that the concomitant over-expression of both GAP43 and CAP-23 (a related growth cone protein) resulted in significant regeneration of the central DRG axons into peripheral nerve transplants, while this failed with the over-expression of each gene alone (Bomze et al., 2001). Still, the most extensive regeneration was observed after a conditioning peripheral axotomy, indicating that over-expression of only two genes (GAP-43 and CAP-23) does not completely substitute for the coordinated expression of a multitude of RAGs that are stimulated by the conditioning peripheral nerve lesion. The results of these above studies illustrate that axonal growth can differ both in its nature (eg. collateral sprouting versus long distance regeneration) and its rate (eg. slow versus rapid growth), and that cell body influences these differences in part by the pattern of RAG expression.  This was perhaps most eloquently demonstrated by Smith and Skene, who found  that the constituitive gene expression of mature DRG neurons facilitated "arborizing" neurite outgrowth, but changes in gene transcription induced by peripheral axotomy could "switch" the neurons into promoting "elongated" axonal outgrowth instead (Smith and Skene, 1997).  As  stated earlier, such a peripheral axotomy is known to initiate important changes in RAG expression.  Conceptually, one of the obvious therapeutic goals for "axonal regeneration  strategies" in the setting of spinal cord injury is to promote the long-distance growth of axons  117  across the injury site to re-innervate distal targets.  It should not, however, be overlooked that  other forms o f axonal regeneration such as the "arborizing" neurite outgrowth illustrated by Smith and Skene (Smith and Skene, 1997) or the collateral sprouting illustrated by Bisby et al (Bisby et al., 1996) may also play an important role in enhancing local plasticity at the spinal cord injury site. The genetic mechanism underlying these forms o f growth therefore also carry some relevance.  A s such, further characterization o f R A G expression within C N S neurons after  injury and the elucidation o f mechanisms by which to stimulate the expression o f those genes necessary for axonal growth o f all forms w i l l be important for the future development o f therapeutic interventions.  118  - CHAPTER 5 AXONAL REGENERATION OF CHRONICALLY INJURED RUBROSPINAL NEURONS  5.1. S U M M A R Y While rubrospinal neuronal survival and size (Chapter 3) and R A G expression (Chapter 4) are thought to be important elements o f the intrinsic regenerative competence, ultimately one is most interested in examining whether axonal regeneration is possible in the chronic injury setting.  B D N F was applied to the spinal cord 2 months after cervical axotomy in 3 different  doses, and an autologous segment o f peripheral nerve was transplanted into the spinal cord injury site.  Alternatively, B D N F was infused into the vicinity o f the rubrospinal neuronal cell bodies  12 months after cervical axotomy and a peripheral nerve autograft was again transplanted.  I  found that B D N F application to the spinal cord did not promote axonal regeneration from chronically injured rubrospinal neurons two months after cervical axotomy ( K w o n et al., 2004a). However, with cell body application o f B D N F as late as 12 months after injury, I observed a significant increase in chronically injured rubrospinal neurons that regenerated axons into this peripheral nerve transplant compared to PBS-treated control animals ( K w o n et al., 2002b). These findings suggest that in the chronic injury state, rubrospinal neurons are capable o f promoting axonal regeneration in response to B D N F applied directly to the cell bodies, but not to their injured axons within the spinal cord.  119  5.2. I N T R O D U C T I O N  Outside o f the efforts to spare spinal cord tissue with neuroprotective therapies from secondary injury, it has long been felt that to effect an improvement in neurologic function in patients with spinal cord injuries (particularly those whose injuries occurred long ago), it would be essential to have axons that are disrupted at the injury site regenerate across it and reconnect with distal targets.  This remains the ultimate objective for axonal regeneration strategies, and,  from a conceptual point o f view, w i l l likely need to be met to some extent i f we are to achieve the goal o f restoring individuals with spinal cord injuries to full physical function in the future. The development o f therapies to promote such axonal growth continues to be a major focus o f neuroscientific attention. A s stated in the Background chapter, there are numerous strategies that have been explored to promote axonal regeneration within the injured spinal cord. The challenge is largely two-fold:  one being the failure to mount and sustain a sufficient R A G expression response  (discussed in detail in Chapter 4), and the other being elements within the injured spinal cord that impede axonal growth, thus establishing a non-permissive environment.  It is widely believed  that the optimal therapeutic result w i l l be achieved by combinatorial interventions that address both o f these challenges.  A s such, in this chapter I have combined the use o f the neurotrophic  factor B D N F (conceptually to augment the intrinsic regenerative competence) with the local transplantation o f autogenous peripheral nerve grafts (conceptually to provide a permissive environment) in order to evaluate axonal regeneration o f chronically injured rubrospinal neurons.  120  5.2.1. Neurotrophic Factors and Axonal Regeneration Because o f their widespread effects within the nervous system, particularly with respect to neuronal survival and axonal growth, neurotrophic factors have represented an appealing therapeutic strategy for spinal cord injury.  Numerous methods o f delivering neurotrophic  factors to the injured spinal cord have been established, including direct injection, within gelfoam, continuous infusion, and gene therapy approaches (both ex vivo and in vivo), the latter aimed at promoting the more prolonged delivery o f neurotrophic factors within the C N S .  The  effectiveness o f neurotrophic factor application as a therapeutic strategy relies heavily upon the bioavailability o f the protein and the presence o f the appropriate receptors on the target tissue. The exogenous application o f B D N F to the injured spinal cord has been evaluated by numerous investigators in the past.  Particularly relevant to this chapter on rubrospinal axonal  regeneration, the study o f L i u and colleagues reported long-distance regeneration o f rubrospinal axons across a cervical hemisection lesion with the acute transplantation o f fibroblasts genetically modified to secrete B D N F ( L i u et al., 1999).  The authors also demonstrate  anterogradely labeled rubrospinal axons crossing through the fibroblast graft and re-entering the host spinal cord distal to the injury site, where some fibers even re-entered the grey matter (possibly contributing to the observed improvements in motor function).  In contrast, however,  the same BDNF-fibroblast transplantation performed 6 weeks after cervical hemisection did not promote rubrospinal axonal regeneration through or distal to the transplant, nor did it promote significant motor recovery (Tobias et al., 2003).  121  5.2.2. Modifying the Inhibitory Environment of the Injured CNS While neurotrophic factors are thought to enhance the neuron's ability to regenerate its injured axon (either directly by acting upon the neuron and its axon or indirectly by acting upon surrounding cells that in turn support axonal growth),  the other principal strategy to promote  axonal regeneration within the injured C N S is to provide the injured axons with a more permissive  environment  for growth.  Conceptually this can be achieved  with cellular  transplantation strategies that "bridge" the injury (Murray, 2004), and by therapies that neutralize the inhibitory elements within the injured C N S , which are largely related to the glial scar and to C N S myelin (reviewed by Silver and Miller, 2004 and Grados-Munro and Fournier, 2003). Amongst the various cellular substrates available for transplantation, the use o f peripheral nerve grafts and their Schwann cells have been extensively studied.  The transplantation o f  peripheral nerve grafts into the spinal cord was rationalized by the recognition that axonal regeneration o f P N S neurons occurred readily within a presumably permissive Schwann cell environment.  O f note, the important study o f Richardson and Aguayo over two decades ago that  illustrated the long-denied regenerative ability o f C N S neurons employed a peripheral nerve graft implanted into the spinal cord (Richardson et al., 1980b).  Since then, numerous authors have  demonstrated the regeneration o f C N S axons into peripheral nerve transplants or Schwann cell grafts applied acutely into the injury site (reviewed by Bunge, 2000).  However, similar to the  decline in effectiveness seen during the chronic injury setting with BDNF-secreting fibroblasts (Tobias et al., 2003), axonal regeneration into peripheral nerve grafts transplanted with a 3 to 4 week delay after injury has also not been met with success (Decherchi and Gauthier, 2000, Houle, 1991).  122  5.3. OVERVIEW OF EXPERIMENTAL QUESTIONS AND HYPOTHESES  In this chapter, I evaluated the regeneration o f rubrospinal axons into peripheral nerve transplants to test the following hypotheses: 1.  Given that B D N F administration to the spinal cord injury site appears to diminish in its  ability to promote axonal regeneration after a delay in intervention (Tobias et al., 2003), and that various doses/concentrations o f B D N F  have been applied to the injured spinal cord,  /  hypothesized that the loss of effectiveness in promoting axonal regeneration might be related to providing the appropriate dose/concentration of the neurotrophic factor. To test this hypothesis, animals underwent a cervical axotomy, then two months later, B D N F in 3 exponentially increasing concentrations (50, 1000, and 20,000 ng/pl) was applied within gelfoam to the refreshed injury site (in addition to PBS-treated controls).  A pre-  degenerated autologous nerve graft was then transplanted into the injury site, and the extent o f axonal regeneration o f both chronically and acutely injured rubrospinal neurons into the nerve graft was evaluated with retrograde tracers.  2. Given that the infusion o f B D N F into the vicinity o f the rubrospinal cell bodies was shown to promote axonal regeneration o f acutely injured rubrospinal neurons into peripheral nerve transplants (Kobayashi et al., 1997), / hypothesized that the cell-body administration of BDNF could promote the axonal regeneration of chronically injured rubrospinal neurons. To test this hypothesis, animals underwent a cervical axotomy, then twelve months later, B D N F was infused through an osmotic minipump cannula stereotactically placed just lateral to the red nucleus. A pre-degenerated autologous nerve graft was then transplanted into the injury  123  site, and the extent o f axonal regeneration o f both chronically and acutely injured rubrospinal neurons into the nerve graft was evaluated with retrograde tracers.  124  5.4. RESULTS 5.4.1. Axonal Regeneration Two Months Post-Axotomy with BDNF Applied to Cord The thoracolumbar descending population o f rubrospinal neurons was retrogradely labeled with FluoroGold injected at T l .  Seven days later, the dorsolateral aspect o f the cervical  spinal cord at C3/4 was cut to unilaterally sever the rubrospinal tract.  T w o months later, the  cervical axotomy site was refreshed, B D N F or P B S applied in gelfoam (as described in detail in the methods section) and a 35 m m segment o f predegenerated tibial nerve was inserted into the spinal cord injury site and secured with 10-0 prolene sutures.  T w o months later, B D A was  applied to the free end o f the nerve graft to retrogradely label neurons whose axons grew to the end o f the peripheral nerve transplant.  A week later the animals were sacrificed and counts o f  double-labeled  and  (chronically  injured)  single-labeled (acutely  injured)  neurons  were  performed. Regeneration of Chronically Injured Neurons - Double Labeled After  intervention with P B S or B D N F  at the injury  site and peripheral nerve  transplantation, the number o f double-labeled rubrospinal neurons was counted to quantify neurons that were injured two months previously (labeled with FluoroGold), and that were able to extend an axon to the tip o f the nerve transplant (to obtain the B D A ) .  (Figure 5.1)  The  numbers o f double labeled neurons in each group were 6.2±1.5, 6.9±2.6, 10.5±5.5, and 10.5±3.1 for the P B S , L o w , Medium, and H i g h B D N F groups respectively. (Figure 5.2) There was no significant difference in double labeled cells between the groups oh one way A N O V A  (p=0.71).  Even pooling all o f the BDNF-treated animals and comparing the number o f double-labeled cells (9.25±2.1; n=20) with that o f the P B S treated animals (6.2±1.5; n=6) did not reveal a significant difference (p=0.44).  The data suggested that perhaps there was a B D N F effect with the higher  125  doses, but when pooling the medium and high dose B D N F groups (10.5±2.9; n=13) and comparing this to the P B S alone (6.2±1.5, n=6) or to the pooled P B S and low dose B D N F group (6.5±1.52; n=13) there were no significant differences (p=0.17 and p=0.12 respectively).  This  indicates that B D N F , at any o f the three doses, did not promote axonal regeneration o f chronically injured rubrospinal neurons two months after axotomy when applied to the injury site. Regeneration of Acutely Injured Neurons - Single Labeled The refreshment injury which extends the spinal cord lesion rostrally is likely to injure for the first time a number o f axons that terminated just proximal to the original injury (ie. above C3/4).  Such axons would not have picked up the FluoroGold injection at T l , and thus, i f  successfully regenerating through the transplanted peripheral nerve graft, would only be single labeled with the B D A .  The single-labeled neurons were counted, i n part to determine i f B D N F  had an acute effect on the regenerative capacity o f these neurons, and in part to confirm that the microsurgical placement o f the nerve graft was such that at least some form o f axonal regeneration was possible through the cord-graft interface. The numbers o f single labeled neurons in each group were 13±6.0, 22.1±5.0, 29.3±5.2, and 29.0±3.4 for the P B S , L o w , Medium, and H i g h B D N F groups respectively. There was no significant difference in single-labeled cells between the four groups on one way A N O V A (p=0.10).  However, when pooling the BDNF-treated animals and comparing the number o f  single-labeled cells (26.7±2.6; n=20) with that o f the P B S treated animals (13.0±6.0; n=6) there was a statistically significant difference (p=0.01).  (Figure 5.2) This would suggest that B D N F  did promote the axonal regeneration o f acutely injured rubrospinal axons, in keeping with the data o f L i u et al. in their acute transplantation o f BDNF-secreting fibroblasts ( L i u et al., 1999).  126  There was no significant difference in the number o f single-labeled rubrospinal neurons between the three B D N F groups on one way A N O V A (p=0.46).  127  Figure 5.1.  Double labeling paradigm (FluoroGold and BDA) to evaluate regeneration  into peripheral nerve transplants two months after cervical injury. FluoroGold labeling o f descending rubrospinal neurons (A) is represented here in the green channel (false colour) for illustrative purposes.  Neurons retrogradely labeled with B D A  from extending axons through the peripheral nerve transplant are shown in (B).  Overlaying  these images (C) demonstrates yellow double-labeled neurons (arrows), indicating that they were both injured 2 months prior to implantation and then successfully regenerated through the graft. Note also the presence o f single labeled B D A neurons (asterisks) - such neurons likely had axons that ended just proximal to the original injury and then were axotomized for the first time with the refreshment injury. (Scale Bar = 50 pm)  A  *  FluoroGold  |B B D A  %  f  i  4  m  * C  FluoroGold + BDA  HP  128  Figure 5.2. Axonal regeneration data from animals treated two months after axotomy at the spinal cord injury site with PBS or BDNF at three different doses (LOW, MEDIUM, HIGH). A two-month chronic time point, there was no significant difference in the number o f chronically injured neurons that regenerated into the peripheral nerve transplants (double-labeled cells - light grey) amongst the groups. Even pooling the number o f double-labeled cells from the low, medium, and high dose B D N F groups and comparing against the P B S group did not result in a significantly greater number o f chronically injured neurons regenerating with B D N F treatment (p=0.44).  Conversely, pooling the number o f single-labeled cells from the low,  medium and high dose B D N F groups and comparing this to the P B S group revealed a significantly greater number o f acutely injured neurons regenerating with B D N F  treatment  (p=0.01).  40  35  30  25  • Single Labeled  20  • Double Labeled  15  10  5  0  PBS  LOW BDNF  MED BDNF  HIGH BDNF  129  5.4.1  Axonal Regeneration Twelve Months Post-Axotomy with BDNF Applied to  Brainstem Similar to the axonal regeneration experiments at 2 months post-axotomy, lumbardescending rubrospinal neurons were retrogradely labeled prior to cervical axotomy. In these experiments, the axonal tracer FastBlue was injected at C 8 prior to cutting the dorsolateral funiculus at C3/4.  One year later, I injected FluoroGold below T l to retrogradely label any  residual rubrospinal axons that might have escaped injury with the cervical axotomy.  No  retrograde FluoroGold labeling o f rubrospinal neurons was ever observed, thus confirming the completeness o f the unilateral rubrospinal axotomy.  A predegenerated 35 m m segment o f tibial  nerve was transplanted into the spinal cord injury site and the B D N F applied v i a osmotic minipump into the vicinity o f the rubrospinal cell bodies. D i l or B D A was applied to the free tip o f the graft to retrogradely label axons that regenerated to the end.  (Figures 5.3 and 5.4)  The  animals were sacrificed two months after transplantation to evaluate the number o f double labeled neurons. The number o f double-labeled neurons in the BDNF-treated animals (n=14) was 28.6±8.3, and in the vehicle-treated animals (n=6) was 11.0±4.4.  This was a statistically  significant difference (p=0.038), indicating that cell-body treatment with B D N F did promote the axonal regeneration o f chronically injured rubrospinal neurons. neurons  in the  same  BDNF  The number o f single-labeled  and vehicle-treated animals was 71. 1=1=18.8 and  22.2±6.6  respectively, which again demonstrated a significantly greater number o f regenerating acutely injured rubrospinal neurons with B D N F treatment (p=0.013)  Figure 5.5 The spinal cord o f an  animal with B D N F treatment and B D A at the tip o f the peripheral nerve transplant was sectioned sagittally to illustrate the interface between the cord and peripheral nerve transplant. (Figure 5.6)  130  Figure 5.3. Double labeling paradigm (FastBlue and DO) to evaluate regeneration into peripheral nerve transplants twelve months after cervical injury. In concept, the double-labeling strategy employed here is similar to that used at two months (illustrated in Figure 5.1) except that the rubrospinal neurons were initially labeled with FastBlue instead o f FluoroGold, and in this case, D i l was placed at the tip o f the peripheral nerve graft to retrogradely label neurons that regenerated axons through the transplant.  The FastBlue  image o f rubrospinal neurons labeled retrogradely prior to axotomy (left) was overlaid upon the fluorescent D i l image o f rubrospinal neurons (right).  The overlaid image demonstrates red D i l  single labeled rubrospinal neurons which are presumably acutely injured (arrowheads), double labeled neurons which are chronically injured (arrows). Scale bar, 50 pm.  and  131  Figure 5.4. Double labeling paradigm (FastBlue and BDA) to evaluate regeneration into peripheral nerve transplants twelve months after cervical injury. In this case, B D A was applied to the free tip o f the peripheral nerve graft to retrogradely label rubrospinal neurons with axons regenerating through the transplant.  Similar to Figure 5.1.  the images are imported into an R G B figure, with the image o f the BDA-labeled rubrospinal neurons in the red channel and the FastBlue in the blue channel. therefore appear pink in the overlaid image.  The BDA-labeled neurons  Note the presence o f both double-labeled neurons  (arrow) and single-labeled neurons (asterisks), the former representing chronically injured neurons, the latter representing acutely injured neurons that regenerated through the graft.  FastBlue  I FastBlue + B D A  132  Figure 5.5. Axonal regeneration data from animals treated with cell body administration of BDNF or vehicle 12 months after axotomy. A t the 12 month time point, the animals treated with B D N F infusions to the vicinity o f the red nucleus achieved significantly greater numbers o f both double and single labeled neurons than vehicle treatment (p<0.05 for both).  100  90 80 70 60 • Single L a b e l e d  50  • Double L a b e l e d  40 30 20 10  Vehicle  BDNF  133  Figure 5.6.  Sagittal section of spinal cord at the interface between the cord and peripheral  nerve graft in a 12 month chronically injured animal.  For illustrative purposes, the spinal cord of an animal treated with BDNF 12 months after cervical axotomy was sagittally sectioned to examine the interface between the cord and graft. (A) This animal had B D A placed at the tip of the peripheral nerve transplant and demonstrated the greatest number of double-labeled neurons of all the BDNF-treated animals. Sections were processed with the A B C kit to visualize the B D A labeled axons, and then counterstained with cresyl violet. Note the large B D A labeled axons (arrows) in the position of the rubrospinal tract (B) entering into the peripheral nerve transplant (C).  Scale bar, 50 pm.  A  Peripheral Nerve Transplant  Spinal Cord  B  \ •J-  -  -  A  t  r..  134  5.5.  DISCUSSION  5.5.1. C h a p t e r S u m m a r y  The results from these peripheral nerve transplant experiments demonstrate that rubrospinal neurons chronically injured at the cervical spinal cord level can regenerate their axons in response to B D N F administered directly to the cell bodies (12 months post-injury) but not to their injured axons (2 months post-injury).  These findings are consistent with the reversal of  neuronal atrophy and the upregulation of GAP-43 and T a l tubulin expression with cell body and not spinal cord application of BDNF (discussed in Chapter 3 and 4).  5.5.2.  A x o n a l T r a c i n g for the Evaluation of A x o n a l Regeneration After C h r o n i c Injury  A number of retrograde tracers were used in these experiments to evaluate axonal regeneration of chronically injured rubrospinal neurons. As we and others have pointed out, the in vivo study of axonal regeneration can be a challenging task (Kwon et al., 2002d, Steward et al., 2003). One of the major drawbacks with injury models that employ partial spinal cord lesions is the potential for axonal sparing. The rubrospinal tract runs in the dorsolateral aspect of the spinal cord; in this position, it can .be reliably transected with an incision through the dorsolateral funiculus. This is an injury model that we (Kobayashi et al., 1997, Kwon et al., 2002b, Kwon et al., 2002c) and others (Liu et al., 2002, Liu et al., 1999, Tobias et al., 2003) have used extensively to study rubrospinal regeneration.  The partial nature of this dorsolateral funiculus injury is particularly  useful for chronic regeneration experiments, as the animals must survive with the injury for often prolonged periods of time.  Such animals quickly regain normal bowel/bladder function and  grossly normal lower extremity locomotor function.  It is acknowledged, however, that with  variability in the method of performing the axotomy and the stretching of the axons, some  135  sparing o f axons might occur.  In my 12 month chronic experiments, this issue was addressed  with the second retrograde tracer injection at T l (FluoroGold);  with this, I did not see  retrogradely labeled rubrospinal neurons in the injured red nucleus, indicating that the performance o f my dorsolateral funiculus incision was consistent and sufficiently aggressive to reliably cut the entire rubrospinal tract unilaterally This confirmatory step was performed in the 12 month chronic injury experiments;  the experiments at 2 months post-axotomy were  performed subsequent to this, and I felt that my cumulative experience with the injury model made it unnecessary to continue with this second retrograde tracer injection to rule out sparing. The defining feature o f the double-labeling tracer paradigm in the peripheral nerve transplant experiments is that it allows one to distinguish axonal regeneration originating from versus chronically injured neurons.  acutely  In this paradigm, axons descending to the thoracolumbar  spinal cord receive the tracer and their neurons are retrogradely labeled; they are then cut at C3/4.  If they then successfully regenerate through the peripheral nerve graft and pick up the  second retrograde tracer placed at its free end, they w i l l then be "double-labeled".  Neurons  that are single-labeled with only the second retrograde tracer and not the first likely had axons that ended just proximal to the original C3/4 axotomy and were injured for the first time during the refreshment injury and insertion o f the nerve graft. Being able to make this distinction between axonal regeneration from acutely and chronically injured axons is obviously important i f we are to evaluate the effectiveness o f therapies for chronic spinal cord injury.  Unfortunately, such a distinction is often difficult to  make from studies o f therapeutic interventions at chronic time points. For example, Tuszynski and colleagues transplanted fibroblasts genetically modified to secrete N G F into a dorsal hemisection spinal cord injury model, 3 months after original injury (Grill et al., 1997). They  136  reported impressive invasion o f cerulospinal and primary afferent sensory axons but not o f corticospinal axons - growth that was qualitatively similar to that which they observed in an acute injury paradigm (Tuszynski et al., 1996).  However, these chronic experiments did not  employ a double-labeling tracing paradigm, and as such, it is difficult to tell whether the axons they observed growing into the graft were actually chronically injured or i f they were acutely injured at the time o f the graft placement (Grill et al., 1997).  This absence o f a double-labeling  tracing paradigm has made the distinction between acute and chronic neuronal regeneration similarly difficult in other chronic injury studies, including studies o f BDNF-secreting fibroblasts inserted into a cervical hemisection injury 5 weeks after injury (Jin et al.,.2000), minced autologous peripheral nerve inserted into a dorsal hemisection injury 25 weeks after injury (Ferguson et al., 2001), and olfactory ensheathing cell transplants inserted into a thoracic transection 4 weeks after injury ( L u et al., 2002). In this regard, I have demonstrated that some rubrospinal neurons, injured 12 months previously, were capable o f regenerating through peripheral nerve transplants.  The B D N F  infusion within the vicinity o f the rubrospinal cell bodies enhanced the regenerative capacity o f these chronically injured neurons, as evidenced by the significantly greater numbers o f doublelabeled neurons in BDNF-treated compared to vehicle-treated animals. Interestingly, there were a small number o f double-labeled neurons in the animals treated with vehicle solution alone. The results o f Houle would suggest that no regeneration occurs from rubrospinal neurons into peripheral nerve transplants injured 1 month prior (Houle, 1991).  M y somewhat contradictory  observations may be related to the placement o f the minipump cannula and infusion into the vicinitiy o f the red nucleus, with the resultant inflammatory reaction promoting a growth response from the rubrospinal neurons.  Such a growth response to inflammation was illustrated  137  by L u and Richardson, who incited an inflammatory reaction within the D R G by injecting it with Corynebacterium parvum or macrophages and then demonstrated an increased D R G axonal regeneration propensity through a crush injury o f the dorsal root ( L u and Richardson, 1991). Benowitz and colleagues have demonstrated a similar phenomenon in the eye, whereby an injury to the lens incites an inflammatory reaction (with macrophages being a critical component) that increased G A P - 4 3 expression within retinal ganglion cells and promoted axonal regeneration in the crushed optic nerve (Leon et al., 2000, Y i n et al., 2003).  I have not further characterized the  complex nature o f the inflammatory response incited by the cannula o f the osmotic minipump and how this might affect the growth propensity o f rubrospinal neurons, although I recognize that such study might help to delineate some o f the pathways by which inflammation influences axonal regeneration. Perhaps better characterized is the effect o f the second axotomy on chronically injured rubrospinal neurons. A t two months post-axotomy, I observed that the G A P - 4 3 and T a l tubulin I S H signals were increased to a similar extent in all animals, whether they received the repeated applications o f B D N F (in one o f three concentrations) or P B S within gelfoam at the spinal cord injury site (Chapter 4).  I similarly found a small increase in cell size i n all o f these animals  compared to animals that did not have a refreshment injury (Chapter 3).  Storer and Houle have  also demonstrated that this second axotomy promotes a significant rise i n G A P - 4 3 and pTI tubulin expression.  I would postulate that this growth response may be responsible for the  regeneration o f chronically injured rubrospinal neurons two months post-axotomy.  The fact  that there was no difference in double-labeled neurons between all animal groups on one way A N O V A or even between the P B S and all pooled B N D F treated animals suggests that this observed regeneration was related to the effects o f the second axotomy.  138  5.5.3. BDNF and the Promotion of Axonal Regeneration A s discussed briefly in Chapter 3, numerous investigators have applied B D N F to the injured spinal cord either by direct infusion/application or by genetically modified cell lines (ex vivo gene therapy). Such studies have demonstrated  a reduction in retrograde rubrospinal  atrophy and loss ( L i u et al., 2002, Diener and Bregman, 1994), the promotion o f rubrospinal axonal regeneration ( L i u et al., 1999), and the improvement o f motor function ( L i u et al., 1999, N a m i k i et al., 2000, Jakeman et al., 1998, L i et al., 2003, Ikeda et al., 2002) in animals treated during the acute phase o f injury with B D N F .  The inference from these studies reporting  improved motor function is that the B D N F application encouraged some form o f axonal growth or plasticity or accommodation which then mediated the observed neurologic recovery. Determining the anatomic correlate behind functional recovery, however, is an extremely difficult process and in most cases can only be postulated, recognizing that lower animals such as rodents have remarkable spinal cord plasticity and intrinsic locomotor ability to compensate for partial or even complete spinal cord injuries (Barbeau and Rossignol, 1994).  Generally  speaking, the exact contribution that a single neuronal system such as the rubrospinal system makes to a particular motor function is only known to a rudimentary extent (Kuchler et al., 2002). My  current experiments  examined only axonal regeneration  within the peripheral  nerve  transplant and did not evaluate intraspinal regeneration o f rubrospinal axons (ie. across the injury site) nor did it evaluate functional recovery.  A s indicated previously, intraspinal regeneration o f  rubrospinal axons was observed through a graft o f BNDF-secreting fibroblasts when implanted acutely after spinal cord injury ( L i u et al., 1999) but not when implanted 6 weeks after spinal cord injury (Tobias et al., 2003).  M y observations o f axonal regeneration 12 months after  139  axotomy with cell body treatment o f rubrospinal neurons builds on the work o f Kobayashi et al. who demonstrated increased axonal regeneration with such cell body treatment in an acute spinal cord injury model (Kobayashi et al., 1997).  Conceptually, the cell body application o f B D N F  may be more directly able to effect the necessary changes within the neuronal soma at a chronic time point than the spinal cord application o f B D N F .  Such a statement is supported by my  observations that at two months post-axotomy, spinal application o f B D N F did not stimulate rubrospinal axonal regeneration while cell body application twelve months after axotomy did. The influence o f T r k B receptor expression on these findings is discussed further in Chapter 6. Nevertheless, while the cell body treatment provides proof o f principle that axonal regeneration might be stimulated by intervening at this level, a method for inducing such changes with an intervention at the spinal cord would clearly be more desirable.  Intuitively, the surgical  manipulation o f the already injured spinal cord i n order to apply a therapy (eg. B D N F ) would be far less dangerous to an individual patient than an intracranial, intraparenchymal injection into the otherwise uninjured brain.  Interestingly, an in vivo gene therapy strategy was recently  described by K o d a et al., who administered a BDNF-encoding adenovirus vector into an acute thoracic spinal cord transection (Koda et al., 2004).  Using both anterograde and retrograde  labeling, the authors reported rubrospinal axonal regeneration across the injury site.  They also  reported marker gene (lacZ) expression within the rubrospinal neurons, demonstrating successful retrograde transfection o f these neurons with the implication that their B D N F production was increased.  Even though the authors o f this study report axonal regeneration across a complete  thoracic spinal cord transection, the description o f the spinal cord after injury raises questions about the completeness o f the injury, particularly around the periphery.  Nonetheless, the ability  to achieve gene expression changes and possibly also increased B D N F secretion within the cell  140  bodies with an intervention directed to the acute injury site is indeed interesting, and hopefully further studies o f this treatment w i l l be performed at chronic time points to establish whether retrograde transfection o f chronically injured rubrospinal neurons to enhance their B D N F production w i l l be possible. The discussion surrounding the successful axonal regeneration o f rubrospinal neurons with acute B D N F fibroblast engraftment compared to 6 week post-injury intervention ( L i u et al., 1999, Tobias et al., 2003) and the failure o f B D N F in 3 doses to promote axonal regeneration 2 months post-injury described herein would imply that the sooner the B D N F were applied, the better.  Bregman and colleagues have demonstrated that this might not be the case, and that the  situation may in fact be somewhat more complicated (Coumans et al., 2001, L i u et al., 1999). Following a complete thoracic spinal cord transection injury, they transplanted fetal tissue and infused B D N F at a concentration o f 1000 ng/pl v i a osmotic minipump either acutely, or at 2 or 4 weeks post-injury.  They found that the growth o f serotonergic axons into the transplant was  greatest in animals that received the transplant and B D N F 2 weeks after injury, rather than acutely.  Retrograde labeling o f rubrospinal neurons was also demonstrated in animals receiving  delayed transplants, although a quantification o f rubrospinal growth at each time point o f intervention (acute, 2 weeks, 4 weeks) was not provided.  It is important to note that although  total serotonergic fiber length distal to the lesion was improved in animals treated 2 and 4 weeks post-injury compared to those treated acutely, the response at 4 weeks post-injury was notably less than at 2 weeks post-injury, suggesting that while a two week delay in intervention appeared favorable, further delaying the treatment would not be beneficial.  A s an explanation for the  improved response at 2 weeks post injury, they proposed that the inflammatory response might be more active acutely than "subchronically" and hence the spinal cord environment more  141  amenable to axonal regeneration at the latter time points.  Also, they proposed that injured  neurons themselves at the latter time points might mount an augmented regenerative response after a second axotomy, a hypothesis that would be supported to some extent by the observations of Storer and Houle who found that rubrospinal upregulation o f G A P - 4 3 and p i l tubulin was actually greater and more rapid after a second cervical axotomy than after the initial injury (Storer and Houle, 2003).  While the findings o f Coumans et al. raises questions about the  optimal time period to intervene for the promotion o f axonal regeneration (Coumans et al., 2001), they cannot necessarily be extrapolated to neuroprotective therapies, which are generally shown to be most effective when applied as soon as possible after injury.  5.5.4.  Peripheral Nerve Transplants and Rubrospinal A x o n a l Regeneration  The  long-recognized permissiveness o f peripheral nerves to axonal growth may be  largely attributable to the Schwann cells, and the relative ease with which they can be acquired and expanded in culture has made them attractive candidates for promoting regeneration in spinal cord injured patients (Bunge, 2000).  A n important study i n 1996 by Cheng et al. (Cheng  et al., 1996) demonstrated corticospinal tract regeneration and functional recovery after bridging a rat spinal cord transection with 18 tiny intercostal nerve grafts stabilized with a fibrin glue containing acidic fibroblast growth factor.  One o f the authors o f this paper has recently reported  the application o f this intercostal nerve graft transplant paradigm in a chronic thoracic transection injury model, in which axonal regeneration and partial functional recovery was observed in treated animals (Fraidakis et al., 2004).  To extend these research findings into the  clinical setting, a human case report o f such autologous nerve grafting with acidic fibroblast growth factor was recently published (Cheng et al., 2004). The authors reported on a patient with an incomplete thoracic spinal cord laceration (stabbing victim) who underwent this grafting  142  procedure  approximately 4  years  after  injury and  improvement in lower extremity motor function.  subsequently  achieved  measureable  While the implication o f this study is that  axonal regeneration occurred through the nerve graft, it is difficult to rule out that the recovery was mediated by fibers from the intact part o f the spinal cord. It should be recognized that cellular substrates, including Schwann cells, used for transplantation represent more than just a passive substitute for the non-permissive C N S environment and its inhibitory elements. Cells used in transplantation paradigms constituitively express, or can be genetically modified to secrete a host o f neurotrophic factors and thus they themselves can influence the regenerative competence o f injured C N S neurons (Zompa et al., 1997).  Schwann cells are certainly no exception to this, as they are known to express  neurotrophic factors, cell adhesion molecules, and extracellular matrix molecules important for axonal regeneration (Guenard et al., 1993) and have also been genetically modified to increase their secretion o f such neurotrophic factors as B D N F (Menei et al., 1998) and N G F (Tuszynski et al., 1998). A s such, while conceptually providing a permissive environment for axonal growth (in essence, overcoming the extrinsic barriers to axonal growth) the application o f cellular substrates such as Schwann cells within peripheral nerve transplants is not mutually exclusive from the strategy o f enhancing the intrinsic regenerative capacity o f C N S neurons (with neurotrophic factors, for example). In this regard, the transplantation o f Schwann cells or peripheral nerves would seemingly be an ideal "combinatorial" therapeutic strategy to promote axonal regeneration, addressing both some o f the intrinsic and extrinsic obstacles. The corollary to this however, is that as permissive as an environment as they might be, Schwann cells (and other cellular substrates for that matter) still ultimately require that the neurons themselves mount a regenerative response in response to  143  the injury and treatment.  The chronic spinal cord injury setting, therefore, imposes a particular  challenge, as illustrated by my findings that rubrospinal axonal regeneration was not stimulated by the application o f B D N F to the spinal cord injury site, despite the varying doses o f B D N F applied.  These findings are consistent with the failed rubrospinal regeneration in response to  BDNF-secreting fibroblasts 6 weeks after injury (Tobias et al., 2003). A l s o consistent with this, Decherchi et al. found that the implantation o f an autologous peripheral nerve transplant 3 weeks after cervical spinal cord injury led to 80% less axonal regeneration than when the graft was implanted acutely (147±44.5 double-labeled cells acutely versus 21.7 ± 5 . 6 double-labeled cells chronically) (Decherchi and Gauthier, 2000).  Houle similarly found that a delay in autologous  peripheral nerve transplantation o f 4 weeks resulted in no axonal regeneration o f rubrospinal neurons (or any other supraspinal neuron for that matter) into the transplant (Houle, 1991). While these reports describe the failure o f axonal regeneration from chronically injured rubrospinal neurons in response to spinal cord application o f B D N F and peripheral nerve transplants, the change in responsiveness over time to particular therapies is not a phenomenon exclusive to the rubrospinal system and to these particular therapies. One o f the most studied experimental therapies in spinal cord injury research is the neutralization o f inhibitory epitopes within C N S myelin with antibodies.  Pioneering work by Schwab and colleagues demonstrated  long-distance corticospinal axonal regeneration and subsequent functional recovery in response to the acute administration o f IN-1 antibody to mitigate the inhibitory effects o f a protein now known as N O G O - A (Schnell and Schwab, 1990, Bregman et al., 1995, Chen et al., 2000). However, a delay i n applying the IN-1 antibody by 8 weeks after injury led to 98% less corticospinal regeneration than at 2 weeks post-injury (von Meyenburg et al., 1998).  A l o n g the  same lines, Houle and Y e reported that the administration o f b F G F to a cervical hemisection  144  injury 8 weeks after injury promoted 50% less regeneration o f various supraspinal neurons than the same intervention at 4 weeks post-injury (Houle and Y e , 1997). This disparity between the extent o f axonal regeneration observed with acute versus delayed application o f B D N F and acute versus delayed transplantation o f a peripheral nerve graft illustrate that a therapy may lose its effectiveness i n promoting axonal regeneration with a delay in intervention.  Obviously, this has fairly important implications for individuals with chronic  spinal cord injuries whose hopes for an imminent therapy for their condition are generally founded i n the encouraging results o f studies that report axonal regeneration in acute injury models.  A s such, this provides strong rationale to evaluate therapies at chronic time points after  spinal cord injury.  M y findings o f rubrospinal axonal regeneration 12 months post-injury with  cell body application o f B D N F is promising proof o f principle that axonal regeneration may be possible after prolonged delays in intervention.  O f the significant issues to be yet resolved is  how such an intervention can be applied in a less invasive fashion, and what combinatorial therapies are required to promote the regeneration o f chronically injured rubrospinal neurons across the injury site and into the distal spinal cord.  In the context o f combinatorial therapies, a  recent study by Bunge and colleagues has demonstrated that after a contusion injury, the application o f a Schwann cell graft (as a strategy to "bridge" the inhibitory spinal cord environment) in combination with means o f elevating intracellular c A M P levels (as a strategy to boost the neuronal regenerative propensity) promoted regeneration o f serotonergic axons and locomotor recovery (Pearse et al., 2004).  Another recent study by Tuszynski and colleagues  also demonstrated the potential utility o f combinatorial approaches ( L u et al., 2004).  These  authors reported that the combined application o f c A M P and N T - 3 in conjunction with the transplantation o f bone marrow stromal cells into a dorsal column injury promoted axonal  145  regeneration beyond the spinal cord injury site. application o f c A M P or N T - 3 alone.  Such regeneration was not observed with the  These exciting findings which demonstrate the potential  for combinatorial therapies in the acute injury setting may prove to be beneficial for promoting regeneration o f chronically injured neurons.  146  - CHAPTER 6 TRKB RECEPTORS WITHIN CHRONICALLY INJURED RUBROSPINAL NEURONS AND AXONS  6.1. S U M M A R Y  In this chapter, I review the expression o f full length T r k B receptors within the chronically injured rubrospinal system, with the assumption that the presence o f T r k B receptors would make the rubrospinal system most directly responsive to the exogenous application o f BDNF.  Using immunohistochemistry, I documented the presence o f full length T r k B receptors  on the cell bodies o f rubrospinal neurons both 2 and 12 months after cervical axotomy ( K w o n et al., 2002b).  Conversely, I found that anterogradely labeled rubrospinal axons at the cervical  injury site did not contain full length T r k B immunoreactivity ( K w o n et al., 2004a).  These  differences in the expression o f full length T r k B receptors on the injured axons within the spinal cord and the neuronal cell bodies are consistent with the lack o f B D N F effectiveness applied to the spinal cord and its effectiveness when infused into the vicinity o f the red nucleus.  147  6.2. INTRODUCTION The effectiveness  o f the  exogenous  administration  o f neurotrophic  factors  therapeutic strategy for C N S injury ultimately depends on the presence o f the  as  a  appropriate  receptors within the target tissue. The Trk neurotrophin receptors are a family o f highly related tyrosine kinases that demonstrate specificity i n their binding to members o f the classic neurotrophin family (discussed in more detail i n the Background chapter, section 1.6.1). T r k B receptors bind with high affinity to B D N F and N T - 4 / 5 , and as such their expression is o f particular relevance to these experiments in which I have applied B D N F to promote axonal regeneration o f the chronically injured rubrospinal system.  T r k B receptors contain both an extra  and intracellular domain, with the kinase moieties found intracellularly (reviewed by Barbacid (Barbacid, 1994)).  Both full length and truncated forms o f the T r k B receptor have been  described, with the latter lacking the intracellular kinase domain while maintaining the extracellular binding domain (and thus remaining specific to B D N F and NT-4/5 binding) (Middlemas et al., 1991).  6.2.1. Expression of TrkB Receptors in the Uninjured CNS The expression o f T r k B receptor m R N A has been widely demonstrated in the brain and spinal cord (Klein et al., 1989, K l e i n et al., 1990), and immunoreactivity to the extracellular domain o f the T r k B receptor has also been shown to be widespread i n the C N S (Yan et al., 1997). O f specific relevance to my experiments, Kobayashi et al. used i n situ hybridization to show that virtually all uninjured rubrospinal neurons o f the adult rat express m R N A for fulllength T r k B receptors (Kobayashi et al., 1997), while robust immunoreactivity to  the  extracellular domain o f the T r k B receptor was demonstrated by Y a n et al. on the cell bodies o f  148  adult rat rubrospinal neurons (Yan et al., 1997).  Within the spinal cord, i n situ hybridization  studies have identified m R N A o f full length T r k B receptors in the neurons o f grey mater, but not in the glial cells o f the white matter (Frisen et al., 1992, Liebl et al., 2001, K i n g et al., 2000).  A  predominantly non-neuronal expression o f the truncated isoforms o f T r k B is seen within the uninjured spinal cord (King et al., 2000, Frisen et al., 1993). immunoreactivity is observed within the  A similar cellular pattern o f  spinal cord to antibodies directed against  the  intracellular kinase domain o f the T r k B receptor (Skup et al., 2002).  6.2.2. Expression of TrkB Receptors in the Injured CNS While the expression o f T r k B receptors on the native rubrospinal system is obviously o f importance, it is the expression o f these receptors in response to spinal cord injury that is most relevant to the effectiveness o f exogenous B D N F administration as a potential treatment strategy. Additionally, in the context o f how such a neurotrophic intervention might be influenced after injury, it is worth noting the changes that occur at both the supraspinal and the spinal cord level as they pertain to the neuronal cell bodies and injured axons respectively.  Kobayashi et al.  demonstrated with in situ hybridization that one week after cervical axotomy, the expression o f full length T r k B receptor m R N A i n injured rubrospinal neurons had decreased by approximately 30% compared to uninjured, and that this decline i n expression continued i n the second week as neuronal atrophy progressed (Kobayashi et al., 1997). Later time points beyond 2 weeks were not evaluated.  Frisen and colleagues demonstrated i n both cats and rats that within 3 weeks o f a  spinal cord axotomy, the m R N A expression o f truncated T r k B receptors increased significantly in the scar tissue and white matter adjacent to the injury (Frisen et al., 1992, Frisen et al., 1993). The authors noted that the m R N A increase was specifically o f the truncated form o f T r k B , was restricted to glial cells, and was limited to the area immediately surrounding the axotomy, with  149  no alterations i n T r k B expression in sections above or below the injury (Frisen et al., 1993). Similar findings were reported by Liebl et al using a contusion injury model, in which they reported a high level o f truncated T r k B m R N A expression in ependymal cells and astrocytes that lined the injury site, with the absence o f full length T r k B expression at the injury site (Liebl et al., 2001).  Consistent with this, K i n g et al. demonstrated a dramatic increase i n truncated T r k B  receptor immunoreactivity at the interface between damaged and undamaged spinal cord after a hemisection injury ( K i n g et al., 2000). This increase began within days o f injury, peaked at 2 weeks post injury, and remained for at least 4 weeks (later time points were not evaluated). Conceptually, i f the expression o f full length T r k B receptors were to diminish over time in injured rubrospinal neurons (as shown by (Kobayashi et al., 1997), this neuronal system could become less responsive to the exogenous application o f B D N F . The aforementioned changes in T r k B expression within the spinal cord after injury, with an increase in truncated and decrease in full length isoforms, might also influence the effectiveness o f B D N F applied exogenously to this region.  What these studies have not demonstrated, however, is the immunoreactivity to full  length T r k B o f the rubrospinal neurons both at the level o f their cell bodies and in their axons within the spinal cord.  Recognizing the demonstrated decrease i n the full length T r k B m R N A  expression in rubrospinal neurons after injury (Kobayashi et al., 1997), it might be convenient to consider that the localization o f the protein would be diminished throughout the neuron and axon.  However, L u et al. demonstrated i n the corticospinal system that after subcortical injury,  immunoreactivity to full length T r k B receptors was found on the cell bodies o f corticospinal neurons, but was absent on their projecting axons at the level o f the injury ( L u et al., 2001). In this chapter I immunohistochemically evaluated full length T r k B receptor expression in the chronically injured rubrospinal system, working under the assumption that the expression  150  of full length T r k B receptors is important for the responsiveness o f rubrospinal neurons and axons to B D N F application, and acknowledging that the expression o f T r k B receptors (both full length and truncated isoforms) appears to change after cervical axotomy and may potentially differ between the neuronal cell body and its projecting axon.  O f particular interest in this  research question was to seek some rationale for the observed differences between cell body and spinal cord application o f B D N F in terms o f the effects on rubrospinal neuronal atrophy (Chapter 3), R A G expression (Chapter 4), and axonal regeneration (Chapter 5).  151  6.3. OVERVIEW OF EXPERIMENTAL QUESTIONS AND HYPOTHESES In this chapter, I evaluated full length T r k B receptor immunoreactivity on the cell bodies and axons o f chronically injured rubrospinal neurons to test the following following hypotheses: 1.  Given that the cell body administration o f B D N F to chronically injured rubrospinal neurons  reversed neuronal atrophy, increased G A P - 4 3 and T a l tubulin expression, and promoted axonal regeneration, / hypothesized that chronically injured rubrospinal neurons maintain full length TrkB receptors on their cell bodies as the means of remaining responsive to this neurotrophic factor. To test this hypothesis, animals underwent a cervical axotomy, then two and twelve months later the presence o f full length T r k B receptors on the injured rubrospinal neurons was evaluated with a polyclonal antibody to the intracellular kinase domain o f the receptor (SC-12, Santa Cruz Biotechnology, Santa Cruz, C A ) . 2.  Given that the spinal cord administration o f B D N F at three exponentially different doses did  not reverse neuronal atrophy, increase G A P - 4 3 and T a l tubulin expression, or promote axonal regeneration o f chronically injured rubrospinal neurons, and that others too have demonstrated the ineffectiveness o f B D N F administration to the spinal cord, / hypothesized that full length TrkB receptors are not maintained on the axons of chronically injured rubrospinal neurons at the injury site. To test this hypothesis, animals underwent a cervical axotomy, and then two months later the injured and uninjured rubrospinal tracts were anterogradely labeled.  Immunohistochemistry  with the a polyclonal antibody to the intracellular kinase domain o f the receptor (SC-12, Santa Cruz Biotechnology, Santa Cruz, C A ) was then performed on horizontal sections o f the spinal cord to evaluate T r k B immunoreactivity on rubrospinal axons adjacent to the injury site.  152  6.4. R E S U L T S  To evaluate T r k B receptor expression on rubrospinal cell bodies, immunohistochemistry on 20 micron thick cryostat sections o f the caudal half o f the red nucleus was performed, using a rabbit polyclonal T r k B antibody specific for the intracellular domain o f the receptor, a biotinylated secondary antibody, and the A B C kit for visualization.  T o evaluate T r k B receptor  expression on the injured axons, the rubrospinal axons were first anterogradely labeled with B D A (both on the injured and uninjured side). 20 micron thick horizontal sections were taken through the spinal cord injury site, so that both the injured and uninjured rubrospinal tracts could be visualized on the same section. Immunohistochemistry to the T r k B receptor was then carried out as above, but with a fluorescent secondary antibody (Alexa 488, Molecular Probes, Eugene Or). Colocalization between the T r k B and B D A (as visualized with streptavidin conjugated to Cy3) was evaluated with confocal microscopy. Twelve months after cervical axotomy, immunoreactivity to full length T r k B receptors was observed on the cell bodies o f both injured and uninjured rubrospinal neurons (Figure 6.1). These animals had B D N F or vehicle alone infused into the vicinity o f the rubrospinal neurons via osmotic minipump. Despite the atrophy o f the vehicle treated animals, the immunoreactivity to T r k B was apparent. Similar immunoreactivity was observed on the cell bodies o f injured and uninjured rubrospinal neurons 2 months after cervical axotomy. to the spinal cord injury site within gelfoam.  These animals had B D N F or P B S applied  Again, there was significant atrophy o f the injured  neurons, but despite this atrophy they appeared to maintain full length T r k B immunoreactivity (Figure 6.2).  The T r k B immunoreactivity on the injured rubrospinal neurons did not appear to  153  be grossly different between the P B S and BDNF-treated animals (see Figure 6.2), although the quantification o f the immunoreactivity was not attempted. Anterograde labeling o f the rubrospinal tract on both sides o f the spinal cord was consistently achieved with the B D A injection performed using the coordinates as described by Houle et al (Houle and Jin, 2001).  (Figure 6.3)  After anterogradely labeling the rubrospinal  tract (both injured and uninjured), there appeared to be very consistent co-localization between the B D A labeled rubrospinal axons and the full length T r k B immunoreactivity on the uninjured side o f the spinal cord at the C3/4 level. Conversely, on the injured side o f the spinal cord at this level, T r k B immunoreactivity was not observed to colocalize with the anterogradely labeled axons, suggesting that at the injury site, chronically injured rubrospinal axons had lost their T r k B receptors. (Figure 6.4)  Overall, T r k B staining on the injured side o f the spinal cord appeared  to be substantially less than that observed on the uninjured side (See Figure 6.4 - Panels B versus E , and Figure 6.8 - Panel A versus B ) .  A t 63x magnification, it was frequently not possible to  see multiple BDA-labeled axons on the horizontal sections, but on cross sectional analysis at the C I level, colocalization between the B D A and T r k B was seen on every anterogradely labeled axon. (Figure 6.5) The lack o f T r k B receptors on rubrospinal axons adjacent to the injury site was seen in all groups o f animals, while robust colocalization between axons and T r k B was seen on the uninjured axons in all groups o f animals.  Control slides in which the T r k B primary  antibody was incubated with its blocking peptide in a 1:1 ratio or in which the T r k B primary antibody was not applied demonstrated no specific binding (Figure 6.5).  154  Figure 6.1. Full length TrkB receptor immunohistochemistry is maintained on rubrospinal neuronal cell bodies 12 months after axotomy. Full length T r k B receptor immunoreactivity is demonstrated here on the cell bodies o f rubrospinal neurons from animals treated with cell body administration o f the vehicle solution (top row) or B D N F (bottom row), twelve months after cervical axotomy. Note that the vehicletreated injured rubrospinal neurons remain extremely atrophic (A) compared to the contralateral uninjured  neurons (B) yet  maintain  immunoreactivity  for  full  length  TrkB  receptors.  Conversely, note the reversal o f atrophy in injured rubrospinal neurons treated with B D N F (C), making them comparable in size to the contralateral uninjured neurons (D). Scale bar, 50 pm.  Injured  Contralateral Uninjured  B  O JC  > wa  D m •*•> LL  OQ  SrjQ CN  155  Figure 6.2. Full length TrkB receptor immunohistochemistry is maintained on rubrospinal neuronal cell bodies 2 months after axotomy. Full length T r k B receptor immunoreactivity is demonstrated here on the cell bodies o f rubrospinal neurons from animals treated with spinal cord administration o f P B S (top row) or B D N F (bottom row), two months after cervical axotomy.  Similar to that seen at 12 months  post-axotomy (Figure 6.1), injured neurons are atrophic compared to their uninjured contralateral counterparts yet maintain T r k B immunoreactivity.  Note that B D N F treatment did not reverse  the atrophy, but T r k B immunoreactivity on the cell bodies is again maintained.  A l l BDNF  treated animals at each o f the three concentrations o f B D N F (50, 1,000, and 20,000 ng/pl) demonstrated similar T r k B immunoreactivity. Scale bar, 50 pm.  Injured  Uninjured  156  Figure 6.3. Anterograde labeling of with BDA provides consistent visualization of the rubrospinal tract within the dorsolateral funiculus of the spinal cord. A 5 x (A) and lOx (B) image of the spinal cord in cross section demonstrates the B D A labeling of the rubrospinal tract within the dorsolateral funiculus. This cross section is from the spinal cord just rostral to the segment of cord harvested for horizontal sections. Scale bar, 2 0 0 pm.  B  157  Figure 6.4. T r k B immunoreactivity is not maintained on the rubrospinal axons at the site of spinal cord injury, 2 months after the injury  Colocalization o f T r k B immunoreactivity (green) and B D A anterograde labeling (red) o f the rubrospinal tract two months after injury.  Panels A to F are taken from a PBS-treated  animal, with panels A to C demonstrating an anterogradely labeled axon at the injury site and panels D to F demonstrating an anterogradely labeled axon on the uninjured side o f the same section.  For  both  injured  and  uninjured,  the  BDA  labeled  axon,  the  TrkB  immunohistochemistry, and the merged, overlaid images are shown. The arrowheads (injured) and arrows (uninjured) are reference points to highlight the absence o f T r k B colocalization on the injured axon and the close co-localization o f T r k B on the uninjured  axon.  The  immunoreactivity to T r k B appeared to be less on the injured side o f the spinal cord at the injury site (B) compared to the opposite, uninjured side o f the spinal cord (E) (Scale bar = 25 pm).  159  Figure 6.5. T r k B immunoreactivity closely colocalizes w i t h B D A labeled rubrospinal axons on cross-sectional images of the spinal cord at C I , well p r o x i m a l to the injury site. These are the B D A (A), T r k B (B) and overlaid images o f a cross section of the uninjured rubrospinal tract at approximately C I (well proximal to the axotomy).  Note that in panel A ,  only a fraction o f the axons pick up the anterograde B D A tracer. However, every anterogradely labeled axon colocalizes with T r k B on the merged image (C) (small arrows). pm).  B TrkB  A BDA  i  9-  - * •*  «/  i  1 j  (Scale bar = 25  Figure 6.6. Control TrkB immunohistochemistry slides demonstrate no specific binding. After incubation of the primary TrkB antibody with its blocking peptide, there is no TrkB staining of the tissue (A).  Also, with no primary antibody, there is no unspecific green staining  from the secondary antibody (B). (Scale bar = 25 pm).  A  Blocking Peptide  B  No Primary Antibody  161  6.5 D I S C U S S I O N  6.5.1. C h a p t e r S u m m a r y In this chapter, I sought to characterize the localization o f full length T r k B receptors i n the chronically injured rubrospinal system, i n part to seek some biological rationale for the differences in effectiveness between cell body and spinal cord application o f B D N F .  I found  that anterogradely labeled rubrospinal axons at the spinal cord injury site did not contain full length T r k B receptors, while the uninjured axons on the contralateral side o f the spinal cord did, as evidenced by the strong colocalization between T r k B and B D A on the uninjured side o f the spinal cord.  Additionally, I found that the cell bodies o f injured rubrospinal neurons maintained  full length T r k B immunoreactivity at two and twelve months after cervical axotomy. In Chapters 3, 4, and 5,1 observed that the application o f B D N F to the injured spinal cord 2 months after cervical axotomy was not effective in reversing rubrospinal neuronal atrophy, did not increase G A P - 4 3 and T a l tubulin expression, and did not promote axonal regeneration into peripheral nerve transplants. Conversely, the application o f B D N F to the cell bodies at an even more chronic time point (12 months post-axotomy) was effective in reversing neuronal atrophy, increasing G A P - 4 3 and T a l tubulin expression, and promoting axonal regeneration peripheral nerve transplants.  into  The expression o f full length T r k B receptors on the rubrospinal  cell bodies but not on the rubrospinal axons at the chronic spinal cord injury site provides some explanation for these findings.  Furthermore, the expression o f full length T r k B receptors on the  uninjured rubrospinal axons on the contralateral side o f the spinal cord might explain why B D N F applied acutely after injury to the spinal cord appears to be effective in promoting axonal regeneration ( L i u et al., 1999).  162  6.5.2. Anterograde Labeling of Rubrospinal Tract Anterograde labeling o f the rubrospinal tract was performed via an intraparenchymal injection o f B D A to the medial aspect o f the red nucleus where the rubrospinal axons emerge and project into the spinal cord. The coordinates for this injection were those published by Houle et al. in their study o f rubrospinal axonal dieback from a cervical hemisection injury (Houle and Jin, 2001).  These authors demonstrated effective anterograde labeling o f the rubrospinal tract  and were able to quantify the extent o f axonal dieback weeks after the cervical spinal cord injury. M y analysis o f cross sections o f the spinal cord demonstrated pronounced anterograde labeling in the dorsolateral funiculus o f the spinal cord, confirming that the technique worked in my hands as well.  O n these cross sections, however, it appeared that the B D A tracer was picked up  by only a fraction o f the total number o f rubrospinal axons.  Given that there are thousands o f  neurons i n the red nucleus, one would expect there to be many more axons labeled at the upper part o f the cervical spinal cord i f the B D A tracer had been picked up by the entire population o f axons.  Indeed, on the horizontal sections, at 63x magnification, it was often not possible to find  multiple B D A labeled axons in the same field o f view.  One can note on these horizontal  sections, particularly on the uninjured side o f the spinal cord, that there appears to be "streaks" o f green T r k B staining that run in parallel with the anterogradely labeled axons (see Figure 6.4, panel E). These streaks o f T r k B staining are likely representative o f rubrospinal axons that were simply not labeled with B D A , as is suggested by the cross sectional imaging. (Figure 6.5)  163  6.5.3. T r k B Receptors on the A x o n s and C e l l Bodies of R u b r o s p i n a l Neurons M y observations that T r k B receptors are maintained on the cell body but are not found on the axons at the spinal cord injury site are consistent with the reported findings o f L u et al. in their evaluation o f corticospinal neurons ( L u et al., 2001).  Using very similar techniques in  which corticospinal axons were anterogradely labeled with B D A and colocalization with T r k B immunoreactivity was evaluated with confocal microscopy, these authors demonstrated the maintenance o f T r k B receptors on the corticospinal neuronal cell bodies but not on the axons in the spinal cord after a dorsal hemisection injury. not promote  the  B D N F applied to the injured spinal cord did  growth o f corticospinal axons  (devoid o f T r k B receptors),  but  when  administered subcortically - presumably making the neurotrophic factor available to the cell bodies which possessed T r k B receptors - the B D N F was effective at preventing retrograde neuronal death after axotomy.  This correlation between the presence o f T r k B receptors and  responsiveness to B D N F in the injured corticospinal system is very consistent with my findings in the injured rubrospinal system. Given the changes that I observed in T r k B immunoreactivity within the injured spinal cord, it is interesting to consider how long it takes for such changes to occur.  Conceptually, i f  the loss o f T r k B receptors at the injury site occurs over a long period o f time, that time period would define a window o f opportunity to apply neurotrophic factors to the as yet responsive axons.  Kobayashi et al. demonstrated with in situ hybridization that T r k B receptor expression  within rubrospinal neurons decreased by 30% within one week o f cervical axotomy (Kobayashi et al., 1997).  Clearly, changes occur quickly in T r k B transcription, but without doing  immunohistochemical evaluations on the injured spinal cord at various time points after injury, it is not possible to know how that change in gene expression is reflected by changes in protein  164  levels at the distal tip o f the axon.  O f note, L i e b l et al. reported that T r k B expression within  rubrospinal neurons did not decrease significantly after thoracic contusion injury o f the spinal cord, but in this study, with a dorsal contusion injury, it is uncertain how many o f the laterally place rubrospinal axons were in fact disrupted to begin with (Liebl et al., 2001).  Alternatively,  the disparity between the results o f Kobayashi and Liebl could be explained by different responses to injury at the cervical and thoracic levels.  While my cervical partial transection  model is less representative o f a clinical spinal cord injury than a contusion injury, the certainty with which the rubrospinal tract is cut allows us to answer the question o f what is happening at the cell body level in response to injury. Furthermore, i f T r k B receptors are lost on the axons at the injury site, it would be interesting to know how far back along the axon such changes exist. Are the TrkB receptors lost for a few hundred microns, millimeters,  or along the entire axon up to the axon hillock?  I f the  receptors were lost for only a short distance, than perhaps spinal cord application o f B D N F might work i f one could get the neurotrophic factor to diffuse proximally enough to access the remaining T r k B receptors.  Alternatively, i f the receptors were lost all the way up to the axon  hillock, then no amount o f B D N F diffusion from the injury site would likely be effective. Unfortunately, my current experiments do not allow us to definitively answer this question.  The  greatest difficulty arises in distinguishing between those axons on the injured side o f the spinal cord that are themselves injured, and those that are actually terminating on their targets proximal to the injury site.  I evaluated T r k B receptor expression on axons adjacent to the injury site  itself, as I felt that only these axons could reliably be defined as actually being injured.  More  proximal to this, when the injury site cannot be visualized, it is more difficult to know i f a given B D A labeled axon is injured or is terminating prior to reaching the axotomy site.  This problem  165  is schematically illustrated in Figure 6.7.  One could argue that you could simply follow an  axon from the injury site back towards the cell body, but on horizontal sections, the axons often move out o f the plane o f the section within a few hundred microns, making it impossible to follow a given axon for a prolonged length o f spinal cord.  The cross sectional images at C I  suggest that there is less T r k B receptor expression within the dorsolateral funiculus on the injured side compared to uninjured.  (Figure 6.8) Nevertheless, colocalization o f B D A labeled  axons and T r k B receptors is also seen on the injured side o f the spinal cord well proximal to the injury (around C I ) , which again raises the problem illustrated in Figure 6.7.  A thorough  evaluation o f serial cross sections moving back from the spinal cord injury site might be helpful in resolving this question.  166  Figure 6.7. The current experimental paradigm of evaluating injured rubrospinal axons on horizontal sections makes it difficult to determine how far proximally the loss of TrkB receptors occurs. W i t h the lack o f T r k B colocalization with rubrospinal axons at the injury site, it would be interesting to determine how far proximally this loss o f receptors occurs.  Unfortunately, the  problem with looking proximally i n the spinal cord is determining whether the actual axon being evaluated is injured or uninjured further caudally.  Looking at these two axons proximal to the injury, it is not possible to distinguish between the intact and the injured axon.  Injured axon at the axotomy site  167  Figure 6.8. Immunoreactivity to full length TrkB appears to be less on the injured side of the spinal cord compared to uninjured well proximal to the injury site (at CI). With regards to the question of how far proximally the loss o f T r k B receptors extends away from the injury site, at C I , there appears to be less T r k B immunoreactivity on the injured than uninjured side o f the spinal cord.  This would suggest that the loss o f T r k B receptors  extends well proximal to the injury site, although it is impossible to know on the injured side o f the spinal cord which axons are intact or cut distally (as described in Figure 6.6).  Serial  analysis o f cross sections more proximal to the injury site would be helpful to resolve this issue. Scale bar, 25 pm.  168  - CHAPTER 7GENERAL DISCUSSION  7.1. S U M M A R Y  In this thesis, I have described a number o f experiments that have been performed to evaluate the rubrospinal system in a chronic injury state after cervical axotomy.  For the most  part, the experiments were performed at two and twelve months after axotomy, and they attempted to determine changes that occurred over time at both the level o f the rubrospinal cell bodies and at their injured axons.  Brain derived neurotrophic factor ( B D N F ) was applied to  either the rubrospinal cell bodies or to their axons to determine the responsiveness o f this neuronal system in the chronic injury state. M y findings can be summarized as follows.  Two months after cervical axotomy,  rubrospinal neurons undergo significant atrophy and exhibit limited expression o f GAP-43 and T a l tubulin, genes thought to be important for axonal regeneration.  Rubrospinal neurons  appear to maintain full length T r k B receptors on their cell bodies, and while their uninjured axons within the cervical spinal cord also contain T r k B receptors, the injured axons at the level o f the spinal cord axotomy do not. Consistent with this, B D N F applied to the spinal cord injury site at three exponentially increasing concentrations did not reverse rubrospinal cell atrophy, did not stimulate GAP-43 and T a l tubulin expression, and did not promote axonal regeneration o f rubrospinal axons into the permissive environment o f a peripheral nerve transplant.  A t 12  169  months after cervical axotomy, a stereologic evaluation o f rubrospinal neurons demonstrates that rubrospinal neurons are in fact alive, but very atrophic.  Similar to the findings at 2 months post-  injury, the rubrospinal neurons 12 months post-injury display limited expression o f G A P - 4 3 and T a l tubulin but do maintain full length T r k B receptors on their cell bodies. A t this chronic time point, the administration o f B D N F to the injured cell bodies reversed neuronal  atrophy,  stimulated G A P - 4 3 and T a l tubulin expression, and promoted axonal regeneration  into  peripheral nerve transplants. While much o f the discussion o f the results has been covered in the individual chapters, a number o f broad issues surrounding the nature o f my experimental model and the implications o f the findings warrant further discussion.  In the following sections, I w i l l attempt to place these  findings into a larger context, and consider some o f the future directions for such research.  170  7.2. MODELING OF CHRONICITY IN SPINAL CORD INJURY  Currently, there are over 250,000 individuals living in North America who have suffered a spinal cord injury some time ago, whose neurologic status is essentially stable, and who are now considered to be chronically injured (Sekhon and Fehlings, 2001).  This number o f chronically  injured individuals by itself provides compelling rationale to study axonal regeneration strategies in chronic injury paradigms.  These patients, particularly those with complete loss o f motor and  sensory function whose neurologic deficits are stable (ie. no longer expected to improve on their own), are considered to be the most likely candidates for initial clinical trials into experimental therapies that might induce some axonal regeneration, as it felt that they have the "least to lose, and the most to gain".  Given this fact, it is somewhat ironic that all axonal regeneration  strategies are tested experimentally in the acute injury setting, and few are ever evaluated after a delay in intervention to model a chronic injury situation.  It may be tempting to consider  intervening with an axonal regeneration therapy acutely in patients with complete motor and sensory paralysis, given that their prognosis for distal lower extremity recovery is expected to be poor (Marino et al., 1999).  Despite this, these patients often do achieve some local motor  recovery, mediated by preserved spinal cord tissue around the zone o f injury, even in the absence o f distal recovery. injuries.  This is an issue o f critical importance to those with cervical spinal cord  For example, for someone who presents acutely with a C 6 level o f complete  quadriplegia but with a flicker o f triceps movement (mediated by the C 7 nerve root), the very real possibility that he or she may naturally regain functional triceps power over time is enormously important, even though the chance o f recovering lower extremity function is negligible (functional triceps power would enable the patient to transfer in and out o f bed independently and use a non-motorized wheelchair).  A s such, until the patient  spontaneousy  171  achieved his/her full recovery potential (of course, with the help o f standard medical, surgical, and rehabilitative care) and establishes a stable neurologic impairment, it would be extremely risky to undertake an intervention that could potentially diminish perserved tissue at the injury site. A n example o f this would be the surgical manipulation required to transplant a cellular substrate into the injured cord.  This spontaneous recovery typically occurs over the first 18  months that follow the traumatic incident, again, reinforcing the need for research in spinal cord injury models with a delay in intervention (Marino et al., 1999). Having outlined the compelling need for experimental research that accounts for a delay in therapeutic intervention, it is nonethelesss unclear how to reproduce the chronic condition in a rat model o f spinal cord injury.  One o f the issues o f some uncertainty is the actual definition o f  "chronic" as it applies to the neurobiology o f spinal cord injury.  A t an operational level, for the  purposes o f animal modeling and testing o f experimental treatments, it might be reasonable to suggest that an injury becomes chronic when treatments that work acutely no longer remain effective.  While this may have some conceptual appeal, this time frame o f effectiveness may  vary widely amongst therapies.  Alternatively, the definition o f chronicity could apply to the  molecular, biochemical, and morphologic changes that occur within the relevant neuronal systems after spinal cord injury. I performed my experiments largely at two different time points after injury: 2 months and 12 months.  Clearly, waiting 12 months post-injury is, from a practical perspective, an  extremely difficult endeavour, as the animal care is costly and the animals, having passed over half their lifespan, are relatively fragile to the anesthetic and surgical procedures necessary to test these interventions.  I therefore proposed 2 months post-axotomy as a reasonable period o f time  to wait before intervening with the B D N F treatment.  With respect to changes that occur at the  172  spinal cord injury site, H i l l et al. reported the establishment o f a clearly defined cavity 3 weeks after a contusion spinal cord injury, which was fully developed and stable at 14 weeks postinjury and remained so for the subsequent 6 months ( H i l l et al., 2001).  Following a sharp  hemisection injury, Houle and Jin reported dieback o f rubrospinal, vestibulospinal, and reticulospinal axons that occurred primarily within the first week post-injury and then remained fairly stable for the next 13 weeks (Houle and Jin, 2001).  In terms o f the molecular changes  that occur at the level o f the cell body, G A P - 4 3 and T a l tubulin expression increases acutely after cervical axotomy but then diminishes over the subsequent 4 to 7 weeks (Fernandes et al., 1999, Fernandes and Tetzlaff, 2000, Kobayashi et al., 1997, Tetzlaff et al., 1991) More recently, Storer and Houle have demonstrated that 4 weeks after cervical axotomy, the acute increases in G A P - 4 3 and p i l tubulin expression have abated and the levels o f expression are either at or below that o f the contralateral uninjured neurons (Storer and Houle, 2003).  I feel, therefore, that  the selection o f a two-month post-axotomy period in the rat provides a reasonable reproduction o f a chronic spinal cord injury, both with respect to the changes that occur at the level o f the cell body and at the spinal cord.  Additionally, the results o f my experiments in which the  rubrospinal system was essentially unresponsive to B D N F applied at the spinal cord two months after injury provides some support to the operational definition o f chronicity at this time point. O f note, a similar failure to upregulate G A P - 4 3 and p i l tubulin expression in rubrospinal neurons or to reverse their atrophy was recently reported by Storer et al. when applying B D N F to the injury site only 4 weeks post-axotomy (Storer et al., 2003). Given that our laboratory and others have characterized some o f the changes that occur in the rubrospinal system two months after cervical axotomy, and have identified aspects o f the system that influence its responsiveness to neurotrophic therapy, we would propose that it  173  represents a reasonably useful "chronic" spinal cord injury model to study the effectiveness o f axonal regeneration strategies.  Useful features o f this as a model include the assuredness in  performing a complete transection o f the rubrospinal tract, the ability to both anterogradely and retrogradely label the system, and the ability to use changes i n neuronal cross sectional area, R A G expression, and axonal regeneration into peripheral nerve transplants as outcome measures. The generalizability o f the findings in the rubrospinal system to other neuronal systems after chronic injury is uncertain, but is certainly relevant to any clinical application o f an experimental therapy for patients with chronic spinal cord injury.  174  7.3. ADMINISTRATION OF BDNF AS A THERAPEUTIC STRATEGY FOR SPINAL CORD INJURY  In these experiments, we applied B D N F to the brainstem or spinal cord in an effort to establish its effect on the chronically injured rubrospinal system and ultimately to shed insights into the challenges o f promoting axonal regeneration in patients with chronic spinal cord injury. Such in vivo exogenous administration o f B D N F as a therapeutic strategy raises interesting questions.  A s full length T r k B receptors appear to be important for mediating the biological  effect o f B D N F , my line o f reasoning was to study T r k B receptor expression within the rubrospinal system.  The findings o f full length T r k B receptors on the rubrospinal cell bodies  and on uninjured rubrospinal axons fits into a fairly rational model i n which the administration o f B D N F binds to T r k B receptors and exerts its effects on gene expression, neuronal size, and axonal regeneration.  This is supported by my observations after B D N F  rubrospinal cell bodies 12 months post-axotomy.  application to  Conversely, the direct administration o f  B D N F to injured rubrospinal axons that lack full length T r k B receptors fails to elicit a change in gene expression, neuronal atophy, and axonal regeneration.  This is supported by my  observations after B D N F application to the spinal cord 2 months post-axotomy. While all o f this seems fairly rationale, it is recognized that the infusion or application o f such enormous doses o f B D N F is a crude experimental paradigm, although likely more selective than the systemic and intrathecal administration o f B D N F that, for example, has been tested in patients with amyotrophic lateral sclerosis (Ochs et al., 2000, B D N F Study Group, 1999).  The  side effects reported in these studies (eg. diarrhea, muscle weakness, agitation, and sleep disturbances) highlight the widespread distribution o f T r k B receptors both within and outside the nervous system.  One could also postulate that the B D N F that we infused into the vicinity o f the  175  red nucleus stimulated surrounding glial cells or neurons which then secreted other factors (possibly even B D N F ) that acted in a paracrine fashion to effect the changes we observed in the 12 month chronically injured rubrospinal neurons.  For example, microglia/macrophages that are  attracted to the cannula might have been stimulated by the B D N F to secrete other neurotrophic factors, as they have been shown to do in vitro (Elkabes et al., 1996).  Indeed, the small extent  of axonal regeneration observed in chronically injured animals that received intracranial infusions o f vehicle solution alone suggest that elements o f an inflammatory or glial response to cannula insertion may have stimulated the rubrospinal neurons.  The failure to observe a  reversal o f atrophy, an increase in R A G expression, and the promotion o f axonal regeneration with the spinal cord application o f B D N F in the chronic setting could be similarly explained by a failure to elicit such a response in surrounding tissue.  Resolving the actual mechanism by which  the B D N F is acting would o f course shed further light into the obstacles that must be overcome to promote axonal regeneration in a chronic injury setting.  176  7.4. F U N C T I O N O F T R K B R E C E P T O R S I N T H E C H R O N I C A L L Y I N J U R E D RUBROSPINAL SYSTEM  If we were to make the reasonable assumption that it is i n fact the presence o f full length T r k B receptors in the rubrospinal system that makes it responsive to B D N F , it is interesting to consider how they are actually working to effect this response, particularly given that we are applying the neurotrophic factor to the cell body or to the axon.  H o w does B D N F activation o f  full length T r k B receptors on the cell body translate into the changes in cell size, gene transcription, and axonal regeneration that we observed?  H o w different is this from the  activation o f these receptors on the axons (for example, in the acute injury setting)?  A s long as  the T r k B receptors are present, would cell body and spinal cord administration o f B D N F have the same effect? M u c h has been published on the downstream signalling that follows Trk receptor activation, reflecting a widespread interest in the biologic mechanisms by which neurotrophic factors exert their actions.  Most o f this literature is on the T r k A receptor, but there is likely  much overlap in the intracellular pathways activated by T r k A and T r k B receptors. Neurotrophin binding to the extracellular domain o f Trk receptors induces receptor dimerization and kinase activation.  The phosphorylation o f specific cytoplasmic tyrosine residues then attracts P L C - y  and adaptor proteins such as S H C and S H 2 - B (Patapoutian and Reichardt, 2001). A t w a l et al. demonstrated that for T r k B receptors, S H C binding is coupled to the activation o f the extracellular signal regulated kinase ( E R K ) protein kinase pathway and to the activation o f the survival factor A k t v i a the phosphatidylinositol-3-OH kinase (PI3K) pathway (Atwal et al., 2000).  Downstream effects o f the activation o f these pathways include the elevation o f  177  intracellular calcium, or changes in gene transcription.'  The exact molecular mechanisms by  which this ultimately translates into the outcome measures that we studied - notably, the increases in cross sectional area o f neurons, I S H for G A P - 4 3 and T a l tubulin, and axonal regeneration - is not entirely clear. It is, nonetheless, intriguing to consider that the intracellular signalling pathways initiated in rubrospinal neurons by B D N F administration at the cell body might actually be different than what occurs when it is delivered to the injured spinal cord - ie. that the activation o f T r k B receptors on the cell bodies initiates different signalling pathways than activation o f T r k B receptors on the axons. actually  represent a  If this were the case, the cell body administration o f B D N F would  fundamentally  different  treatment paradigm  than  the  spinal cord  administration o f B D N F (and not one distinguished only by the effectiveness after a delay in intervention).  Indeed, while such in vivo data is not currently available, Watson et al. studied  D R G neurons in compartmented cultures and reported that the different intracellular signalling pathways could be initiated by the same stimulus depending on i f the stimulus occurred at the cell body or at the axonal level (Watson et al., 2001).  In this study, the authors found that  neurotrophic factor administration to the cell bodies activated C R E B v i a the stimulation o f both E r k l / 2 and Erk5.  Neurotrophic factor administration to the axons activated both E r k l / 2 and  Erk5 locally within the axon, but retrograde C R E B activation at the cell body caused by Erk5 only, implying that changes in gene transcription at the nuclear level were mediated by different intracellular pathways depending on where the neurotrophic stimulation occurred (cell body versus axon) (Watson et al., 2001).  Conversely, local activation o f E r k l / 2 at the level o f the  axon may itself contribute to axonal outgrowth independent o f a cell body response, possibly as the result o f the phosphorylation o f microtubule-associated proteins and modulation o f other  178  cytoskeletal proteins (Atwal et al., 2000).  These studies point to differences between the  intracellular sequelae o f neurotrophic factor administration at either the cell body or axon - a distinction that certainly could have some relevance to my current observations within the rubrospinal system.  M y findings at the two month time point after axotomy would suggest that  the lack o f a regenerative response seen with spinal cord application o f B D N F was related to the loss o f T r k B receptors. However, even i f T r k B receptors were still maintained on the chronically injured axons, one could also consider the possibility that for the outcome measures that we were studying (neuronal cross sectional area, G A P - 4 3 and T a l tubulin expression, and axonal regeneration), T r k B activation at the cell body (via B D N F administration) simply activated the necessary signalling pathways in the chronic setting that T r k B activation at the axons did not. A l o n g the same lines, it is interesting to consider how such a retrograde signal (eg. T r k B activation v i a B D N F ) at the axon is transferred back to the cell body in the acute, let alone the chronic state.  Ultimately, this has a profound influence on the therapeutic strategy o f providing  neurotrophic factors directly to the injured spinal cord, assuming that successful long-distance axonal regeneration requires some form o f participation from the cell body.  A number o f  potential mechanisms have been proposed for the means by which neurotrophic factor stimulation at the axonal level initiates a signal that is retrogradely transmitted back to the cell body (reviewed by M i l l e r and Kaplan, 2001 and 2002 and by Ginty and Segal, 2002).  Binding  of the neurotrophic factor to the Trk receptor may cause internalization o f the Trk receptor, with or without the neurotrophic factor, into a vesicle which is then retrogradely transported back to the cell body (Watson et al., 1999, Bhattacharyya et al., 1997).  The internalization o f the Trk  receptor occurs in such a manner as to leave the intracellular domains o f the receptor on the outside o f the vesicle where they can continue to initiate downstream signalling (thus being  179  described as "signalling endosomes" (Beattie et al., 1996, Y e et al., 2003).  Alternatively, there  may be a rapid "wave" o f Trk receptor activation initiated by the neurotrophin binding but then propogated along the course o f the axon without vesicular transport (Senger and Campenot, 1997).  Finally, intracellular signalling proteins such as PI-3 kinase might be activated at the  axon level and serve as the retrograde activation signal to the cell body (Kuruvilla et al., 2000). While these methods o f retrogade signalling are not mutually exclusive, it is possible that these pathways themselves are affected i n the chronic injury state.  For example, given all the  other molecular changes that have occurred in chronically injured rubrospinal neurons after cervical axotomy, what is to say that the retrograde transport mechanisms required to move signalling endosomes back to the cell body have remained intact?  It is interesting, however, to  note that Storer and Houle found that while the application o f B D N F to the spinal cord 1 month after axotomy did not lead to changes in rubrospinal cross sectional area or G A P - 4 3 expression, the application o f G D N F did (Storer et al., 2003).  G D N F binds to G D N F - F a m i l y Receptor-a 1  ( G F R a l ) then complexes with Receptor Tyrosine Kinase (RET), which, like T r k B receptors, possesses a number o f intracellular tyrosine kinase domains that serve as docking sites for adaptor proteins (Airaksinen and Saarma, 2002).  Similar to T r k B receptors, phosphorylation o f  R E T allows for S H C binding and subsequent activation o f many o f the same intracellular signalling cascades, including the PI3K, E R K , and M E K 5 (Takahashi, 2001).  If one were to  make the assumption that these kinase pathways function similarly whether they are activated by the autophosphorylation o f T r k B receptors or the autophosphorylation o f R E T , then the reversal o f rubrospinal atrophy with the application o f G D N F (Storer et al., 2003) would indicate that the mechanisms by which retrograde signalling is accomplished are indeed intact in this chronic setting (one month post-injury).  180  The implications o f this discussion on the development o f therapeutic strategies involving neurotrophic application in the chronic injury setting are clear:  i f the failure o f B D N F  application is solely related to the expression o f T r k B receptors on the axons at the chronic injury site, then there would be a compelling rationale to pursue a strategy by which T r k B expression might be augmented, possibly by genetic manipulation.  If, however, dysfunction o f other  intracellular mechanisms downstream from Trk activation would preclude effective retrograde transmission o f such a signal, then establishing neurotrophic factor therapy at the chronic injury setting becomes exponentially more complex.  Targeting the cell body directly, as I have done  with the intracranial infusion o f B D N F , might then represent a more appealing strategy. From a purely practical point o f view, however, the clinical application o f such an intervention would be difficult to justify, as it would require inserting a needle through otherwise uninjured brain tissue. A l s o , it would be difficult to anticipate the effect o f B D N F or the inflammatory response to the insertion device on other neuronal systems nearby.  Clearly, an intervention at the spinal cord  that could retrogradely effect the necessary changes within the cell body for axonal outgrowth would be more desirable. The recent report o f K o d a et al in which a B D N F - e n c o d i n g adenovirus was applied to the acute spinal cord injury site and was found to successfully transfect rubrospinal neurons (Koda et al., 2004) demonstrates that technologies may become available to effect the necessary regenerative changes at the cell body level with an intervention directed at the chronically injured spinal cord.  181  7.5. FUTURE DIRECTIONS This research has provided some insight into the chronic spinal cord condition, particularly as it applies to the rubrospinal system.  While these findings shed some light on the  challenges that may be faced in the development o f therapeutic strategies to promote axonal regeneration in the chronic injury setting, clearly, much remains to be done to realize this goal.  1. What is happening to TrkB expression within the injured rubrospinal system? Given the interest in applying neurotrophic factors within the spinal cord, it would be useful to better understand what is happening to T r k B expression within the injured rubrospinal axons.  A s stated in chapter 6, it is unclear how far back towards the cell body the loss o f T r k B  receptors on injured rubrospinal axons extends.  For this, it would be useful to sample the  chronic injury site in cross section instead o f longitudinally (as was performed in my experiments) in order to evaluate the anterogradely labeled rubrospinal axons serially as one moves away from the injury site.  In such an experiment, one could determine at fairly precise  distances  how  from  the  injury  site  many  B D A labeled axons  also  contained  TrkB  immunoreactivity, and how many did not. For example, i f all B D A labeled axons 2 m m rostral to the injury contained T r k B immunoreactivity, then one would infer that the loss o f T r k B receptors is only a very local phenomenon confined to the injury site alone.  If, however, the  ratio B D A and T r k B labeled axons compared to the total number o f B D A labeled axons was low or continued to decrease as one moved more rostrally, it might be inferred that the T r k B receptors were lost along the entire length o f the axon. (Figure 7.1)  182  Figure 7 . 1 .  Experimental paradigm to characterize the loss of T r k B  receptors on  rubrospinal axons.  Serial cross sectional analysis o f anterogradely labeled rubrospinal axons with T r k B immunohistochemistry may help to determine whether the loss o f T r k B receptors occurs only adjacent to the axotomy site (below, left) or along the entire length o f the axon (below, right). B y counting the number o f anterogradely labeled axons and then determining how many o f them are co-labeled with T r k B , one may be able to make this distinction.  If the loss o f receptors is  restricted only to the injury site, then presumably, further rostrally, all o f the labeled axons w i l l also contain T r k B immunoreactivity (below left).  If, however, the loss o f receptors occurs  along the entire length o f the axon, back to the axon hillock, as one moves rostrally, there w i l l be a combination o f both B D A labeled axons with T r k B (those that aren't injured distally) and B D A labeled axons without T r k B (those that are injured distally).  T r k B l o s t o n l y at the distal tip of a x o n ( a d j a c e n t t o injury)  Axotomy  T r k B lost a l o n g entire length o f a x o n  Axotomy •  Serial c r o s s - s e c t i o n s of spinal c o r d , starting f r o m the a x o t o m y site a n d m o v i n g rostrally  183  2.  What is the true therapeutic potential of cell body application of BDNF in the chron  injury state?  M y experiments demonstrated that cell body application o f B D N F 12 months after cervical axotomy could promote axonal regeneration into a free-ending peripheral nerve transplant.  Clearly, it would be interesting to know whether this intervention can lead to  intraspinal regeneration or sprouting, either alone or in conjunction with a strategy to address the inhibitory injury environment, such as a bridging peripheral nerve graft or olfactory ensheathing cells.  B D N F infusion into the parenchyma o f the motor cortex, for example, was found to  promote sprouting o f corticospinal fibers after thoracic spinal cord injury, although regeneration into peripheral nerve transplants was not observed (Hiebert et al., 2002).  Pilot studies in our lab  are underway with the transplantation o f bridging peripheral nerve grafts within the spinal cord to evaluate the ability o f cell body application o f B D N F to drive rubrospinal axonal regeneration into these grafts and back into the injured spinal cord.  Furthermore, it would be o f interest to  evaluate whether a combinatorial strategy such as this could lead to some functional recovery, although in such experiments it is often difficult to ascribe a certain functional change to the regeneration o f a specific axonal tract.  Nonetheless, the role o f the rubrospinal tract in upper  and lower extremity function is a topic o f interest in our lab and in others ( M u i r and Whishaw, 2000, Whishaw et al., 1990) and as this is better elucidated, it the interpretation o f such studies w i l l become easier.  184  3.  T h e cell body infusion of B D N F is highly invasive and clearly invokes some damage to  surrounding parenchyma - is there a more sophisticated way to invoke the same effects?  Clearly, while the findings o f rubrospinal cell survival and axonal regeneration with cell body application o f B D N F are promising, this really represents a proof o f concept experiment. Our laboratory has performed pilot studies with a B D N F encoding adeno-associated virus and has found that the intracranial injection into the vicinity o f the red nucleus appeared to effect a trophic response i n cervically axotomized rubrospinal neurons with less o f an inflammatory response in the surrounding brainstem parenchyma.  Nevertheless, it would obviously be more  desirable to achieve the same responses by intervening at the spinal cord injury site. A s stated earlier, the application o f a BDNF-encoding adenovirus to the spinal cord injury site after acute injury achieved some transfection o f rubrospinal neurons; it is anticipated that these investigators are currently looking into the applicability o f such technology at a chronic time point.  185  7.6.  CONCLUSIONS  M u c h remains to be answered about the neurobiology o f chronic spinal cord injury and the development o f axonal regeneration strategies to effect functional recovery.  The need is  compelling nonetheless, as the population o f individuals with chronic spinal cord injuries only increases each year.  The survival o f chronically injured rubrospinal neurons and their potential  to mount a regenerative response long after injury given the appropriate stimulus is encouraging nonetheless, and such findings w i l l hopefully stimulate further work i n this challenging but important line o f research.  186  BIBLIOGRAPHY Abercrombie M (1946) Estimation o f nuclear population from microtome sections. Anat Rec 94: 239-247. Aigner L , Arber S, Kapfhammer JP, Laux T, Schneider C , Botteri F , Brenner H R , Caroni P (1995) Overexpression o f the neural growth-associated protein G A P - 4 3 induces nerve sprouting in the adult nervous system o f transgenic mice. Cell 83: 269-278. Aigner L , Caroni P (1995) Absence o f persistent spreading, branching, and adhesion in G A P 43-depleted growth cones. J C e l l B i o l 128: 647-660. Airaksinen M S , Saarma M (2002) The G D N F family: signalling, biological functions and therapeutic value. Nat Rev Neurosci 3: 383-394. A l i s k y J M , Tolbert D L (1994) Differential labeling o f converging afferent pathways using biotinylated dextran amine and cholera toxin subunit B . J Neurosci Methods 52: 143-148. Andersen L B , Schreyer D J (1999) Constitutive expression o f G A P - 4 3 correlates with rapid, but not slow regrowth o f injured dorsal root axons in the adult rat. E x p Neurol 155: 157-164. Anderson P N , Lieberman A R (2000) Intrinsic determinants o f differential axonal regeneration by adult mammalian central nervous system neurons. In: Degeneration and Regeneration in the Nervous System (Saunders N R , Dziegielewska K M , eds), pp 53-75. Amsterdam: Harwood Academic Publishers. Ankeny D P , McTigue D M , Guan Z , Y a n Q, Kinstler O, Stokes B T , Jakeman L B (2001) Pegylated brain-derived neurotrophic factor shows improved distribution into the spinal cord and stimulates locomotor activity and morphological changes after injury. E x p Neurol 170: 85100. Antal M , Sholomenko G N , Moschovakis A K , Storm-Mathisen J, Heizmann C W , Hunziker W (1992) The termination pattern and postsynaptic targets o f rubrospinal fibers in the rat spinal cord: a light and electron microscopic study. J C o m p Neurol 325: 22-37. A t w a l J K , Massie B , M i l l e r F D , Kaplan D R (2000) The TrkB-Shc site signals neuronal survival and local axon growth via M E K and P l 3-kinase. Neuron 27: 265-277. Ausubel F M , Brent R, Kingston R E , Moore D D , Seidman J G , Smith K A , Struhl K (1987) Current protocols in molecular biology. N e w York. Bamber N I , L i H , L u X , Oudega M , Aebischer P, X u X M (2001) Neurotrophins B D N F and N T - 3 promote axonal re-entry into the distal host spinal cord through Schwann cell-seeded mini-channels. Eur J Neurosci 13: 257-268. Barbacid M (1994) The Trk family o f neurotrophin receptors. J Neurobiol 25: 1386-1403.  187  Barbacid M (1995) Neurotrophic factors and their receptors. Curr Opin C e l l B i o l 7: 148-155. Barbeau H , Rossignol S (1994) Enhancement o f locomotor recovery following spinal cord injury. Curr Opin Neurol 7: 517-524. Barde Y A , Davies A M , Johnson J E , Lindsay R M , Thoenen H (1987) Brain derived neurotrophic factor. Prog Brain Res 71: 185-189. Barde Y A , Edgar D , Thoenen H (1982) Purification o f a new neurotrophic factor from mammalian brain. E M B O J 1: 549-553. Barron K D , Banerjee M , Dentinger M P , Scheibly M E , Mankes R (1989) Cytological and cytochemical ( R N A ) studies on rubral neurons after unilateral rubrospinal tractotomy: the impact o f G M 1 ganglioside administration. J Neurosci Res 22: 331-337. Baxter G T , Radeke M J , K u o R C , Makrides V , Hinkle B , Hoang R, Medina-Selby A , Coit D , Valenzuela P, Feinstein S C (1997) Signal transduction mediated by the truncated trkB receptor isoforms, t r k B . T l and trkB.T2. J Neurosci 17: 2683-2690. B D N F Study Group (1999) A controlled trial o f recombinant methionyl human B D N F in A L S : The B D N F Study Group (Phase III). Neurology 52: 1427-1433. Beattie E C , Zhou J, Grimes M L , Bunnett N W , Howe C L , Mobley W C (1996) A signaling endosome hypothesis to explain N G F actions: potential implications for neurodegeneration. C o l d Spring Harb Symp Quant B i o l 61: 389-406. Becker T, Bernhardt R R , Reinhard E , Wullimann M F , Tongiorgi E , Schachner M (1998) Readiness o f zebrafish brain neurons to regenerate a spinal axon correlates with differential expression o f specific cell recognition molecules. J Neurosci 18: 5789-5803. Belhaj-Saif A , Karrer J H , Cheney P D (1998) Distribution and characteristics o f poststimulus effects in proximal and distal forelimb muscles from red nucleus i n the monkey. J Neurophysiol 79: 1777-1789. Benes F M , Lange N (2001) Two-dimensional versus three-dimensional cell counting: a practical perspective. Trends Neurosci 24: 11-17. Benowitz L I , Routtenberg A (1997) G A P - 4 3 : an intrinsic determinant o f neuronal development and plasticity. Trends Neurosci 20: 84-91. Bernstein-Goral H , Bregman B S (1997) Axotomized rubrospinal neurons rescued by fetal spinal cord transplants maintain axon collaterals to rostral C N S targets. E x p Neurol 148: 13-25. Berry M , Carlile J, Hunter A (1996) Peripheral nerve explants grafted into the vitreous body o f the eye promote the regeneration o f retinal ganglion cell axons severed i n the optic nerve. J Neurocytol 25: 147-170.  188  Bhattacharyya A , Watson F L , Bradlee T A , Pomeroy S L , Stiles C D , Segal R A (1997) Trk receptors function as rapid retrograde signal carriers in the adult nervous system. J Neurosci 17: 7007-7016. Biffo S, Offenhauser N , Carter B D , Barde Y A (1995) Selective binding and internalisation by truncated receptors restrict the availability o f B D N F during development. Development 121: 2461-2470. Bisby M A , Tetzlaff W (1992) Changes i n cytoskeletal protein synthesis following axon injury and during axon regeneration. M o l Neurobiol 6: 107-123. Bisby M A , Tetzlaff W , Brown M C (1996) G A P - 4 3 m R N A in mouse motoneurons undergoing axonal sprouting in response to muscle paralysis o f partial denervation. Eur J Neurosci 8: 1240-1248. Blaha G R , Raghupathi R, Saatman K E , Mcintosh T K (2000) Brain-derived neurotrophic factor administration after traumatic brain injury in the rat does not protect against behavioral or histological deficits. Neuroscience 99: 483-493. Blits B , Oudega M , Boer G J , Bartlett B M , Verhaagen J (2003) Adeno-associated viral vectormediated neurotrophin gene transfer in the injured adult rat spinal cord improves hind-limb function. Neuroscience 118: 271-281. Bomze H M , Bulsara K R , Iskandar B J , Caroni P, Pate Skene J H (2001) Spinal axon regeneration evoked by replacing two growth cone proteins in adult neurons. Nat Neurosci 4: 38-43. Bregman B S , Broude E , McAtee M , Kelley M S (1998) Transplants and neurotrophic factors prevent atrophy o f mature C N S neurons after spinal cord injury. E x p Neurol 149: 13-27. Bregman B S , Kunkel-Bagden E , Schnell L , D a i H N , Gao D , Schwab M E (1995) Recovery from spinal cord injury mediated by antibodies to neurite growth inhibitors. Nature 378: 498501. Bregman B S , McAtee M , Dai H N , K u h n P L (1997) Neurotrophic factors increase axonal growth after spinal cord injury and transplantation in the adult rat. E x p Neurol 148: 475-494. Bregman B S , Reier P J (1986) Neural tissue transplants rescue axotomized rubrospinal cells from retrograde death. J Comp Neurol 244: 86-95. Brown L T (1974) Rubrospinal projections in the rat. J Comp Neurol 154: 169-187. Buffo A , Holtmaat A J , Savio T, Verbeek JS, Oberdick J, Oestreicher A B , Gispen W H , Verhaagen J, Rossi F , Strata P (1997) Targeted overexpression o f the neurite growthassociated protein B-50/GAP-43 in cerebellar Purkinje cells induces sprouting after axotomy but not axon regeneration into growth-permissive transplants. J Neurosci 17: 8778-8791.  189  Bunge M B (2000) What types o f bridges w i l l best promote axonal regeneration across an area o f injury in the adult mammalian spinal cord? In: Degeneration and regeneration in the nervous system (Saunders N R , Dziegielewska K M , eds), pp 171-190. Amsterdam: Harwood Academic Publishers. Caroni P (1997) Intrinsic neuronal determinants that promote axonal sprouting and elongation. Bioessays 19: 767-775. Caroni P (2001) N e w E M B O members' review: actin cytoskeleton regulation modulation o f PI(4,5)P(2) rafts. E M B O J 20: 4332-4336.  through  Cavalier-Smith T (1978) Nuclear volume control by nucleoskeletal D N A , selection for cell volume and cell growth rate, and the solution o f the D N A C-value paradox. J C e l l Sci 34: 247278. Chaisuksunt V , Zhang Y , Anderson P N , Campbell G , Vaudano E , Schachner M , Lieberman A R (2000) Axonal regeneration from C N S neurons in the cerebellum and brainstem o f adult rats: correlation with the patterns o f expression and distribution o f messenger R N A s for L I , C H L 1 , c-jun and growth-associated protein-43. Neuroscience 100: 87-108. Chao M V (1994) The p75 neurotrophin receptor. J Neurobiol 25: 1373-1385. Chen D F , Jhaveri S, Schneider G E (1995) Intrinsic changes in developing retinal neurons result in regenerative failure o f their axons. Proc Natl A c a d Sci U S A 92: 7287-7291. Chen M S , Huber A B , van der Haar M E , Frank M , Schnell L , Spillmann A A , Christ F, Schwab M E (2000) N o g o - A is a myelin-associated neurite outgrowth inhibitor and an antigen for monoclonal antibody IN-1 [see comments]. Nature 403: 434-439. Cheng H , Cao Y , Olson L (1996) Spinal cord repair in adult paraplegic rats: partial restoration o f hind limb function [see comments]. Science 273: 510-513. Cheng H , Liao K K , Liao SF, Chuang T Y , Shih Y H (2004) Spinal cord repair with acidic fibroblast growth factor as a treatment for a patient with chronic paraplegia. Spine 29: E284E288. Chong M S , Reynolds M L , Irwin N , Coggeshall R E , Emson P C , Benowitz L I , W o o l f C J (1994) G A P - 4 3 expression in primary sensory neurons following central axotomy. J Neurosci 14: 4375-4384. Coggeshall R E , Chung K (1984) The determination o f an empirical correction factor to deal with the problem o f nucleolar splitting in neuronal counts. J Neurosci Methods 10: 149-155. Coggeshall R E , Chung K , Greenwood D , Hulsebosch C E (1984) A n empirical method for converting nucleolar counts to neuronal numbers. J Neurosci Methods 12: 125-132.  190  Coumans J V , L i n T T , D a i H N , MacArthur L , McAtee M , Nash C , Bregman B S (2001) Axonal regeneration and functional recovery after complete spinal cord transection in rats by delayed treatment with transplants and neurotrophins. J Neurosci 21: 9334-9344. Cowan W M (1998) The emergence o f modern neuroanatomy and developmental neurobiology. Neuron 20: 413-426. Daniel H , Billard J M , Angaut P, Batini C (1987) The interposito-rubrospinal Anatomical tracing o f a motor control pathway in the rat. Neurosci Res 5: 87-112.  system.  David S, Aguayo A J (1981) Axonal elongation into peripheral nervous system "bridges" after central nervous system injury in adult rats. Science 214: 931-933. Decherchi P, Gauthier P (2000) Regrowth o f acute and chronic injured spinal pathways within supra-lesional post-traumatic nerve grafts. Neuroscience 101: 197-210. Diener P S , Bregman B S (1994) Neurotrophic factors prevent the death o f C N S neurons after spinal cord lesions in newborn rats. Neuroreport 5: 1913-1917. Dutcher S K (2003) Long-lost relatives reappear: identification o f new members o f the tubulin superfamily. Curr Opin Microbiol 6: 634-640. Dyer J K , Bourque J A , Steeves J D (1998) Regeneration o f brainstem-spinal axons after lesion and immunological disruption o f myelin in adult rat. E x p Neurol 154: 12-22. Egan D A , Flumerfelt B A , G w y n D G (1977) A x o n reaction i n the red nucleus o f the rat. Perikaryal volume changes and the time course o f chromatolysis following cervical and thoracic lesions. Acta Neuropathol (Berl) 37: 13-19. Elkabes S, D i C i c c o - B l o o m E M , Black IB (1996) Brain microglia/macrophages express neurotrophins that selectively regulate microglial proliferation and function. J Neurosci 16: 2508-2521. Ernfors P, Lee K F , Jaenisch R (1994) M i c e lacking brain-derived neurotrophic factor develop with sensory deficits. Nature 368: 147-150. Ernfors P, Rosario C M , Merlio JP, Grant G , Aldskogius H , Persson H (1993) Expression o f m R N A s for neurotrophin receptors in the dorsal root ganglion and spinal cord during development and following peripheral or central axotomy. Brain Res M o l Brain Res 17: 217226. Fan M , M i R, Y e w D T , Chan W Y (2001) Analysis o f gene expression following sciatic nerve crush and spinal cord hemisection i n the mouse by microarray expression profiling. C e l l M o l Neurobiol 21: 497-508. Fawcett J W (1998) Spinal cord repair: from experimental models to human application. Spinal Cord 36: 811-817.  191  Ferguson I A , Koide T, Rush R A (2001) Stimulation o f corticospinal tract regeneration in the chronically injured spinal cord. Eur J Neurosci 13: 1059-1064. Feringa E R , M c B r i d e R L , Pruitt J N (1988) Loss o f neurons i n the red nucleus after spinal cord transection. E x p Neurol 100: 112-120. Fernandes K J , Fan D P , Tsui B J , Cassar S L , Tetzlaff W (1999) Influence o f the axotomy to cell body distance in rat rubrospinal and spinal motoneurons: differential regulation o f G A P - 4 3 , tubulins, and neurofilament-M. J Comp Neurol 414: 495-510. Fernandes K J , Tetzlaff W (2000) Gene Expression in Axotomized Neurons: Identifying the Instrinsic Determinants o f Axonal Growth. In: Axonal Regeneration in the Central Nervous System ( I n g o g l i a N A , Murray M , eds), pp 219-266. N e w York: Marcel Dekker, Inc. Fiford R J , Bilston L E , Waite P, L u J (2004) A vertebral dislocation model o f spinal cord injury in rats. J Neurotrauma 21: 451-458. Fischer D , He Z , Benowitz L I (2004) Counteracting the Nogo receptor enhances optic nerve regeneration i f retinal ganglion cells are in an active growth state. J Neurosci 24: 1646-1651. Fraidakis M J , Spenger C , Olson L (2004) Partial recovery after treatment o f chronic paraplegia in rat. E x p Neurol 188: 33-42. Frey D , Laux T, X u L , Schneider C , Caroni P (2000) Shared and unique roles o f C A P 2 3 and G A P 4 3 i n actin regulation, neurite outgrowth, and anatomical plasticity. J C e l l B i o l 149: 14431454. Frisen J, Verge V M , Cullheim S, Persson H , Fried K , Middlemas D S , Hunter T, Hokfelt T, Risling M (1992) Increased levels o f trkB m R N A and trkB protein-like immunoreactivity in the injured rat and cat spinal cord. Proc Natl A c a d Sci U S A 89: 11282-11286. Frisen J, Verge V M , Fried K , Risling M , Persson H , Trotter J, Hokfelt T, Lindholm D (1993) Characterization o f glial trkB receptors: differential response to injury in the central and peripheral nervous systems. Proc Natl A c a d Sci U S A 90: 4971-4975. Fryer R H , Kaplan D R , Feinstein S C , Radeke M J , Grayson D R , Kromer L F (1996) Developmental and mature expression o f full-length and truncated T r k B receptors in the rat forebrain. J Comp Neurol 374: 21 -40. Fukuoka T, M i k i K , Yoshiya I, Noguchi K (1997) Expression o f beta-calcitonin gene-related peptide in axotomized rubrospinal neurons and the effect o f brain derived neurotrophic factor. Brain Res 767: 250-258. Funakoshi H , Frisen J, Barbany G , Timmusk T, Zachrisson O, Verge V M , Persson H (1993) Differential expression o f m R N A s for neurotrophins and their receptors after axotomy o f the sciatic nerve. J C e l l B i o l 123: 455-465.  192  Giehl K M , Schacht C M , Y a n Q, Mestres P (1997) G D N F is a trophic factor for adult rat corticospinal neurons and promotes their long-term survival after axotomy in vivo. Eur J Neurosci 9: 2479-2488. Giehl K M , Tetzlaff W (1996) B D N F and N T - 3 , but not N G F , prevent axotomy-induced death o f rat corticospinal neurons in vivo. Eur J Neurosci 8: 1167-1175. Ginty D D , Segal R A (2002) Retrograde neurotrophin signaling: Trk-ing along the axon. Curr Opin Neurobiol 12: 268-274. Goldberg J L , Barres B A (2000) The relationship between neuronal survival and regeneration. A n n u Rev Neurosci 23: 579-612. Goshgarian H G , Koistinen J M , Schmidt E R (1983) C e l l death and changes in the retrograde transport o f horseradish peroxidase in rubrospinal neurons following spinal cord hemisection in the adult rat. J Comp Neurol 214: 251-257. Grados-Munro E M , Fournier A E (2003) Myelin-associated inhibitors o f axon regeneration. J Neurosci Res 74: 479-485. GrandPre T, Nakamura F, Vartanian T, Strittmatter S M (2000) Identification o f the Nogo inhibitor o f axon regeneration as a Reticulon protein. Nature 403: 439-444. G r i l l R J , Blesch A , Tuszynski M H (1997) Robust growth o f chronically injured spinal cord axons induced by grafts o f genetically modified NGF-secreting cells. E x p Neurol 148: 444452. Gris P, Murphy S, Jacob J E , Atkinson I, B r o w n A (2003) Differential gene expression profiles in embryonic, adult-injured and adult-uninjured rat spinal cords. M o l C e l l Neurosci 24: 555567. Guenard V , X u X M , Bunge M B (1993) The use o f Schwann cell transplantation to foster central nervous system repair. Sem Neurosci 5: 401-411. Guillery R W , Herrup K (1997) Quantification without pontification: choosing a method for counting objects in sectioned tissues. J Comp Neurol 386: 2-7. Haapasalo A , Koponen E , Hoppe E , W o n g G , Castren E (2001) Truncated t r k B . T l is dominant negative inhibitor o f trkB.TK+-mediated cell survival. Biochem Biophys Res Commun 280: 1352-1358. Hempstead B L , Martin-Zanca D , Kaplan D R , Parada L F , Chao M V (1991) High-affinity N G F binding requires coexpression o f the trk proto-oncogene and the low-affinity N G F receptor. Nature 350: 678-683. Herdegen T, Skene P, Bahr M (1997) The c-Jun transcription factor—bipotential mediator o f neuronal death, survival and regeneration. Trends Neurosci 20: 227-231.  193  Hiebert G W , Khodarahmi K , M c G r a w J, Steeves J D , Tetzlaff W (2002) Brain-derived neurotrophic factor applied to the motor cortex promotes sprouting o f corticospinal fibers but not regeneration into a peripheral nerve transplant. J Neurosci Res 69: 160-168. H i l l C E , Beattie M S , Bresnahan J C (2001) Degeneration and sprouting o f identified descending supraspinal axons after contusive spinal cord injury in the rat. E x p Neurol 171: 153-169. Ho P R , Coan G M , Cheng E T , N i e l l C , Tarn D M , Zhou H , Sierra D , Terris D J (1998) Repair with collagen tubules linked with brain-derived neurotrophic factor and ciliary neurotrophic factor in a rat sciatic nerve injury model. A r c h Otolaryngol Head Neck Surg 124: 761-766. Hoke A , Gordon T, Zochodne D W , Sulaiman O A (2002) A decline i n glial cell-line-derived neurotrophic factor expression is associated with impaired regeneration after long-term Schwann cell denervation. E x p Neurol 173: 77-85. Holmqvist B I , Ostholm T, Ekstrom P (1992) D i l tracing in combination with immunocytochemistry for analysis o f connectivities and chemoarchitectonics o f specific neural systems in a teleost, the Atlantic salmon. J Neurosci Methods 42: 45-63. Houle J D (1991) Demonstration o f the potential for chronically injured neurons to regenerate axons into intraspinal peripheral nerve grafts. E x p Neurol 113: 1-9. Houle J D , Jin Y (2001) Chronically injured supraspinal neurons exhibit only modest axonal dieback in response to a cervical hemisection lesion. E x p Neurol 169: 208-217. Houle J D , Tessler A (2003) Repair o f chronic spinal cord injury. E x p Neurol 182: 247-260. Houle J D , Y e J H (1997) Changes occur in the ability to promote axonal regeneration as the post-injury period increases. Neuroreport 8: 751-755. Houle J D , Y e J H (1999) Survival o f chronically-injured neurons can be prolonged by treatment with neurotrophic factors. Neuroscience 94: 929-936. Huigrok T J H , Cella F (1995) Precerebellar nuclei and red nucleus. In: The Rat Nervous System (Paxinos G , ed), pp 277-308. San Diego: Academic Press. Huisman A M , Kuypers H G , Verburgh C A (1982) Differences i n collateralization o f the descending spinal pathways from red nucleus and other brain stem cell groups in cat and monkey. Prog Brain Res 57: 185-217. Ikeda O, Murakami M , Ino H , Yamazaki M , K o d a M , Nakayama C , M o r i y a H (2002) Effects of brain-derived neurotrophic factor ( B D N F ) on compression-induced spinal cord injury: B D N F attenuates down-regulation o f superoxide dismutase expression and promotes upregulation o f myelin basic protein expression. J Neuropathol E x p Neurol 61: 142-153.  194  Ikeda O, Murakami M , Ino H , Yamazaki M , Nemoto T, K o d a M , Nakayama C , M o r i y a H (2001) Acute up-regulation o f brain-derived neurotrophic factor expression resulting from experimentally induced injury in the rat spinal cord. Acta Neuropathol (Berl) 102: 239-245. Jakeman L B , Guan Z , W e i P, Ponnappan R, Dzwonczyk R, Popovich P G , Stokes B T (2000) Traumatic spinal cord injury produced by controlled contusion in mouse. J Neurotrauma 17: 299-319. Jakeman L B , W e i P, Guan Z , Stokes B T (1998) Brain-derived neurotrophic factor stimulates hindlimb stepping and sprouting o f cholinergic fibers after spinal cord injury. E x p Neurol 154: 170-184. Jeffery N D , Fitzgerald M (2001) Effects o f red nucleus ablation and exogenous neurotrophin-3 on corticospinal axon terminal distribution in the adult rat. Neuroscience 104: 513-521. Jenkins R, Tetzlaff W , Hunt SP (1993) Differential expression o f immediate early genes in rubrospinal neurons following axotomy in rat. Eur J Neurosci 5: 203-209. Jin Y , Tessler A , Fischer I, Houle J D (2000) Fibroblasts genetically modified to produce B D N F support regrowth o f chronically injured serotonergic axons. Neurorehabil Neural Repair 14: 311-317. Jing S, Tapley P, Barbacid M (1992) Nerve growth factor mediates signal transduction through trk homodimer receptors. Neuron 9: 1067-1079. Jones K R , Farinas I, Backus C , Reichardt L F (1994) Targeted disruption o f the B D N F gene perturbs brain and sensory neuron development but not motor neuron development. C e l l 76: 989-999. Jones L L , Oudega M , Bunge M B , Tuszynski M H (2001) Neurotrophic factors, cellular bridges and gene therapy for spinal cord injury. J Physiol 533: 83-89. Jones L L , Tuszynski M H (2001) Chronic intrathecal infusions after spinal cord injury cause scarring and compression. Microsc Res Tech 54: 317-324. Jung M , Petrausch B , Stuermer C A (1997) Axon-regenerating retinal ganglion cells in adult rats synthesize the cell adhesion molecule L l but not T A G - 1 or S C - 1 . M o l C e l l Neurosci '9: 116-131. Kaplan D R , Hempstead B L , Martin-Zanca D , Chao M V , Parada L F (1991a) The trk protooncogene product: a signal transducing receptor for nerve growth factor. Science 252: 554-558. Kaplan D R , Martin-Zanca D , Parada L F (1991b) Tyrosine phosphorylation and tyrosine kinase activity o f the trk proto-oncogene product induced by N G F . Nature 350: 158-160. Kaplan D R , M i l l e r F D (2000) Neurotrophin signal transduction in the nervous system. Curr Opin Neurobiol 10: 381-391.  195  Kennedy P R (1990) Corticospinal, rubrospinal and rubro-olivary projections: a unifying hypothesis. Trends Neurosci 13: 474-479. Kennedy P R , Gibson A R , Houk J C (1986) Functional and anatomic differentiation between parvicellular and magnocellular regions o f red nucleus in the monkey. Brain Res 364: 124-136. K i n g V R , Bradbury E J , M c M a h o n S B , Priestley J V (2000) Changes in truncated trkB and p75 receptor expression in the rat spinal cord following spinal cord hemisection and spinal cord hemisection plus neurotrophin treatment. E x p Neurol 165: 327-341. K l e i n R , Parada L F , Coulier F , Barbacid M (1989) trkB, a novel tyrosine protein kinase receptor expressed during mouse neural development. E M B O J 8: 3701-3709. K l e i n R , Martin-Zanca D , Barbacid M , Parada L F (1990) Expression o f the tyrosine kinase receptor gene trkB is confined to the murine embryonic and adult nervous system. Development 109: 845-850. K l e i n R , Jing S Q , Nanduri V , O'Rourke E , Barbacid M (1991) The trk proto-oncogene encodes a receptor for nerve growth factor. Cell 65: 189-197. K l e i n R, Smeyne R J , Wurst W , Long L K , Auerbach B A , Joyner A L , Barbacid M (1993) Targeted disruption o f the trkB neurotrophin receptor gene results in nervous system lesions and neonatal death. Cell 75: 113-122. Kobayashi N R , Bedard A M , Hincke M T , Tetzlaff W (1996) Increased expression o f B D N F and trkB m R N A in rat facial motoneurons after axotomy. Eur J Neurosci 8: 1018-1029. Kobayashi N R , Fan D P , Giehl K M , Bedard A M , Wiegand SJ, Tetzlaff W (1997) B D N F and NT-4/5 prevent atrophy o f rat rubrospinal neurons after cervical axotomy, stimulate G A P - 4 3 and Talphal-tubulin m R N A expression, and promote axonal regeneration. J Neurosci 17: 9583-9595. Kobbert C , Apps R , Bechmann I, Lanciego J L , M e y J, Thanos S (2000) Current concepts in neuroanatomical tracing. Prog Neurobiol 62: 327-351. K o d a M , Hashimoto M , Murakami M , Yoshinaga K , Ikeda O, Yamazaki M , Koshizuka S, Kamada T, M o r i y a H , Shirasawa H , Sakao S, Ino H (2004) Adenovirus vector-mediated in vivo gene transfer o f brain-derived neurotrophic factor ( B D N F ) promotes rubrospinal axonal regeneration and functional recovery after complete transection o f the adult rat spinal cord. J Neurotrauma 21: 329-337. K o d a M , Murakami M , Ino H , Yoshinaga K , Ikeda O, Hashimoto M , Yamazaki M , Nakayama C , M o r i y a H (2002) Brain-derived neurotrophic factor suppresses delayed apoptosis o f oligodendrocytes after spinal cord injury in rats. J Neurotrauma 19: 777-785. Korsching S (1993) The neurotrophic factor concept: a reexamination. J Neurosci 13: 27392748.  196  K r y l D , Barker P A (2000) TTIP is a novel protein that interacts with the truncated T l T r k B neurotrophin receptor. Biochem Biophys Res Commun 279: 925-930. Kuchler M , Fouad K , Weinmann O, Schwab M E , Raineteau O (2002) R e d nucleus projections to distinct motor neuron pools in the rat spinal cord. J Comp Neurol 448: 349-359. Kuruvilla R, Y e H , Ginty D D (2000) Spatially and functionally distinct roles o f the P I 3 - K effector pathway during N G F signaling in sympathetic neurons. Neuron 27: 499-512. K w o n B K , Borisoff JF, Tetzlaff W (2002a) Molecular targets for therapeutic intervention after spinal cord injury. M o l Intervent 2: 244-258. K w o n B K , L i u J, Messerer C , Kobayashi N R , M c G r a w J, Oschipok L , Tetzlaff W (2002b) Survival and regeneration o f rubrospinal neurons 1 year after spinal cord injury. Proc Natl A c a d Sci U S A 99: 3246-3251. K w o n B K , L i u J, Oschipok L , Teh J, L i u Z W , Tetzlaff W (2004a) Rubrospinal neurons fail to respond to brain-derived neurotrophic factor applied to the spinal cord injury site 2 months after cervical axotomy. E x p Neurol epub Jul 3, 2004. K w o n B K , L i u J, Oschipok L , Tetzlaff W (2002c) Reaxotomy o f chronically injured rubrospinal neurons results in only modest cell loss. E x p Neurol 177: 332-337. K w o n B K , Oxland T R , Tetzlaff W (2002d) A n i m a l models used in spinal cord regeneration research. Spine 27: 1504-1510. K w o n B K , Tetzlaff W (2001) Spinal cord regeneration: from gene to transplants. Spine 26: S13-S22. K w o n B K , Tetzlaff W , Grauer J N , Beiner J, Vaccaro A R (2004b) Pathophysiology and pharmacologic treatment o f acute spinal cord injury. Spine J 4: 451-464. Laferriere N B , MacRae T H , Brown D L (1997) Tubulin differentiating neurons. Biochem C e l l B i o l 75: 103-117.  synthesis  and  assembly  in  Lamballe F , K l e i n R, Barbacid M (1991) trkC, a new member o f the trk family o f tyrosine protein kinases, is a receptor for neurotrophin-3. C e l l 66: 967-979. Laux T, Fukami K , Thelen M , Golub T, Frey D , Caroni P (2000) G A P 4 3 , M A R C K S , and C A P 2 3 modulate PI(4,5)P(2) at plasmalemmal rafts, and regulate cell cortex actin dynamics through a common mechanism. J Cell B i o l 149: 1455-1472. Leibrock J, Lottspeich F , Hohn A , Hofer M , Hengerer B , Masiakowski P, Thoenen H , Barde Y A (1989) Molecular cloning and expression o f brain-derived neurotrophic factor. Nature 341: 149-152. Leon S, Y i n Y , Nguyen J, Irwin N , Benowitz L I (2000) Lens injury stimulates axon regeneration in the mature rat optic nerve. J Neurosci 20: 4615-4626.  197  Lev-Montalcini R, Hamburger V (1951) Selective growth stimulating effects o f mouse sarcoma on the sensory and sympathetic nervous system o f the chick embryo. J E x p Z o o l 116: 321-362. Lev-Montalcini R, Hamburger V (1953) A diffusible agent o f mouse sarcoma, producing hyperplasia o f sympathetic ganglia and hyperneurotization o f viscera i n the chick embryo. J E x p Z o o l 123:233-288. L i L , X u Q, W u Y , H u W , G u P, F u Z (2003) Combined therapy o f methylprednisolone and brain-derived neurotrophic factor promotes axonal regeneration and functional recovery after spinal cord injury in rats. Chin M e d J ( E n g l ) 116: 414-418. Liebl D J , Huang W , Young Y , Parada L F (2001) Regulation o f Trk receptors following contusion o f the rat spinal cord. Experimental Neurology 167: 15-26. L i u Y , Himes B T , Murray M , Tessler A , Fischer I (2002) Grafts o f BDNF-producing fibroblasts rescue axotomized rubrospinal neurons and prevent their atrophy. E x p Neurol 178: 150-164. L i u Y , K i m D , Himes B T , Chow S Y , Schallert T, Murray M , Tessler A , Fischer I (1999) Transplants o f fibroblasts genetically modified to express B D N F promote regeneration o f adult rat rubrospinal axons and recovery o f forelimb function. J Neurosci 19: 4370-4387. L u J, Feron F, Mackay-Sim A , Waite P M (2002) Olfactory ensheathing cells promote locomotor recovery after delayed transplantation into transected spinal cord. Brain 125: 14-21. L u P, Blesch A , Tuszynski M H (2001) Neurotrophism without neurotropism: B D N F promotes survival but not growth o f lesioned corticospinal neurons. J Comp Neurol 436: 456-470. L u P, Yang H , Jones L L , Filbin M T , Tuszynski M H (2004) Combinatorial therapy with neurotrophins and c A M P promotes axonal regeneration beyond sites o f spinal cord injury. J Neurosci 24: 6402-6409. L u X , Richardson P M (1991) Inflammation near the nerve cell body enhances axonal regeneration. J Neurosci 11: 972-978. Maier D L , M a n i S, Donovan S L , Soppet D , Tessarollo L , McCasland JS, M e i r i K F (1999) Disrupted cortical map and absence o f cortical barrels in growth-associated protein (GAP)-43 knockout mice. Proc Natl Acad Sci U S A 96: 9397-9402. Maisonpierre P C , Belluscio L , Friedman B , Alderson R F , Wiegand SJ, Furth M E , Lindsay R M , Yancopoulos G D (1990) N T - 3 , B D N F , and N G F in the developing rat nervous system: parallel as well as reciprocal patterns o f expression. Neuron 5: 501-509. Marino R J , Ditunno JF, Jr., Donovan W H , Maynard F , Jr. (1999) Neurologic recovery after traumatic spinal cord injury: data from the M o d e l Spinal Cord Injury Systems. A r c h Phys M e d Rehabil 80: 1391-1396.  198  Martin-Zanca D , Hughes S H , Barbacid M (1986) A human oncogene formed by the fusion o f truncated tropomyosin and protein tyrosine kinase sequences. Nature 319: 743-748. Mason M R , Campbell G , Caroni P, Anderson P N , Lieberman A R (2000) Overexpression o f G A P - 4 3 in thalamic projection neurons o f transgenic mice does not enable them to regenerate axons through peripheral nerve grafts. E x p Neurol 165: 143-152. Mason M R , Lieberman A R , Anderson P N (2003) Corticospinal neurons up-regulate a range o f growth-associated genes following intracortical, but not spinal, axotomy. Eur J Neurosci 18: 789-802. M c B r i d e R L , Feringa E R , Garver M K , Williams J K , Jr. (1989) Prelabeled red nucleus and sensorimotor cortex neurons o f the rat survive 10 and 20 weeks after spinal cord transection. J Neuropathol E x p Neurol 48: 568-576. M c B r i d e R L , Feringa E R , Garver M K , Williams J K , Jr. (1990) Retrograde transport o f fluorogold in corticospinal and rubrospinal neurons 10 and 20 weeks after T-9 spinal cord transection. E x p Neurol 108: 83-85. M c l l w a i n D L (1991) Nuclear and cell body size in spinal motor neurons. A d v Neurol 56: 6774. McKerracher L , David S, Jackson D L , Kottis V , Dunn R J , Braun P E (1994) Identification o f myelin-associated glycoprotein as a major myelin-derived inhibitor o f neurite growth. Neuron 13: 805-811. McKerracher L , Essagian C , Aguayo A J (1993) Marked increase in beta-tubulin m R N A expression during regeneration o f axotomized retinal ganglion cells i n adult mammals. J Neurosci 13: 5294-5300. M c P h a i l L T , M c B r i d e C B , M c G r a w J, Steeves J D , Tetzlaff W (2004) A x o t o m y abolishes N e u N expression in facial but not rubrospinal neurons. E x p Neurol 185: 182-190. Menei P, Montero-Menei C , Whittemore S R , Bunge R P , Bunge M B (1998) Schwann cells genetically modified to secrete human B D N F promote enhanced axonal regrowth across transected adult rat spinal cord. Eur J Neurosci 10: 607-621. Middlemas D S , Lindberg R A , Hunter T (1991) trkB, a neural receptor protein-tyrosine kinase: evidence for a full-length and two truncated receptors. M o l C e l l B i o l 11: 143-153. M i l l e r F D , Kaplan D R (2001) O n Trk for retrograde signaling. Neuron 32: 767-770. M i l l e r F D , Kaplan D R (2002) Neurobiology. T R K makes the retrograde. Science 295: 14711473. M i l l e r F D , Naus C C , Durand M , B l o o m F E , Milner R J (1987) Isotypes o f alpha-tubulin are differentially regulated during neuronal maturation. J C e l l B i o l 105: 3065-3073.  199  M i l l e r F D , Tetzlaff W , Bisby M A , Fawcett J W , Milner R J (1989) Rapid induction o f the major embryonic alpha-tubulin m R N A , T alpha 1, during nerve regeneration in adult rats. J Neurosci 9: 1452-1463. Morgenstern D A , Asher R A , Fawcett J W (2002) Chondroitin sulphate proteoglycans in the C N S injury response. Prog Brain Res 137: 313-332. M o r i F , Himes B T , Kowada M , Murray M , Tessler A (1997) Fetal spinal cord transplants rescue some axotomized rubrospinal neurons from retrograde cell death in adult rats. E x p Neurol 143: 45-60. M o r r o w D R , Campbell G , Lieberman A R , Anderson P N (1993) Differential regenerative growth o f C N S axons into tibial and peroneal nerve grafts in the thalamus o f adult rats. E x p Neurol 120: 60-69. M u i r G D , Whishaw IQ (2000) Red nucleus lesions impair overground locomotion in rats: a kinetic analysis. Eur J Neurosci 12: 1113-1122. M u l l e n R J , Buck C R , Smith A M (1992) N e u N , a neuronal specific nuclear protein in vertebrates. Development 116: 201-211. Murer M G , Y a n Q, Raisman-Vozari R (2001) Brain-derived neurotrophic factor in the control human brain, and i n Alzheimer's disease and Parkinson's disease. Prog Neurobiol 63: 71-124. Murray H M , Gurule M E (1979) Origin o f the rubrospinal tract o f the rat. Neurosci Lett 14: 1923. Murray M (2004) Cellular transplants: steps toward restoration o f function in spinal injured animals. Prog Brain Res 143: 133-146. N a m i k i J, K o j i m a A , Tator C H (2000) Effect o f brain-derived neurotrophic factor, nerve growth factor, and neurotrophin-3 on functional recovery and regeneration after spinal cord injury in adult rats. J Neurotrauma 17: 1219-1231. Nathan P W , Smith M C (1982) The rubrospinal and central tegmental tracts in man. Brain 105: 223-269. Neumann S, W o o l f C J (1999) Regeneration o f dorsal column fibers into and beyond the lesion site following adult spinal cord injury. Neuron 23: 83-91. Nobunaga A I , G o B K , Karunas R B (1999) Recent demographic and injury trends in people served by the Model Spinal Cord Injury Care Systems. A r c h Phys M e d Rehabil 80: 1372-1382. N o v i k o v a L , N o v i k o v L , Kellerth J O (1997) Persistent neuronal labeling by retrograde fluorescent tracers: a comparison between Fast Blue, Fluoro-Gold and various dextran conjugates. J Neurosci Methods 74: 9-15.  200  N o v i k o v a L N , N o v i k o v L N , Kellerth J O (2000) Survival effects o f B D N F and N T - 3 on axotomized rubrospinal neurons depend on the temporal pattern o f neurotrophin administration. Eur J Neurosci 12: 776-780. N o v i k o v a L N , N o v i k o v L N , Kellerth J O (2002) Differential effects o f neurotrophins on neuronal survival and axonal regeneration after spinal cord injury i n adult rats. J Comp Neurol 452: 255-263. Ochs G , Perm R D , Y o r k M , Giess R, Beck M , Tonn J, Haigh J, Malta E , Traub M , Sendtner M , Toyka K V (2000) A phase I/II trial o f recombinant methionyl human brain derived neurotrophic factor administered by intrathecal infusion to patients with amyotrophic lateral sclerosis. Amyotroph Lateral Scler Other Motor Neuron Disord 1: 201-206. Offenhauser N , M u z i o V , Biffo S (2002) B D N F binding to truncated t r k B . T l does not affect gene expression. Neuroreport 13: 1189-1193. Patapoutian A , Reichardt L F (2001) Trk receptors: mediators o f neurotrophin action. Curr Opin Neurobiol 11: 272-280. Pearse D D , Pereira F C , Marcillo A E , Bates M L , Berrocal Y A , Filbin M T , Bunge M B (2004) c A M P and Schwann cells promote axonal growth and functional recovery after spinal cord injury. Nat M e d 10: 610-616. Pearson E C , Bates D L , Prospero T D , Thomas J O (1984) Neuronal nuclei and glial nuclei from mammalian cerebral cortex. Nucleosome repeat lengths, D N A contents and H I contents. Eur J Biochem 144: 353-360. Pena E , Berciano M T , Fernandez R, Ojeda J L , Lafarga M (2001) Neuronal body size correlates with the number o f nucleoli and Cajal bodies, and with the organization o f the splicing machinery i n rat trigeminal ganglion neurons. J Comp Neurol 430: 250-263. Piehl F , Frisen J, Risling M , Hokfelt T, Cullheim S (1994) Increased trkB m R N A expression by axotomized motoneurones. Neuroreport 5: 697-700. Plunet W , K w o n B K , Tetzlaff W (2002) Promoting axonal regeneration i n the central nervous system by enhancing the cell body response to axotomy. J Neurosci Res 68: 1-6. Prinjha R , Moore S E , Vinson M , Blake S, M o r r o w R , Christie G , Michalovich D , Simmons D L , Walsh F S (2000) Inhibitor o f neurite outgrowth in humans. Nature 403: 383-384. Raineteau O, Fouad K , Noth P, Thallmair M , Schwab M E (2001) Functional switch between motor tracts in the presence o f the m A b FN-l in the adult rat. Proc Natl A c a d Sci U S A 98: 6929-6934. Ramon y Cajal S (1928) Degeneration and regeneration o f the nervous system. London: Oxford University Press.  201  Richardson P M , Issa V M (1984) Peripheral injury enhances central regeneration o f primary sensory neurones. Nature 309: 791-793. Richardson P M , Issa V M , Aguayo A J (1984) Regeneration o f long spinal axons in the rat. J Neurocytol 13: 165-182. Richardson P M , McGuinness U M , Aguayo A J (1980) Axons from C N S neurons regenerate into P N S grafts. Nature 284: 264-265. Rose C R , B l u m R, Pichler B , Lepier A , Kafitz K W , Konnerth A (2003) Truncated T r k B - T l mediates neurotrophin-evoked calcium signalling in glia cells. Nature 426: 74-78. Rosenzweig E S , M c D o n a l d J W (2004) Rodent models for treatment o f spinal cord injury: research trends and progress toward useful repair. Curr Opin Neurol 17: 121-131. Ruitenberg M J , Blits B , Dijkhuizen P A , te Beek E T , Bakker A , van Heerikhuize JJ, Pool C W , Hermens W T , Boer G J , Verhaagen J (2004) Adeno-associated viral vector-mediated gene transfer o f brain-derived neurotrophic factor reverses atrophy o f rubrospinal neurons following both acute and chronic spinal cord injury. Neurobiol D i s 15: 394-406. Sato S, Burgess S B , M c l l w a i n D L (1994) Transcription and motoneuron size. J Neurochem 63: 1609-1615. Sayer F T , Oudega M , Hagg T (2002) Neurotrophins reduce degeneration o f injured ascending sensory and corticospinal motor axons in adult rat spinal cord. E x p Neurol 175: 282-296. Schabitz W R , Berger C , Kollmar R, Seitz M , Tanay E , Kiessling M , Schwab S, Sommer C (2004) Effect o f Brain-Derived Neurotrophic Factor Treatment and Forced A r m Use on Functional Motor Recovery After Small Cortical Ischemia. Stroke. Schlessinger J, U l l r i c h A (1992) Growth factor signaling by receptor tyrosine kinases. Neuron 9: 383-391. Schmidt E E , Schibler U (1995) C e l l size regulation, a mechanism that controls cellular R N A accumulation: consequences on regulation o f the ubiquitous transcription factors O c t l and N F Y and the liver-enriched transcription factor D B P . J Cell B i o l 128: 467-483. Schnell L , Schwab M E (1990) Axonal regeneration in the rat spinal cord produced by an antibody against myelin-associated neurite growth inhibitors. Nature 343: 269-272. Schreyer D J , Skene J H (1993) Injury-associated induction o f G A P - 4 3 expression displays axon branch specificity in rat dorsal root ganglion neurons. J Neurobiol 24: 959-970. Sekhon L H , Fehlings M G (2001) Epidemiology, demographics, and pathophysiology o f acute spinal cord injury. Spine 26: S2-12. Senger D L , Campenot R B (1997) Rapid retrograde tyrosine phosphorylation o f trkA and other proteins in rat sympathetic neurons in compartmented cultures. J C e l l B i o l 138: 411-421.  202  Sharma H S , Westman J, Gordh T, A i m P (2000) Topical application o f brain derived neurotrophic factor influences upregulation o f constitutive isoform o f heme oxygenase in the spinal cord following trauma an experimental study using immunohistochemistry in the rat. A c t a Neurochir Suppl 76: 365-369. Silver J, M i l l e r J H (2004) Regeneration beyond the glial scar. Nat Rev Neurosci 5: 146-156. Skene J H (1989) Axonal growth-associated proteins. A n n u Rev Neurosci 12: 127-156. Skup M , Dwornik A , Macias M , Sulejczak D , Wiater M , Czarkowska-Bauch J (2002) Longterm locomotor training up-regulates T r k B ( F L ) receptor-like proteins, brain-derived neurotrophic factor, and neurotrophin 4 with different topographies o f expression in oligodendroglia and neurons in the spinal cord. E x p Neurol 176: 289-307. Smith D S , Skene J H (1997) A transcription-dependent switch controls competence o f adult neurons for distinct modes o f axon growth. J Neurosci 17: 646-658. Spencer T, Filbin M T (2004) A role for c A M P in regeneration o f the adult mammalian C N S . J Anat 204: 49-55. Steeves J D , Tetzlaff W (1998) Engines, accelerators, and brakes on functional spinal cord repair. A n n N Y Acad Sci 860: 412-424. Steward O, Zheng B , Tessier-Lavigne M (2003) False resurrections: distinguishing regenerated from spared axons in the injured central nervous system. J Comp Neurol 459: 1-8.Storer P D , Dolbeare D , Houle J D (2003) Treatment o f chronically injured spinal cord with neurotrophic factors stimulates betall-tubulin and G A P - 4 3 expression in rubrospinal tract neurons. J Neurosci Res 74: 502-511. Storer P D , Houle J D (2003) betall-tubulin and G A P 43 m R N A expression i n chronically injured neurons o f the red nucleus after a second spinal cord injury. E x p Neurol 183: 537-547. Stripling T (1990) The cost o f economic consequences Paraplegia News 8: 50-54.  o f traumatic spinal cord injury.  Strittmatter S M , Fankhauser C , Huang P L , Mashimo H , Fishman M C (1995) Neuronal pathfinding is abnormal in mice lacking the neuronal growth cone protein G A P - 4 3 . Cell 80: 445-452. Strominger R N , McGiffen J E , Strominger N L (1987) Morphometric and experimental studies o f the red nucleus in the albino rat. Anat Rec 219: 420-428. Takahashi M (2001) The G D N F / R E T signaling pathway and human diseases. Cytokine Growth Factor Rev 12: 361-373. ten Donkelaar H J (1988) Evolution o f the red nucleus and rubrospinal tract. Behav Brain Res 28: 9-20.  203  Tetzlaff W , Alexander S W , M i l l e r F D , Bisby M A (1991) Response o f facial and rubrospinal neurons to axotomy: changes in m R N A expression for cytoskeletal proteins and G A P - 4 3 . J Neurosci 11: 2528-2544. Tetzlaff W , Bisby M A (1990) Cytoskeletal protein synthesis and regulation o f nerve regeneration in P N S and C N S neurons o f the rat. Restorative Neurology and Neuroscience 1: 189-196. Tetzlaff W , Kobayashi N R , Giehl K M , Tsui B J , Cassar S L , Bedard A M (1994) Response o f rubrospinal and corticospinal neurons to injury and neurotrophins. Prog Brain Res 103: 271286. Theriault E , Tator C H (1994) Persistence o f rubrospinal projections following spinal cord injury in the rat. J Comp Neurol 342: 249-258. Tobias C A , Shumsky JS, Shibata M , Tuszynski M H , Fischer I, Tessler A , Murray M (2003) Delayed grafting o f B D N F and N T - 3 producing fibroblasts into the injured spinal cord stimulates sprouting, partially rescues axotomized red nucleus neurons from loss and atrophy, and provides limited regeneration. E x p Neurol 184: 97-113. Toma J G , Rogers D , Senger D L , Campenot R B , M i l l e r F D (1997) Spatial regulation o f neuronal gene expression in response to nerve growth factor. Dev B i o l 184: 1-9. Tuszynski M H (1999) Neurotrophic Factors. In: C N S Regeneration: Basic Science and Clinical Advances (Tuszynski M H , Kordower J H , eds), pp 109-158. San Diego: Academic Press. Tuszynski M H , Gabriel K , Gage F H , Suhr S, Meyer S, Rosetti A (1996) Nerve growth factor delivery by gene transfer induces differential outgrowth o f sensory, motor, and noradrenergic neurites after adult spinal cord injury. E x p Neurol 137: 157-173. Tuszynski M H , Weidner N , M c C o r m a c k M , M i l l e r I, Powell H , Conner J (1998) Grafts o f genetically modified Schwann cells to the spinal cord: survival, axon growth, and myelination. C e l l Transplant 7: 187-196. van der Zee C E , Hagg T (2002) Delayed N G F infusion fails to reverse axotomy-induced degeneration o f basal forebrain cholinergic neurons in adult p75(LNTR)-deficient mice. Neuroscience 110: 641-651. Vaudano E , Campbell G , Anderson P N , Davies A P , Woolhead C , Schreyer D J , Lieberman A R (1995) The effects o f a lesion or a peripheral nerve graft on G A P - 4 3 upregulation in the adult rat brain: an in situ hybridization and immunocytochemical study. J Neurosci 15: 3594-3611. Vercelli A , Repici M , Garbossa D , Grimaldi A (2000) Recent techniques for tracing pathways in the central nervous system o f developing and adult mammals. Brain Res B u l l 51:11-28. Verge V M , Tetzlaff W , Bisby M A , Richardson P M (1990) Influence o f nerve growth factor on neurofilament gene expression in mature primary sensory neurons. J Neurosci 10: 2018-2025.  204  von Meyenburg J, Brosamle C , Metz G A , Schwab M E (1998) Regeneration and sprouting o f chronically injured corticospinal tract fibers in adult rats promoted by N T - 3 and the m A b I N - 1 , which neutralizes myelin-associated neurite growth inhibitors. E x p Neurol 154: 583-594. Wang K C , Koprivica V , K i m J A , Sivasankaran R, Guo Y , Neve R L , He Z (2002) Oligodendrocyte-myelin glycoprotein is a Nogo receptor ligand that inhibits neurite outgrowth. Nature 417: 941-944. Wang X M , Terman JR, Martin G F (1999) Rescue o f axotomized rubrospinal neurons by brainderived neurotrophic factor ( B D N F ) in the developing opossum, Didelphis virginiana. Brain Res Dev Brain Res 118: 177-184. Watson F L , Heerssen H M , Bhattacharyya A , Klesse L , L i n M Z , Segal R A (2001) Neurotrophins use the Erk5 pathway to mediate a retrograde survival response. Nat Neurosci 4: 981-988. Watson F L , Heerssen H M , Moheban D B , L i n M Z , Sauvageot C M , Bhattacharyya A , Pomeroy S L , Segal R A (1999) Rapid nuclear responses to target-derived neurotrophins require retrograde transport o f ligand-receptor complex. J Neurosci 19: 7889-7900. Webster D M , Rogers L J , Pettigrew J D , Steeves J D (1990) Origins o f descending spinal pathways in prehensile birds: do parrots have a homologue to the corticospinal tract o f mammals? Brain Behav E v o l 36: 216-226. Webster D M , Steeves J D (1988) Origins o f brainstem-spinal projections i n the duck and goose. J Comp Neurol 273: 573-583. West M J (1999) Stereological methods for estimating the total number o f neurons and synapses: issues o f precision and bias [see comments]. Trends Neurosci 22: 51-61. West M J , Slomanka L (2001) 2-D versus 3-D cell counting—a debate. What is an optical disector? Trends Neurosci 24: 374-380. Whishaw IQ, Tomie J A , Ladowsky R L (1990) Red nucleus lesions do not affect limb preference or use, but exacerbate the effects o f motor cortex lesions on grasping in the rat. Behav Brain Res 40: 131-144. Wilkins A , Chandran S, Compston A (2001) A role for oligodendrocyte-derived IGF-1 in trophic support o f cortical neurons. G l i a 36: 48-57. Wilkins A , Majed H , Layfield R , Compston A , Chandran S (2003) Oligodendrocytes promote neuronal survival and axonal length by distinct intracellular mechanisms: a novel role for oligodendrocyte-derived glial cell line-derived neurotrophic factor. J Neurosci 23: 4967-4974. Woolhead C L , Zhang Y , Lieberman A R , Schachner M , Emson P C , Anderson P N (1998) Differential effects o f autologous peripheral nerve grafts to the corpus striatum o f adult rats on the regeneration o f axons o f striatal and nigral neurons and on the expression o f G A P - 4 3 and the cell adhesion molecules N - C A M and L l . J Comp Neurol 391: 259-273.  205  X u X M , Martin G F (1990) The response o f rubrospinal neurons to axotomy in the adult opossum, Didelphis virginiana. E x p Neurol 108: 46-54. Y a n Q, Radeke M J , Matheson C R , Talvenheimo J, Welcher A A , Feinstein S C (1997) Immunocytochemical localization o f T r k B in the central nervous system o f the adult rat. J Comp Neurol 378: 135-157. Y e H , Kuruvilla R, Zweifel L S , Ginty D D (2003) Evidence in support o f signaling endosomebased retrograde survival o f sympathetic neurons. Neuron 39: 57-68. Y e J H , Houle J D (1997) Treatment o f the chronically injured spinal cord with neurotrophic factors can promote axonal regeneration from supraspinal neurons. E x p Neurol 143: 70-81. Y i n Y , C u i Q, L i Y , Irwin N , Fischer D , Harvey A R , Benowitz L I (2003) Macrophage-derived factors stimulate optic nerve regeneration. J Neurosci 23: 2284-2293. Zhou L , Connors T, Chen D F , Murray M , Tessler A , Kambin P, Saavedra R A (1999) Red nucleus neurons o f Bcl-2 over-expressing mice are protected from cell death induced by axotomy. Neuroreport 10: 3417-3421. Zompa E A , Cain L D , Everhart A W , Moyer M P , Hulsebosch C E (1997) Transplant therapy: recovery o f function after spinal cord injury. J Neurotrauma 14: 479-506.  

Cite

Citation Scheme:

    

Usage Statistics

Country Views Downloads
China 12 12
Canada 8 4
Ukraine 8 0
United States 6 0
Czech Republic 1 0
Mexico 1 0
City Views Downloads
Shenzhen 9 12
Vancouver 7 4
Unknown 6 6
Ashburn 4 0
Odesa 3 0
Beijing 3 0
Monterrey 1 0
Kansas City 1 0
Redmond 1 0
Ancaster 1 0

{[{ mDataHeader[type] }]} {[{ month[type] }]} {[{ tData[type] }]}
Download Stats

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
http://iiif.library.ubc.ca/presentation/dsp.831.1-0092332/manifest

Comment

Related Items