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The effect of growth factors on bulbospinal neurite outgrowth in an in vitro embryonic chick model Salie, Rishard 2002

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T H E E F F E C T O F G R O W T H F A C T O R S ON BULBOSPINAL NEURITE O U T G R O W T H IN AN IN VITRO EMBRYONIC CHICK M O D E L by RISHARD SALIE B.Sc. Queen's University, 1997 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF T H E REQUIREMENTS FOR T H E D E G R E E OF M A S T E R OF SCIENCE in T H E F A C U L T Y OF G R A D U A T E STUDIES Graduate Program,in Neuroscience We accept this thesis as conforming to the renuired standard UNIVERSITY OF BRITISH COLUMBIA November, 2001 © Rishard S a l i e , 2001 UBC Special Collections - Thesis Authorisation Form Page 1 of 1 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of the requirements f o r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree that the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r reference and study. I f u r t h e r agree that permission f o r extensive copying of t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the head of my department or by h i s or her r e p r e s e n t a t i v e s . I t i s understood that copying or p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be allowed without my w r i t t e n permission. The U n i v e r s i t y of B r i t i s h Columbia Vancouver, Canada Department http://www.library.ubc.ca/spcoll/thesauth.html 11/20/01 Abstract Injury to the spinal cord of higher vertebrates damages motor axons that connect the brain and brainstem with their targets in the spinal cord. Axotomized central nervous system (CNS) neurons often experience degeneration of the distal axon, retraction of the proximal end, and atrophy of the cell body. Injured neurons in the C N S do not experience significant functional regeneration, so spinal cord insult often results in permanently compromised locomotor ability. The capability of a severed axon to re-grow is thought to depend on the interplay between intrinsic and external cues. Regenerative failure in the mature axon is thought to be the result of: a) failure to survive primary and secondary damage, b) glial scarring, c) presence of inhibitory growth substrates that prevent neuronal extension, and d) decreased availability of neurotrophic factors and permissive substrates supporting neuronal process extension. Application of trophic factors to axotomized neurons has been shown to enhance survival and neurite outgrowth. Although brainstem-spinal connections play the pivotal role in motor dysfunction, we still know relatively little about the trophic sensitivity of these populations. The experiments presented in this study will help to elucidate the role trophic molecules play in process extension in brainstem-spinal neuron populations. Using an assay specifically developed to examine the effect of trophic molecules on neuronal process extension, this study explores the response of bulbospinal populations to various trophic factors. Already investigated in our laboratory using these techniques are members of the fibroblast growth factor family, (FGF-1 , -2 , -5 and -9) . Several growth factors were initially examined for potential trophic effects on the projection neurons of the brainstem. Nerve growth factor (NGF), glial derived neurotrophic factor (GDNF) and epidermal growth factor (EGF) did not effect process outgrowth in the bulbospinal neurons of the vestibular complex (vestibulospinal neurons). Brain derived neurotrophic factor (BDNF) and insulin-like growth factor (IGF-1) significantly enhance mean process length in both the vestibulospinal neurons and projection neurons from the raphe nuclei (raphespinal neurons). Neurotrophin-3 (NT-3) and neurotrophin-4 (NT-4) require further study to determine their contribution to process elongation. In order to examine the mechanism of trophic factor effects, immunohistochemistry to the cognate receptors for BDNF and IGF-1 was performed. At the developmental stages used in the study, it was determined that receptors for BDNF and IGF-1 were present both on bulbospinal neurons and on surrounding cells with a non-neuronal morphology. It is hoped that this study will contribute to the growing pool of knowledge on spinal cord injury, and may one day play a role in development of a multi-faceted therapy for spinal cord injury. TABLE OF CONTENTS Abstract : ii Table of Contents iv List of Tables vii List of Figures viii List of Abbreviations x Chapter 1: General Introduction and Literature Review 1 1.1 Foreword 1 1.2 The Etiology of Spinal Cord Injury 2 1.3 Locomotion and the Hierarchy of Motor Control 3 1.4 Why Does the CNS Not Regenerate 4 1.4.1 Cell Survival 5 1.4.2 The Glial Scar 7 1.4.3 The Hostile CNS Environment 8 1.4.4 The Adult CNS Lacks Adequate Trophic Support 9 1.5 Human Vs. Chick Brainstem: Relevant Anatomy and Funicular Organization 10 1.5.1 The Vestibulospinal Tracts 11 1.5.1.1 The Lateral Vestibulospinal Tract 11 1.5.1.2 The Medial Vestibulospinal Tract 12 1.5.2 The Pontine Reticulospinal Tract 13 7.5.3 The Raphespinal Tract 15 7.5.4 Interaction of Descending Systems in Posture, Movement and Locomotion 16 1.6 The In Vitro Embryonic Chick Brainstem Explant Culture System.... 18 1.7 Experimental Goals and Hypotheses 23 Chapter 2: Confirmation and Characterization of the Model 24 2.1 Introduction 24 2.2 Methods and Materials 24 2.2.7 Animals 24 2.2.2 Retrograde Tracing 25 2.2.3 Dissection and Cryosectioning 25 2.2.4 Cell Counting 26 2.3 Results 27 2.3.1 Implant Site 27 2.3.2 Cell Body Labelling 27 iv 2.3.3 Nuclear Counts 35 2.4 Discussion 36 2.4.1 Implant Site 36 2.4.2 Cell Body Labelling 36 2.4.3 Nuclear Counts 37 2.5 Conclusions 38 Chapter 3: Growth Factor Screens 41 3.1 Introduction 41 3.1.1. The Neurotrophin Family 41 3 .12 Thep75NTR 42 3.7.3 The Trk Receptors 46 3.7.4 The Insulin-like Growth Factor Family 48 3.7.5 The IGF-1 Receptor 51 3.1.6 GDNF and the GDNF Receptors 54 3.7.7 EGF and the EGF Receptor 57 3.2 Methods and Materials 57 3.2.7 Explant Culture 58 3.2.2 Analysis of Explant Cultures 59 3.3 Results 60 3.3.7 The Effect of Growth Factors on Mean Neurite Length 61 3.4 Discussion. 64 3.4.1 NGF Does not Affect Vestibulospinal Outgrowth 65 3.4.2 GDNF Does not Affect Vestibulospinal Outgrowth 65 3.4.3 NT-3 Effects Require a More Thorough Examination 66 3.4.4 BDNF and NT-4 Increase Mean Neurite Length 67 3.4.5 IGF-1 Increases Mean Neurite Length 67 3.4.6 EGF Does not Affect Vestibulospinal Outgrowth 68 3.5 Conclusions 69 Chapter 4: BDNF and TrkB in Bulbospinal Neurite Outgrowth 70 4.1 Introduction 70 4.2 Methods and Materials 71 4.2.7 Immunohistochemistry.. 71 4.3 Results 72 4.3.7 The Effect of BDNF on Vestibulospinal Neurons 74 4.3.2 The Effect of BDNF on Raphespinal Neurons 79 4.3.3 TrkB Immunoreactivity 79 4.3.1.1 TrkB Immunoreactivitv in the Vestibular Complex 83 4.3.1.2 TrkB Immunoreactivity in the Raphe Nuclei 88 4.3.2 P75NTR Immunoreactivity 88 4.3.2.1 p 7 5 N T R Immunoreactivitv in the Vestibular Complex 88 4.3.2.2 p 7 5 N T R Immunoreactivitv in the Raphe Nuclei 93 v 4.4 Discussion 96 4.5 Conclusions 98 Chapter 5: IGF-1 and the IGF-1R in Bulbospinal Neurite Outgrowth 99 5.1 Introduction 99 5.2 Methods and Materials 100 5.3 Results 100 5.3.1 The Effect of IGF-1 on Vestibulospinal Neurons 103 5.3.2 The Effect of IGF-1 on Raphespinal Neurons 103 5.3.3 IGF-1 R Immunoreactivity 108 5.3.3.1 IGF-1 R Immunoreactivity in the Vestibular Complex 108 5.3.3.2 IGFR Immunoreactivity in the Raphe Nuclei 113 5.4 Discussion 118 5.5 Conclusions 119 Chapter 6: General D iscuss ion 120 6.1 Drawbacks to the Model 120 6.1.1 Use of a Developmental System to Study Spinal Cord Regeneration 120 6.1.2 Use of a Single Growth Factor 121 6.1.3 Mixed Subpopulations 123 6.1.4 Bias 123 6 .15 Fasiculation or Collateralizaton ? 123 6.1.6 Increased Survival or Enhanced Outgrowth 124 6.2 Regarding the Cell Populations Used 124 6.3 Unmasking Potential Growth Factor Effects by Minimizing Baseline Growth 125 6.4 Positive Controls 126 6.5 Variation in Neurite Outgrowth 127 6.6 Trophic Factor Therapy: A Potential Contributor to SCI Treatment.. 128 6.7 Conclusions 128 6.8 Future Experiments 130 Bibliography 131 Appendix 149 vi LIST OF T A B L E S Table 1: Quantification of Dil labeled neurons in the vestibular and raphe nuclear groups 35 Table 2: Summary table for effects of NGF, BDNF, NT-4 and G D N F on vestibulospinal neurite outgrowth 61 Table 3: Summary table for effects of NT-3, IGF-1 and E G F on vestibulospinal neurite outgrowth 64 Table 4a: Summary table for effects of titred BDNF on vestibulospinal neurite outgrowth 149 Table 4b: Summary table for effects of titred BDNF on vestibulospinal neurite outgrowth 149 Table 5a: Summary table for effects of titred BDNF on raphespinal neurite outgrowth 152 Table 5b: Summary table for effects of titred BDNF on raphespinal neurite outgrowth 152 Table 6a: Summary table for effects of titred IGF-1 on vestibulospinal neurite outgrowth 155 Table 6b: Summary table for effects of titred IGF-1 on vestibulospinal neurite outgrowth 155 Table 7a: Summary table for effects of titred IGF-1 on raphespinal neurite outgrowth 158 Table 7b: Summary table for effects of titred IGF-1 on raphespinal neurite outgrowth 158 Table 8: Summary Table for Effects of Titred E G F on vestibulospinal neurite outgrowth 161 vii LIST OF F IGURES Figure 1: Implant site location 21 Figure 2: Features of the Dil implant site 29 Figure 3: The Dil labelled brainstem 31 Figure 4: Dil labelling of brainstem nuclear populations 34 Figure 5: The effect of neurotrophins on pontine reticular neurons 40 Figure 6: The neurotrophin family 44 Figure 7: Trk signaling pathways 50 Figure 8: The insulin-like growth factor family 53 Figure 9: IGF-1 R signaling pathways 56 Figure 10: Growth factor screens on vestibulospinal neurons 63 Figure 11: BDNF increases vestibulospinal neurite length 73 Figure 12: TrkB labelling (with Hoechst staining) 76 Figure 13: Titered effects of BDNF on vestibulospinal neurons 78 Figure 14: BDNF increases raphespinal neurite length 80 Figure 15: Titered effects of BDNF on raphespinal neurons 82 Figure 16: TrkB expression in the vestibular complex 85 Figure 17: TrkB expression on vestibulospinal fibers 87 Figure 18: TrkB expression in the raphe nuclei 90 Figure 19: p 7 5 N T R expression in the vestibular complex 92 Figure 20: p 7 5 N T R expression in the raphe nuclei 95 Figure 21: Micrographs of IGF-1 treated vestibular explants 102 Figure 22: IGF-1 increases vestibulospinal neurite outgrowth 104 viii Figure 23: Titered effects of IGF-1 on vestibulospinal neurons 106 Figure 24: IGF-1 increases raphespinal neurite outgrowth 107 Figure 25: Titered effects of IGF-1 on raphespinal neurite outgrowth.... 110 Figure 26: IGF-1 R expression in the vestibular complex 112 Figure 27: IGF-1 R expression on vestibulospinal fibers 115 Figure 28: IGF-1 R expression in the raphe nuclei 117 Figure 29: BDNF increases vestibulospinal neurite outgrowth 150 Figure 30: BDNF increases raphespinal neurite outgrowth 153 Figure 31: IGF-1 increases vestibulospinal neurite outgrowth 156 Figure 32: IGF-1 increases raphespinal neurite outgrowth 159 ix LIST OF ABBREVIATIONS 5-HT Serotonin BDNF Brain Derived Neurotrophic factor BSA Bovine Serum Albumin CNS Central Nervous System CNTF Ciliary Neurotrophic Factor CPG Central Pattern Generator DAG Diacylglycerol Dil 1,1' dioctadecyl-3,3,3',3' tetramethylindocarbocyanine perchlorate DNA Deoxyribonucleic Acid DRG Dorsal Root Ganglion ECM Extracellular Matrix ER Endoplasmic Reticulum FCS Fetal Calf Serum FGF-2 Fibroblast Growth Factor-2 GEF Guanine Exchange Factor GPI Glycophosphatidylinositol HBSS Hank's Buffered Saline Solution HRP Horse Radish Peroxidase IGF-1 Insulin Like Growth Factor-1 IGF-1 R IGF-1 Receptor IP3 4,5-triphosphate IRS Insulin Receptor Substrate M-6-P Mannose 6-Phosphate MAG Myelin Associated Glycoprotein NDS Normal Donkey Serum NGF Nerve Growth Factor NT-3 Neurotrophin 3 NT-4/5 Neurotrophin 4/5 p 7 5 N T R p75 Neurotrophin Receptor PBS Phosphate Buffered Saline PI-3K Phosphatidylinositol-3 Kinase PIP-2 4,5-bisphospate PLC Phospholipase C PNS Peripheral Nervous System RAGs Regeneration Associated Genes Rgc Medullary Reticular Gigantocellular RPC Caudal Pontine Reticular Rpc Medullary Reticular Parvocellular RPgc Pontine Reticular Gigantocellular RPpc Pontine Reticular Parvocellular RST Reticulospinal Tract RTK Receptor Tyrosine Kinase TNF Tumor Necrosis Factor Trk Tropomyosin Receptor Kinase VeD Decendens Vestibular Nucleus VeL Lateral Vestibular Nucleus VeM Medial Vestibular Nucleus x Chapter 1: General Introduction and Literature Review 1.1 Foreword Tissues in the human body are often capable of significant repair when damaged. Cells are replaced or repaired, and often the function of the individual is unimpaired. Tragically, neurons of the central nervous system (CNS) lack the capacity to regenerate under typical physiological conditions. Upon injury to the brain or spinal cord, damaged neurons upregulate genes associated with process extension. Unfortunately, this response is transient, and is followed by degeneration of the distal axon, retraction of the proximal axon, and atrophy of the cell body. This lack of regeneration is likely due to a combination of factors, and is reflected by drastic alteration in behavior, as the sensorimotor information flow is disrupted. In developing and some neonatal organisms, regenerative phenomenon is observed upon injury to the C N S . This has been attributed to a combination of intrinsic and environmental factors. Studies of neurodevelopment provide important clues to aid in the re-creation of an environment that is supportive of axon growth. The vast body of literature in this field, and the research it represents, reflects the extensive gains in knowledge made in the area of spinal cord injury. The first chapter attempts to concisely review the literature as it pertains to the experiments presented in this work. Topics discussed include: the role of the brainstem in motor control, the etiology of SCI, potential causes of C N S regeneration failure as well as strategies to promote regeneration, and the growth factors used in this study as well as their cognate receptors. A section comparing and contrasting brainstem anatomy and funicular 1 trajectory between humans and chickens (the experimental model used in this study) is also included. 1.2 The Etiology of Spinal Cord Injury Spinal cord injuries are tragedies that have dramatic implications for the entire life of the sufferer. Injuries are believed to occur at an annual rate of 20-40/million people in first world countries (Kraus et al., 1975; Shingu et al., 1995), and to date have seen little success in treatment, save from a preventative approach. It has long been thought that the vertebrate C N S was completely incapable of regeneration following injury (Cajal, 1928; Feig inetal . , 1951; Windle, 1956; Bjorklund et al., 1971). This observation is counterintuitive, as the vertebrate peripheral nervous system is capable of reinnervating target tissues unassisted following axonal injury (Cajal, 1928). The boom of recent research in this field has shown that this initial assumption, that C N S axons have no growth potential, is in fact incorrect. Under experimental conditions, axonal regrowth has been observed in numerous cases (Hasan et al., 1993; Asada et al., 1998; Ramon-Cueto et al., 2000). Neurons exhibit signs of terminal sprouting upon injury (Bjorklund et al., 1971), however this outgrowth is transient. Following this abortive sprouting, axotomized C N S neurons undergo degeneration of the distal axon, retraction of the proximal axon, and atrophy of the soma (Fishman and Mattu, 1993; Kobayashi et al., 1997). To further appreciate the impact of damage to the spinal cord it is important that we have at least a rudimentary understanding of brain and brainstem functions relative to locomotion. 2 1.3 Locomotion and the Hierarchy of Motor Control Locomotion is a complex process that generally requires supraspinal input for both its initiation, and sustained motion (Kuypers and Lawrence, 1967; Jordan, 1991). In humans, bipedal motion requires a delicate interaction of various sensory and motor components. Cortex, brainstem, cerebellum and spinal cord all contribute to the initiation and co-ordination of locomotion. Cortical projections synapse on brainstem nuclei (corticobulbar), as well as directly onto the lower motor neurons of the spinal cord (corticospinal). In many animals the corticospinal tract plays a major role in locomotive behavior. In humans the major function of the corticospinal tract is fine control of the muscles of the fingers and wrists (Heffner and Masterton, 1983). Brainstem projections to the spinal cord (bulbospinal) also play an important role in locomotion (Barnes, 1984). In many animals, the brainstem and spinal cord have a reasonable degree of independence in coordinating motion. The posture of decerebate cat seems fairly normal, under non-stressful conditions (Sherrington, 1898; Grillner, 1973; Grillner, 1985), and spinalized cats will exhibit stepping behavior when placed on a treadmill with their weight supported (Brown, 1911). Chickens continue to display stepping behavior long after they have been decapitated. These examples serve to illustrate the hierarchy involved in motor control. If the flow of motor information between supraspinal neurons and their targets in the spinal cord is disrupted, it is obvious that motor control of the organism will suffer. This raises an obvious question: Why does the C N S not regenerate? 3 1.4 Why Does the CNS Not Regenerate? While C N S regeneration is not observed in mammals or even in the majority of adult vertebrates, it is not foreign to all organisms. Salamanders are capable of naturally recovering locomotor function after spinal cord transection in the adult organism (Butlerand Ward, 1967; Simpson, 1983; Davis etal . , 1990). Although function is restored after transection, histological observations show a markedly smaller cord with few neurons and reduced white matter (Butler and Ward, 1967; Stensaas, 1983). Interestingly, the regenerative environment of the salamander is very similar to environments encountered during the embryonic development of higher vertebrates. The protein vimentin, generally found on immature glial cells, is also expressed by radial glia in the adult newt. The C N S myelin of the urodeles lacks neurite growth inhibitors found in mammals (Becker et al., 1999). Transected neurons in urodeles have been observed to regenerate at least 10mm (Davis et al., 1989). Regeneration in an embryonic environment has also been shown in higher organisms. Chick spinal cord transected prior to E13 shows significant functional regeneration (Keirstead et al., 1992; Hasan et al., 1993). Injury to the immature C N S of the marsupial opossum (Monodelphis domestica) does not result in permanent loss of locomotor function. In fact, complete crush as well as full transection injuries to the immature opossum spinal cord as late as postnatal day 7 results in excellent functional recovery (Saunders et al., 1998). Retrograde tracing post-injury determined that fibers projecting from brainstem neurons had made connections to the lumbar cord (Saunders et al., 1998). 4 The lack of C N S regeneration in adult vertebrates is likely due, in part, to changes in the C N S environment as the organism matures. There are several factors that may contribute to this regenerative failure. After the primary injury to the C N S , secondary cell death via necrosis and/or apoptosis may add to the damage. Formation of a glial scar at the injury site is also thought to impede C N S regeneration. Myelin and associated molecules have also been shown to be inhibitory to axonal regeneration. In addition to this, it is believed that the adult C N S lacks appropriate trophic support both to prevent neuronal atrophy and cell death, and to promote axon growth. The next section will provide a more detailed account of each of these factors, as well as briefly outlining potential strategies to overcome these barriers. 1.4.1 Cell Survival It is obvious that if a cell does not survive the trauma of the initial injury (primary cell death) it will be unable to undergo any type of regeneration. Cells that have not been killed by the initial lesion are also at risk from changes in the cellular environment, which may induce secondary cell death (reviewed in Tator and Fehlings, 1991). Several factors are believed to play a role in secondary damage. Firstly, there is hemorrhage at the site of the lesion (Dohrmann and Wick, 1971). This is accompanied by invasion of macrophages and other lymphatic/blood borne cells (Blight, 1985; Blight, 1992). Secondly, damage to the vasculature supplying the region results in hypoxia and ischemia (Dohrmann and Wick, 1971; Stys, 1998), while ionic gradients, which allow for the electrophysiological properties of the nervous system, are also disrupted. Oxygen radicals, formed by the release of previously intracellular iron stores (Liu et al.,1999), -» 5 and hydrolytic enzymes (including proteases) attack the cell (Iwasaki et al., 1987). Finally, depending on the extent of the damage done to the axon, the mechanisms for fast and slow axonal transport may be disrupted (Sahenk and Mendell, 1980; Kristensson and Olsson, 1975). This may deprive the cell body of essential survival related molecules from the target tissue. The phenomenon of secondary cell death is characterized by a continuum of varied characteristics. At one end is necrotic cell death (reviewed in Martin, 2001) resulting in cellular lysis further poisoning the cellular milieu with ions, proteins and debris. The other end is apoptotic cell death, a form of cellular suicide which is characterized by ejection of mitochondrial cytochrome C, cleavage of the DNA into fragments, condensation of the cell into small bundles and a lack of intracellular release into the E C M . Apoptotic cell death is mediated by several families of proteins. Among these are the caspases (cysteine-dependant aspartate specific protease), proteases thought to be related to the ced-3 family in C. Elegans (reviewed in Eldadah and Faden, 2000; Thornberry and Lazebnik,1998). There are 14 members of this family known to date. These can generally be classified into three major sub-groups. Initiator caspases act along the cell surface and within the mitochondria transducing signals to trigger apoptosis. Executioner caspases play a role in effecting the final demise of a cell going through apoptosis. Membrane 'blebbing', chromosomal condensation and DNA fragmentation are all in part mediated by the executioner caspases. Inflammatory mediator caspases bear homology to caspase-1, which is active in converting pro-interleukin-1 p into its cleaved form. Other factors than the caspase proteins mediate apoptotic activity. Proteins such as the Bcl-2 family are also known to inhibit apoptotic 6 death by repressing the activity of specific caspase proteins (Clem et al., 1998). There are two major pathways that are currently believed to mediate caspase induced cell death. Death receptors such as the Fas receptor and the TNFct receptor transduce extracellular death signals (Raoul et al., 1999; Casha et al., 2001). Mitochondrial cytochrome c ejection (Kluck et al., 1997) also has the ability to trigger apoptosis. A third pathway which appears to originate in the ER can also trigger cell death (Beresford et al., 2001). It is thought that manipulation of the pathways involved in secondary cell death will help ensure the survival of neurons after CNS injury. 1.4.2 The Glial Scar Upon damage to the CNS there is formation of a glial scar (reviewed in Fawcett and Asher, 1999). This scar is a dynamic environment with varied cellular and molecular phenomenon occurring at various time points. Invading macrophages and microglia from the blood and surrounding tissue are first to arrive at the lesion site, mere hours after the damage is incurred (Kreutzberg, 1996). This initial reaction is followed by recruitment of oligodendrocyte progenitors between 3 and 5 days (Levine and Nishiyama, 1996; Keirstead et al., 1998). Astrocytes also migrate to the area and proliferate, filling in the void left by the damage (Dusart et al., 1991). In studies where CNS regeneration is observed, fibers are generally unable to cross the barrier created by scarring (Bahr et al., 1995; Davies et al., 1996; Davies et al., 1997) without assistance (Kawaja and Gage, 1991). The mechanism by which the glial scar inhibits axon regeneration has not yet been determined, however, there are several theories. The first is that the E C M generated by 7 glial cells in the scar lack either the surface molecules required for contact mediated growth, or the trophic support molecules necessary to sustain growth (Fawcett and Asher, 1999). The second hypothesis is that the glial scar acts as a mechanical barrier to axonal elongation. The majority of the scar environment consists of a meshwork of glial processes that are linked by junctions (Eng et al., 1987; Reier and Houle, 1988). It has been suggested that the growth cones cannot pass through this tight network of processes. Finally, it has been theorized that that the glial scar is not directly inhibitory to axonal regeneration, but is a rich source of trophic support. The axons grow into the scar, but do not exit. It seems likely that the inhibitory effect of the glial scar is not completely due to a single condition, but some combination of the factors mentioned. 1.4.3 The Hosti le C N S Environment Neurons that attempt to regenerate through the C N S face the obstacle of a hostile growth environment. Many studies indicate that certain molecules in the C N S actively inhibit the regrowth of damaged axons. Neurons in culture do not penetrate into explants of optic nerve even with appropriate trophic support (Schwab and Thoenen, 1985). Several inhibitors of neurite outgrowth have been identified including Nogo-A (Chen et al., 2000), MAG (Tang et al., 1997), and members of the proteoglycan family (Wilson and Snow, 2000; Asher et al., 2000). Various approaches have been taken to overcome this inhibition. An antibody to the Nogo-A protein, IN-1, has been shown to enhance neurite growth into C N S tissue such as optic nerve (Chen et al., 2000). Development of a promising immunological method to disrupt C N S myelin, has been observed to facilitate brainstem spinal 8 regeneration, with functional synaptic connections (Keirstead et al., 1995). Glycoprotein synthesis inhibitors, p-xylosides, disrupt the presentation of at least two proteoglycans, brevican and versican in the E C M resulting in axon growth cones extending along previously inhibitory oligodendrocytes (Niederost et al., 1999). 1.4.4 The Adult C N S Lacks Adequate Trophic Support The necessity for appropriate trophic support during embryonic neurodevelopment is well known. Neurotrophins (NGF, BDNF, NT-3, NT-4/5) and their cognate receptors (TrkA, TrkB, TrkC, p75 N T R ) have been found to play a role in coordinating neural tube formation (Jungbluth et al., 1997), as well as in the differentiation of avian neural tube progenitor cells (Averbuch-Heller et al., 1994). They have also been found to impact on neuronal survival, reducing cell death (Piontek et al., 1999), and atrophy (Kobayashi et al., 1997) as well as increasing neurite outgrowth (Ebadi et al., 1997; Davies, 2000; Ye and Houle, 1997). Several other factors such as IGF-1, CNTF, and FGF-2 have also been found to promote axonal regeneration in various neuronal populations (Hansson et al., 1986; Nachemson et al., 1990; Houle and Ye, 1997; Pataky et al., 2000). The expression of these trophic factors is highly regulated both spatially and temporally. The receptors for these molecules are also selectively expressed. It is for these reasons that different neuronal populations may have varied requirements of neurotrophic factors to encourage an optimal growth response. It is also uncertain whether effects of trophic molecules are due to an autocrine (acting directly on the projecting neuron) or a 9 paracrine effect (acting on an intermediate cell which in turn influences the projection neuron). Application of these trophic molecules to injured neurons has been shown in some cases to increase the expression of regeneration associated genes (RAGs) such as GAP-43 , select tubulin isoforms (Kobayashi et al., 1997) as well as c-jun (Broude et al., 1999). These intrinsic growth molecules are observed to show increased expression shortly after injury, but are quickly down-regulated (Vaudano et al., 1995; Schmitt et al., 1999). It is possible that administration of trophic factors increases the amount or the duration of R A G expression via intracellular signaling cascades. This study tests the effect of various trophic molecules on descending chick brainstem populations. The next section compares and contrasts human and chick brainstem anatomy and organization. 1.5 Human V s . Ch ick Bra instem: Relevant Anatomy and Funicular Organizat ion The differences in body structure, joint organization, and stance in various animals require some differences in locomotor pathways. Bipedal vertebrates make use of different postures and gaits than quadrupeds, due to the higher center of gravity and the reduction in the number of supports on the ground when walking (Dickinson et al., 2000). The purpose of this section is to compare and contrast the locomotor mechanisms of two bipeds, the human and the chicken (Gallus domesticus). Discussed are the anatomy, funicular trajectory and functionality of vestibular, pontine reticular and raphe neurons, the neuronal populations used in these experiments as 10 well as how these descending pathways interact to influence posture, movement and locomotion. Vestibular and pontine reticular projection neurons belong to the medial system, which controls posture and axial muscles. These neurons have also been associated with locomotor function. Raphespinal neurons, well known for production of serotonin as a neurotransmitter, belong to the monoaminergic system, which generally facilitates motor neuron activation in the spinal cord. 1.5.1 The Vest ibulospinal Tracts Vestibulospinal fibers originate in the vestibular nuclei located in the pons and medulla. The vestibular complex is divided into at least two major tracts that project via the spinal cord (lateral, medial). The lateral vestibulospinal tract serves to facilitate extensor and inhibit flexor motor neurons, via interneurons. The medial vestibulospinal tract terminates directly on medial motor neurons of the neck and back but also projects to interneurons. 1.5.1.1 The Lateral Vestibulospinal Tract This projection originates in the lateral vestibular nucleus from giant cells as well as other cell types (Kneisley et al., 1978), leading to a combination of thick and thin fibers in the tract. In humans the fibers descend ipsilaterally as the anterior portion of the lateral funiculus and project down to the lumbrosacral level. Somatotopic organization is also a feature of the vestibulospinal tract as shown by degeneration studies (Pompeiano and Brodal, 1957). The ventrorostral portion of the 11 lateral vestibulospinal nucleus sends its axons to the cervical cord. The dorsocaudal segment innervates the lumbrosacral spinal cord, and thoracic fibers receive efferents from the intermediate portion of the nucleus. Fibers travelling to the cervical segments of the spinal cord are found at the ventral portion of the lateral funiculus while those to the lumbrosacral cord are in the ventral funiculus. Termination of the lateral vestibulospinal tract is at lamina Vl l (medial and central areas) and lamina VIII (Nyberg-Hansen and Mascitti, 1964). Few of these terminals project directly to motor neurons, with those that do being found in the thoracic cord. It is thought that the lateral vestibular nucleus facilitates reflexes of the spinal cord, as well as the mechanisms that control muscular tension. Stimulation of the lateral vestibular nucleus enhances the activity of extensors by increasing muscle tone in animals at rest and increased extensor activity during the stance phase of stepping (Orlovsky, 1972). Avian lateral vestibulospinal pathways have also been delineated (Wold, 1978; Cabot et al., 1982) and are similar to that of mammals in general. Characterized by ipsilateral, somatotopically organized descent in the ventral funiculus, the chicken lateral vestibulospinal tract descends from the lateral vestibular nucleus. Termination also occurs in lamina Vl l and VIII in the chick (Correia et al., 1983). 1.5.1.2 The Medial Vestibulospinal Tract The human medial vestibulospinal tract originates from the medial vestibular nucleus (Nyberg-Hansen and Mascitti, 1964). Fibers in the medial vestibulospinal tract consist of relatively few, bilateral fibers that descend via the medial longitudinal fasiculus adjacent to the ventral median fissure. Medial vestibulospinal fibers terminate 12 at mid-thoracic levels. This indicates the tracts activity in relaying vestibular information to the neck and upper extremities. Even though the medial vestibulospinal tract terminates much earlier than the lateral vestibulospinal tract, the laminar pattern of termination is still layers Vl l and VIII, and in addition makes connections to the motor neurons of the neck and back (Nyberg-Hansen and Mascitti, 1964). In the chicken, the medial descending nucleus is basically identical to that of the human, with the same origin, descending position and lamina of termination (Wold, 1978; Cabot et al., 1982; Correia et al., 1983). Avian medial vestibulospinal projections terminate before the upper thoracic cord. 1.5.2 The Pontine Ret iculospinal Tract Two major areas of the reticular formation send descending projections to the spinal cord. The pontine reticulospinal tract originates in the pontine tegmentum and the medullary reticulospinal tract begins in the medulla. The reticulospinal projections maintain postural tone in collaboration with the vestibulospinal tracts (Maghoun and Rhines, 1946). The reticulospinal tracts utilize a variety of transmitters such as glutamate, substance P and enkephalin (Haines, 2000). The latter two transmitters reflect the reticulospinal systems potential to modify sensory input (especially pain) at the level of the spine. Only the pontine reticulospinal tract will be considered as medullary reticulospinal cultures were not attempted. Cells in the caudal pontine reticular nucleus (RPC) and in the oral pontine reticular nucleus (RPO) both contribute to the pontine reticular tract (Brodal, 1957). The R P C is located in the area between the caudal pontine tegmentum and the level of the 13 motor trigeminal nucleus. The R P O is located in a more rostral section of the pontine tegmentum and reaches to the caudal mesencephalon. The fibers from the R P C extend from all regions of the nucleus, whereas R P O fibers originate only from the caudal portion of the R P O nucleus. This caudal area of the R P O contains giant cells, more than half of which send projections to the spinal cord (Torvik and Brodal, 1957). The pontine reticulospinal tract is predominantly ipsilateral and descends in the medial part of the ventral funiculus close to the medial longitudinal faciculus. Fibers travel the full length of the spinal cord and synapse on interneurons in lamina VII and Vll l (Nyberg-Hansen, 1966). The few fibers that do cross do so at the level of the spine in the anterior white commissure. The pontine reticulospinal tract is not somatotopically organized. The interneurons project to the alpha and gamma motor neurons of the ventral horn (Petras, 1967). Gamma motor neurons innervate intrafusal muscle fibers, which are responsible for controlling muscle tone. The pontine reticulospinal tract influences gamma motor neurons supplying extensor muscles. The pontine RST is important in maintaining contraction of the axial muscles that keep the spine erect as well as the lower limb extensors (Gahery and Massion, 1981). In the chicken the pontine reticular formation is also ipsilateral from the caudal and magnocellular regions of the reticular formation (Wold, 1978; Cabot et al., 1982). It also receives fibers from the medial two thirds of the pontine reticular formation caudal to the isthmus. The tract descends along the medial edge of the ventral funiculus. It is thought that this is the equivalent of the oral pontine contribution in humans. 14 1.5.3 The Raphespinal Tract In humans, as well as other mammals there are eight raphe nuclei, the raphe obscurus, raphe pallidus, raphe magnus, raphe pontis, raphe dorsalis, centralis superior, linearis intermedius, and linearis rostralis. These nuclei begin at the caudal end of the medulla and extend to the rostral mesencephalon running along the midline of the brainstem. The nuclei encapsulate the majority of serotonergic (5-HT) neurons in the brainstem (Dahlstrom and Fuxe, 1965). It is important to note that not all the raphe nuclei have serotonergic spinal cord projections, and not all projections to the cord from the raphe nuclei are serotonergic. 5-HT acts to increase the excitability of motor neurons they project to, likely to the 5HT-1 b receptor subtype. Fibers project to lamina I, II and V of the dorsal horn, where they modify cutaneous, nociceptive information (Wolstencroft, 1980). The nucleus raphe magnus projects axons which descend through the dorsolateral funiculus (Leichnetz et al., 1978; Martin et al., 1978; Basbaum and Fields, 1979). The raphe pallidus and raphe obscurus nuclei project to the cord via the ventral and ventrolateral funiculi. The projections of the raphe magnus nucleus bilaterally innervate the dorsal horn, while that of the raphe pallidus and obscurus innervate the ventral and intermediate grey as well as the intermediolateral grey (Loewy, 1981; Loewy and McKellar, 1981). Fibers from the raphe magnus project to cervical and lumbar regions (Hayes and Rustioni, 1981) and it has been shown that a single serotonergic raphe neuron can extend collaterals to both lumbar and cervical levels (Martin et al., 1982). Raphespinal activity modifies flexor responses to nociceptive cutaneous inputs, 15 as well as the ascending nociceptive information itself. This allows raphespinal neurons to switch off flexor responses of such cutaneous inputs (Wolstencroft, 1980). In the chicken, there is also a raphespinal tract, analogous to that found in humans. The tract originates from the same nuclei, the raphe magnus, obscurus and pallidus. Most of the neurons in these avian nuclei project to the spinal cord, and are also serotonergic. In the chicken, the projections from the raphe magnus nucleus are both ipsilateral and bilateral (Cabot et al., 1982), while those of the raphe pallidus and obscurus are mainly bilateral. HRP studies clearly show the projections of the raphe magnus descending in the dorsolateral funiculus as do projections from the rostral portion of raphe pallidus (Cabot etal . , 1982). The remainder of the raphe pallidus nucleus and that of the raphe obscurus project in the ventrolateral white matter (Cabot et al., 1982). The projections of the raphe magnus nucleus go to lamina I and II (the dorsal horn) of the spinal grey matter, as well as lamina V, VI and the motor neurons of lamina IX. Raphe serotonergic neurons have been shown to inhibit sympathetic preganglionic neurons (lamina Vll) (Cabot et al., 1979). There is no somatotopy found in the raphespinal system in either humans or chickens. 1.5.4 Interaction of Descending Systems in Posture, Movement and Locomot ion In the previous sections, each of the tracts was discussed independently. This was solely for the purpose of organization, and entirely contrary to the how the systems function. At all times other than when we are reclined, we must maintain a basic level of postural control to keep ourselves upright (Johansson and Magnusson,. 1991). Although this may sound like a simple task, a closer look shows it to be quite complex. 16 The vestibular otoliths and semi-circular canals determine the position of our head in space and make connections to the vestibular nuclei. This information allows us not only conscious perception of our head position, but also signals down the vestibulospinal tract to act on the muscles of the body, head, and neck to maintain balance, as well as mediating the vestibular reflexes. A tilt of the head is detected by the vestibular system, and the vestibulospinal reflexes cause activation of muscle groups that oppose motion to correct forthe tilt (Suzuki and Cohen, 1964). The anatomy of the tracts becomes significant when observing the function. The bilaterally projecting medial vestibulospinal tract does not reach past cervical levels and is responsible for the vestibulocollic reflex (that opposes angular head rotation), while the ipsilateral lateral vestibulospinal tract reorients the torso and limbs to maintain upright body posture. Similarly, the other medial tracts serve to act on known information about body position. The reticulospinal nuclei are major contributors to locomotor activity (Matsuyama and Drew, 2000). Together, with information from the propriospinal neurons, these systems work continuously in concert to keep us upright. Although some of the systems may seem redundant, even simple tests can show us this is not the case. Closing ones eyes increases postural sway when standing upright, and disruption of the fluid in the semi-circular canals by repetitive spinning will result in dizziness. Noxious somatosensory information (i.e. stepping on a nail) results in activation of spinal reflex of withdrawal of the injured limb and extension of the opposing one, but also activates the vestibulospinal reflexes to adjust posture and keep balance during this shift of weight. 17 Locomotion combines the activities of these and other nuclear groups. Constant shifts in posture to move require vast numbers of corrections in balance. Interestingly these adjustments, as well as the complicated task of placing one limb in front of the other are performed automatically. Initiation and cessation of locomotive tasks requires input, but central pattern generators (CPG's) mediate the actual motion. These C P G ' s are found at the level of the spinal cord and the brainstem, and will generate motion independently, even when severed from the remainder of the nervous system (For review see Pinter and Dimitrijevic, 1999). The monoamine tracts generally provide facilitation of motor neuron activation in motion, but also make strong connections to the autonomic nervous system. The somatic origins of the monoamine tracts receive projections from several areas of the limbic system. It is postulated that these systems serve to moderate motivational drive of movement and take part in the fight or flight response, affecting the autonomic systems and facilitating muscle contraction for an attack or an escape situation. 1.6 The In Vitro Embryonic Chick Brainstem Explant Culture System Bulbospinal neurons are inherently difficult to study, due to a lack of specific molecular markers. Raphespinal neurons are primarily serotonergic, however not all neurons from the various raphe nuclei project to the spinal cord, making identification of the neurons by staining for serotonin ambiguous. The other neuronal groups of interest (vestibular, reticular) are presently indistinguishable from other neurons in the brainstem. To allow study of these projection neurons, a novel assay was devised in our laboratory (Pataky et al., 2000). This technique takes advantage of the 18 developmental phenomenon of bulbospinal neurons projecting axons to various levels of the spinal cord to label projecting cells. By embryonic day 3 (E3) neurogenesis of projection neurons in the brainstem is for the most part complete (McConnell and Sechrist, 1980). Innervation of the cervical enlargement is first seen at approximately E5 for pontine reticulospinal, raphespinal, and lateral vestibulospinal nuclear groups (Okado and Oppenheim, 1985). Medial and inferior (decendens) vestibular projections are observed entering 2-3 days later (Glover and Petursdottir, 1991). A crystal of the fluorescent, lipophilic tracer, 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (Dil), is placed just ventral to the cervical spinal cord of the developing chick on embryonic day 5 (Figure 1A). As the growth cones of the invading axons pass the crystal, Dil is incorporated into vesicles and retrogradely transported back to the cell body where it is incorporated into membranes of organelles as well as the plasma membrane. Dil, which fluoresces brightly under green light (549 nm), has been reported to be non-toxic to living cells (Honig and Hume, 1986; Honig and Hume, 1989). The peak of the emission spectra is 564nm and through a rhodamine filter, Dil fluoresces bright red. At E8 the chick brainstems are harvested and micro-dissected to obtain the neuronal groups of interest. These explants are tissue cultured under various treatment conditions. Dil diffuses within the cell membrane to label new projections (Figure 1B). Since these projections cannot be accurately described as either dendrites or axons, they are referred to as "neurites". These neurites are easily observed under an epifluorescent microscope and are photographed for analysis (Figure 1C). The analysis 19 Figure 1: (A) Implant site of the Dil crystal in the embryonic chick, (modified from Pataky et al., 2000). (B) Photomontage of a vestibular explant. Dil labelled neurites are easily visible. (C) Tracing of photomontage in (B) for measurement of neurite length. SC: spinal cord. 20 21 involves calculating the mean neurite length for the projections from the explant. An increase in mean neurite length may be indicative of ameliorated trophic conditions. The embryonic chick model was chosen for several reasons. The most obvious is the embryonic in ovo accessibility of an embryonic system. Intrauterine surgery seems a daunting task, and would likely result in very poor survival rates. Conversely, it is relatively easy to open a live chicken egg, implant a crystal of tracer and reseal it. Survival rate is approximately 85-90% after three days. The neuroanatomy and funicular trajectories of the chick brainstem have been well studied and documented, and as previously discussed its bulbospinal projections are very similar to that of other vertebrates including man (Glover and Petursdottir, 1988). An additional advantage that the chick model has over rodent models is that the chick is bipedal, constantly walking on its hindquarters as opposed to quadrupeds. Lastly, the chick model is inexpensive and requires little space or maintenance. The near identity of the bulbospinal tracts, in ovo access and biped locomotion make embryonic chickens an ideal model animal in which to explore the regeneration of brainstem spinal neurons. 22 1.7 Experimental Goals and Hypotheses The purpose of this project is to identify trophic factors that encourage neurite outgrowth of chick bulbospinal neurons. From the literature reviewed in this chapter, hypotheses can be drawn, then tested in the in vitro embryonic chick brainstem explant culture system. Two major hypotheses will be investigated in this study: Hypotheses: 1. Neurite outgrowth in bulbospinal neurons will be enhanced by appropriate trophic support. 2. Neurons and/or surrounding non-neuronal cells will express receptors for trophic molecules that enhance neurite outgrowth. The experiments performed in this study revolve around confirming the correct utilization of this system, and in determining if these hypotheses are valid. 23 Chapter 2: Confirmation and Characterization of the Model 2.1 Introduction Retrograde labelling of chick bulbospinal neurons involves the implant of a crystal of Dil, which must contact, but not damage the spinal cord of the developing chick. This requires a high degree of precision that can only be attained with much practice. In order to ensure that a sufficient skill level had been attained to successfully proceed with culture experiments, I felt it was important to perform experiments confirming my acquisition of the techniques used in retrograde labelling with Dil. These experiments include visual confirmation that implantation of the Dil crystal was performed correctly, and that neuronal groups were properly labeled. Counts of labeled cell bodies in the two successfully cultured neuronal populations (vestibular, raphe) will be presented and compared to counts in article that initially described the model system (Pataky et al., 2000). 2.2 Methods and Materials 2.2.1 Animals Fertilized White Leghorn Chicken eggs (Gallus domesticus) were purchased from University of Alberta, Poultry Research Facility and stored refrigerated (12°C) for up to two weeks until incubation in a forced-draft incubator (38°C, saturating humidity). All animals were staged according to the method of Hamburger and Hamilton (Hamburger and Hamilton, 1951). 24 2.2.2 Retrograde Tracing Crystals of the lipophillic carbocyanine dye, Dil (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate, (Molecular Probes, Oregon)) were suspended in 95% ethanol and partially dried to a paste. The tip of an insect pin was coated with Dil paste and allowed to air dry. On embryonic day 4.5 (E4.5) of their development (stage i 25-26), a circular hole was cut in the top of the egg above the air sac and the shell piece kept. This hole in the shell provided access to the embryo inside. A small hole in the air sac membrane was made with watchmakers' tweezers (5/45, A. Dumont and Sons, Switzerland) above the embryo, with care not to disrupt the vitelline or the allantoic membranes. The amniotic membrane was peeled away from the dorsal side of the embryo, between the cervical and thoracic levels. The pin was inserted at mid-cervical level between the spinal cord and developing vertebrae, making contact with the ventral edge of the cord. The crystal was held in place with the watchmakers' tweezers and the pin removed. The circular piece of shell was replaced and secured in place using melted paraffin wax. The eggs were returned to the incubator until E8. 2.2.3 Dissection and Cryosectioning In a sterile field, the embryos (E8) were removed from the eggs into Hanks balanced saline solution (HBSS, 4°C, 0.5% glucose, Mg 2 + and C a 2 + f r e e , Gibco-BRL). The embryos were decapitated, and the brainstem dissected out using fine forceps. The meningial layer and associated vasculature were carefully removed from the brainstems. The brainstems were immersion fixed in 2-4% paraformaldehyde (BDH Chemicals, Toronto) in 0.1 M P B S between 4 hours and overnight, briefly rinsed in chick 25 P B S . The brainstems were cryoprotected in 30% sucrose (EM Science) till they no longer floated. The tissue was then fixed in OCT compound and frozen over liquid nitrogen. Tissue was sectioned on a Zeiss Microm cryostat at -20°C into slices between 10-20um, and mounted on warmed (50°C) Superfrost Plus slides (Fisher Scientific). For implant site work, 2 cm of neck (1 cm on either side of the implant site) was removed, fixed, cryoprotected and sliced as above. Sections were photographed using a Zeiss epifluorescence microscope attached to a S P O T digital camera (Diagnostic Instruments Inc.). Images were processed using S P O T software. 2.2.4 Cel l Count ing Cell counts were performed using the equation: P=[(A)(m)]/(L+m) Where P is the average number of nuclear points per section, A is the crude count number on a slide, m is the thickness of the section (um) and L is the average length of the cell body (Abercrombie, 1946). Cell body size was taken as (30um), and 10um sections were taken every third section. Percent discrepancy is defined as: [{(reported value-observed value)/reported value}x100%] Reported values were taken as averages from the nuclear counts given in Pataky et al., 2000. 26 2.3 Resul ts 2.3.1 Implant Site Initially, correct implantation of the crystal of Dil into the spinal cord of the chick is difficult. The crystal is to be implanted just ventral to the spinal cord, in contact with but not puncturing the cord. The technique is quickly learned, and is highly reproducible. It is also well tolerated by the animal (85-90% survival rate after 3 days). Figure 2 shows a control section of spinal cord (top) and a spinal cord that has been implanted with a crystal of Dil (bottom), both at similar mid-cervical levels. The notocord (A) is easily visible in both photographs. The developing spinal cord (B) is also visible and upon close inspection the grey matter of the cord can be identified due to its characteristic butterfly shape. The developing vertebral column (C) is also discernable. The implant site (D) is clearly viewed in the bottom figure where the crystal can be seen to pass between the notocord and the developing spinal cord. It is important to note that the spinal cord in the bottom figure is not damaged, but merely deflected from its original position. 2.3.2 Cel l Body Label l ing Figure 3 depicts the ventral surface of an E8 brainstem labelled with Dil. Labelling is clearly visible in the vestibular complex and throughout the entire reticular formation, both pontine and medullary. Strong labelling is also observed in the raphe nuclei. Also visible is the entry point of the facial and vestibulocochlear (VII and Vlll) nerves. 27 Figure 2: The Dil implant site. Top: Control spinal cord (no implant). Bottom: Implanted cord. Insertion of a Dil crystal (D) between the notocord (A) and spinal cord (B) results in retrograde labelling of bulbospinal neurons. The developing vertebral column is also visible in both figures (C). 28 29 Figure 3: A Dil labelled brainstem viewed from the ventral aspect. Strong labelling is observed in the vestibular complex as well as the in the raphe nuclei. The reticular formation is also labelled at both pontine and medullary levels. Entry points of several cranial nerves are also visible. 30 31 Figure 4a is the same picture as in figure 3 and the red lines indicate level of coronal sections (B-F). Moving from rostral to caudal, beginning with figure 4b, the labelled neurons are from the oral pontine reticular formation nuclei (RPO). It is known that about half these cells project spinally (Torvik and Brodel, 1957). In figure 4c, strong labelling is observed in the cell bodies of neurons from the parvocellular and gigantocellular nuclei of the pontine reticular formation (RPpc and RPgc respectively). In figure 4d, neurons of the vestibular complex can be viewed at the distal ends of the slice. Deiters' nucleus, or the lateral vestibular nucleus (VeL) is heavily stained and lies at the tips of the section. Also visible are the inferior vestibular nucleus (descendens; VeD) and the medial vestibular nucleus (VeM). The caudal tip of the RPgc is also visible. Figure 4e is a section through the medullary region of the reticular formation. Scattered about the midline, labelled cell bodies belong to both the parvocellular (Rpc) and gigantocellular (Rgc) nuclei. Figure 4f shows neurons in a line parallel to the seam of the midline. These belong to one of the multiple raphe nuclei stained by implantation of the crystal. It is important to note that although the various nuclei are well visualized, that experimentally, populations in close proximity are not separated. The entire vestibular complex is explanted, as are parvocellular and gigantocellular groups of pontine reticular nuclei. Although raphe explants are taken from the same general area, no effort is made to identify or separate various raphe nuclei. This was due to intermingling of different nuclear groups and the difficulties involved in working with microscopic explants. 32 Figure 4: Dil labelled E8 brainstem from the ventral aspect (A). Specific nuclear groups from the pontine reticular formation (B,C) and medullary reticular formation (E) are labelled. Vestibulospinal (D) and raphespinal (F) cell bodies are also strongly labelled. 33 2.3.3 Nuclear Counts Cell bodies through the raphe and vestibular nuclei were counted. This was performed for two animals, yielding 4 sets of counts for vestibular neurons (two/animal) and two sets for raphe nuclear groups (one/animal). Results are displayed in Table 1. These were compared to the average of counts taken in (Pataky et al., 2000). Although the percent discrepancy looks high in several of the cases it must be noted that the published counts for the vestibular complex range between 663-1,290 neurons and a range of 797-1550 neurons in the raphe nuclear complex. All values fall within these parameters or just slightly above, confirming the effectiveness of the labeling technique. Table 1: Quantification of Dil labeled neurons in the Vestibular and Raphe nuclear groups from two E8 chick embryos. Values are compared to average of data reported in (Pataky et al. 2000). Animal Nuclear Observed Reported Count % Complex Count Averaged from counts in Discrepancy (Pataky, Borisoff et al. 2000) Vestibular (right) 1224 919 33% 1 Vestibular (left) 1084 919 18% Raphe 1716 1197 43% Vestibular (right) 989 919 8% 2 Vestibular (left) 932 919 1.4% Raphe 1516 1197 27% 35 2.4 D iscuss ion 2.4.1 Implant Site It is important to be accurate in implanting the crystal of Dil for several reasons. Firstly, consistency in the labelling is important for reproducibility of the study. If the crystal is implanted too far caudally, it is possible that projection neurons to the more rostral parts of the cord will not take up the tracer. If the tracer is implanted to far rostrally we run the risk of labelling local interneurons in the lower parts of the brainstem. Too far ventral, and the crystal will not take up the tracer at all, resulting in unlabelled specimens. In my opinion, the greatest danger experimentally lies in accidentally puncturing the cord during the implant procedure (too far dorsal). It has been seen that post injury both C N S and P N S neurons upregulate factors associated with regeneration (Hu-Tsai et al., 1994; Vudano et al., 1995; Houle, et al., 1998; Klocker et al., 2001; Caroni, 1997). It has also been shown that a priming injury can facilitate regenerative response (Meyer et al., 1994; Ambron and Walters, 1996; Lazar et al. 1999; Spector et al., 2000). If the cord was damaged during the implantation these growth programs could be activated altering the results of the neurite outgrowth experiments, as well as changing receptor expression. 2.4.2 Cel l Body Label l ing The arrangement of labelled neurons in the brainstem is in agreement with data previously published (Glover and Petursdottir, 1988; Cabot et al., 1982; Ishikawa et al., 1995; Webster and Steeves, 1991; Webster and Steeves, 1988; Wold, 1978). Labelled 36 neurons are found throughout the reticular formation. This is expected, as the reticular formation neurons are believed to provide much of the supraspinal muscular control in locomotion (Valenzuela et al., 1990). We also see strongly labelled projection neurons in the vestibular nuclei. It has been shown previously that vestibulospinal neurons project to varied levels of the chick spinal cord (Wold, 1978; Webster and Steeves, 1991; Cabot et al., 1982). Neurons of the lateral vestibular nucleus enhance the activity of extensors by increasing muscle tone in the cat and increase extensor activity during the stance phase of stepping (Orlovsky, 1972; Orlovsky and Pavlova, 1972). While the medullary raphe nuclei are labelled with Dil the pontine raphe nuclei do not appear to be labelled. This is due to the restriction of more rostral raphe projections to supraspinal levels of the CNS (above the implant site). Since the projections do not contact the crystal of Dil, the cell bodies are not labelled. The caudal nuclei (raphe magnus, pallidus and obscurus) project toward the spine and serve to increase excitability of motor neurons in the cord via serotonergic connections. Raphespinal neurons are also involved in modulation of pain stimulus (Gao and Mason, 1997). 2.4.3 Nuclear Counts As seen from the results presented, the number of cell bodies labeled by the procedure varies between animals as well as bilaterally in the same animal. It is likely that these variations are the result of small, uncontrollable changes in each implantation of the Dil crystal. Different sized crystals and the human error involved in implantation make variation impossible to avoid. This variability in labeling makes it difficult to correlate the number of labeled cells in an explant, with the number of neurites that are 37 extruded. The problem is compounded by both the lack of standardization of the size of the explant and the inability to duplicate the exact position of coronal slicing. Although the size of each explant is approximately the same, coronal sectioning often results in nuclear groups being cleaved through different planes. It is for these reasons that no studies have been performed relating labeled cell bodies to number of neurites in an attempt to study sprouting phenomenon. In this study only two of the many labelled populations were cultured. Preliminary experiments were done using labeled pontine reticular explants, however the results of these experiments were inconsistent and not reproducible (Figure 5). This is unfortunate because of the important role that the reticular formation plays in locomotion. It is possible that the inconsistencies in these experiments were due to inadvertently taking sections at varied coronal levels containing different subpopulations within the pontine reticular nuclei being plated. 2.5 Conclusions From examination of the implant site, cell body labelling in the brainstem and the counts of labelled neurons, it is safe to conclude that the technical aspects of the Dil implant and labelling procedures have been mastered. This is essential for use of labelled explants for in vitro tissue culture, further described and discussed in the next sections. 38 Figure 5: The Effect of Neurotrophins on Pontine Reticular Neurons. Two trials under identical experimental conditions show radically different results (A,B). Other experiments using neurons of the reticular formation were also highly inconsistent. Due to these inconsistencies experiments on pontine reticular neurons were discontinued. 39 700 i 600 -E 500 -£ 400 C Q> 03 300 -3 200 O C 100 0 The Effect of Neurotrophins on Pontine Reticular Neurons Serum Free Control 5% F C S 75ng/ml N G F Treatment 75ng/ml B D N F 75ng/ml NT-3 75ng/ml NT-4 500 The Effect of Neurotrophins on Pontine Reticular Neurons o c 100 Serum Free Control 5 % F C S 75ng/ml N G F 75ng/ml 75ng/ml N T - 3 75ng/ml NT-4 B D N F treatment 40 Chapter 3: Growth Factor Screens 3.1 Introduction The previous chapter explored technical aspects of the bulbospinal labelling with Dil to ensure proficiency with the method. In this chapter I have used this powerful labelling technique, in conjunction with an explant culture model in order to screen a number of potential candidates for effects on vestibulospinal neurite outgrowth. In order to target potential growth factors for in-depth examination, I screened various growth factors (NGF, BDNF, NT-3, NT-4, GDNF, EGF and IGF-1) for promotional effects on vestibulospinal neurite outgrowth. The initial selection of each of these factors for testing was based on two criterion: 1. Ability to stimulate the MAP Kinase Pathway, believed to influence axonal elongation. 2. Expression of receptors for the growth factor in the brainstem. This section will provide specific rationale for the choice of growth factors used in these experiments. 3.1.1. The Neurotrophin Family The neurotrophin family of molecules has been well studied and appears to play a significant role in the development of the nervous system. The ligands of this family include nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and neurotrophin-4/5 (NT-4/5). These molecules share significant sequence homology, with 50-60% sequence identity between members. 41 They appear to have varied functions and indeed are differentially regulated both spatial ly and temporal ly (Pirvola et a l . , 1997; Go rba and W a h l e , 1999; Buck et a l . , 2000). In vitro exper iments in the chick dorsal root gangl ion ( D R G ) have shown that appl icat ion of N G F to the growth cone will cause it to turn toward the trophic source (Gundersen and Barrett, 1979). Cultured neurons from X e n o p u s laevis spinal cord show similar behavior upon exposure to B D N F and NT-3 (Ming et a l . , 1997). Neurotrophins a lso play roles in cell survival as well as differentiation (Lewin and Barde, 1996). B D N F has been shown to increase dendrit ic complexi ty in N T 2 - N cel ls, suggest ing a role in neuronal morphology (Piontek et a l . , 1999). The receptors that mediate the act ions of these trophic molecules come in two general forms. O n e is the p75 neurotrophin receptor (p75 N T R ) designated for its molecular weight, the others are the t ropomyosin receptor k inase (trk family) of receptors. The p 7 5 N T R has been implicated in cell survival while the trk family of receptors has been assoc ia ted with both survival and neurite outgrowth (Enc inas et a l . , 1999; W a t s o n et a l . , 2001). 3.1.2 Thep75 N T R The p 7 5 N T R is a member of a receptor family involved in programmed cell death pathways and/or the activation of N F - k B . Other members of this family include: A p o -1 /CD95 (Fas) , two receptors for tumor necrosis factor (TNF) and at least 15 others. The p 7 5 N T R is character ized by four cysteine rich extracellular doma ins (Figure 6) and an intracellular segment that lacks intrinsic catalytic activity. It binds all four neurotrophins with approximately equal affinity. 42 Figure 6: The Neurotrophin Family. The p75NTR binds all neurotrophins (NGF, BDNF, NT-3 and NT-4) with similar affinity. TrkA preferentially binds NGF, trkB has high affinity for BDNF and NT-4, while trkC binds NT-3 best. NT-3 also binds to both trkA and trkB with lesser affinity than the preferred ligand. 43 The Neurotrophin Family NGF BDNF NT-4 NT-3 p 7 5 N T R T r k A J r k B j r k C 44 It had long been believed that the role of the p75 was that of a co-receptor to the trk receptors. This was supported by the knockout phenotype that showed mild deficits in the peripheral nervous system (Lee et al., 1992). It was later discovered that p75 N T R did have signaling activity. T9 glioblastoma cells expressing p 7 5 N T R , but not trkA showed a reduced growth rate, and initiated process formation upon NGF exposure. They also showed sphignomyelin hydrolysis leading to the production of ceramide (Dobrowsky et al., 1994). Similar production of ceramide has also been observed in p 7 5 N T R expressing fibroblasts and oligodendrocytes exposed to specific neurotrophins (Dobrowsky et al., 1995; Casaccia-Bonnefil et al., 1996). Ceramide is believed to be part of a second messenger cascade for p 7 5 N T R effects (Barrett, 2000). Mouse Schwann cells, which lack catalytic trk receptors but express p 7 5 N T R show translocation of NF-kB to the nucleus in wildtype cells but not in a p 7 5 N T R knockout mouse when exposed to NGF (Carter et al., 1996). Similar translocation of NF-kB has been observed in fibroblasts expressing p 7 5 N T R . It is believed that NF-kB activation helps to prevent cell death (Liu et al., 1996). The effect of p 7 5 N T R on cell death was discovered when cells transfected with p 7 5 N T R died faster than those with control transfection in the absence of serum (Rabizadeh et al., 1993). Furthermore, the addition of NGF to the cells reduced the levels of death in the p 7 5 N T R transfected cells. These observations suggest that in the absence of bound neurotrophin, that the p 7 5 N T R has the potential to induce cell death. Similar ligand-independent cell death has been observed in other members of this family such as the TNF receptor 1 and CD 95 (Boldin etal . , 1995). 45 3.1.3 The Trk Receptors The Trk receptor family consists of three identified members. TrkA, trkB, and trkC are transmembrane tyrosine kinase receptors, which have similar structures. They are approximately 140kDa, and are characterized by an extracellular domain consisting of two immunoglobulin-like regions, and three leucine rich domains flanked by single cysteine rich clusters (Schneider and Schweiger, 1991) (Figure 6). The second immunoglobulin region determines the binding specificity of the trk receptors with a conserved region that imparts selectivity for all members of the neurotrophin family and a specific sequence which allows preference for a particular family member (Urfer et al., 1995; Urfer etal . , 1998). Among each of the trk receptors, there are various isoforms. The trkA receptor has two such isoforms, one 790 a.a. in length, the other 796 a.a. One major difference between the two is in spatial expression, with the longer protein expressed mainly in neuronal cells and the shorter variant in cells of non-neuronal origin (Urfer et al., 1998). The trkB receptor has a very intricate expression system, with a total of eight known splice variants. These fall into two main subcatagories, the TrkB TK+ receptors that are catalytically active (Klein et al., 1989), and the TrkB TK- receptors that have a truncated cytoplasmic domain and lack kinase activity (Klein et al., 1990). TrkC has at least eight isoforms (reviewed in Barbacid, 1994) which also fall into two distinct subcatagories, four with catalytic activity (Lamballe et al., 1991; Lamballe et al., 1993; Tsoulfas et al. 1993; Valenzuela et al., 1993), and the other subclass lacking the tyrosine kinase domains (Tsoulfas et al., 1993; Valenzuela et al., 1993). 46 The trk receptors undergo dimerization upon activation (Jing et al., 1992) and while homodimerization is known to occur, it is certainly possible that the existence of so many splice variants may allow for diversification of receptor combinations, thus increasing the complexity of receptor function. Dimerization allows for trans phosphorylation of various tyrosine residues of the intracellular (C-terminal) portion of the protein. These phosphotyrosines bind adapter or enzymatic proteins via SH2 domains (Schlessinger and Ullrich, 1992). Several proteins appear to bind directly to the trk receptors. She and several related proteins (N-Shc, Sck) contain a phosphotyrosine binding SH2 domain that allows them to associate with the trk receptors (Dikic et al., 1995; Nakamura et al., 1998; Pelicci et al., 1992). In turn, the She protein recruits the Grb-2/SOS protein complex leading to activation of Ras and the MAP kinase pathway (reviewed in Kaplan and Miller, 2000). FRS2 , an adapter protein anchored to the cell membrane also binds phosphotyrosine on active trk receptors (Meakin et al., 1999). In fact, there is evidence that FRS2 and She compete for the same phosphotyrosine adding to the complexity of the system (Meakin et al., 1999). FRS2 recruits the Grb-2/SOS complex as well as SHP-2 (Hadari et al., 1998). The M A P kinase cascade has been associated with neurite outgrowth (Stephens et al., 1994). Phospholipase Cy (PLCy) has also been associated with the trk receptors (Ohmichi et al., 1991). P L C cleaves 4,5-bisphosphate (PIP2) into diacylglycerol (DAG) and 4,5-triphosphate (IP3). DAG in turn activates protein kinase C (PKC), while IP3 increases intracellular Ca2+ levels modifying various cellular functions Trk receptors also indirectly activate PI-3K, a molecule that has been associated with neuronal survival. Grb-2 activates Grb associated binder-1 (Gab-1) which activates 47 PI-3K (Holgado-Madruga et al., 1997). PI-3K is believed to activate PKB/AKT kinase, which has been recently associated with neuronal survival (Franke et al., 1997; Crowder and Freeman, 1998) via inactivation of proapoptotic members of the Bcl-2 family (Datta et al., 1997). It also phosphorylates the transcription factor forkhead, which controls gene expression of cell death genes such as the Fas ligand. These signalling cascades have been summarized in Figure 7. 3.1.4 The Insulin-like Growth Factor Family It has been suggested that the insulin-like growth factor (IGF) family has important roles in development of the nervous system. Evidence to support this hypothesis includes a reduction in total neuron numbers in vivo, upon knock-out of the IGF-1 gene (Beck et al., 1995). Increased in vitro neurogenesis in the neuroepithelia of chick retina upon exposure to IGF-1 has also been observed (Drago et al., 1991). IGF-1 has also been shown to promote survival in spinal motor neurons (Hughes et al., 1993) and enhance of neurite outgrowth in sympathetic neurons (reviewed in Zackenfels etal . , 1995). Ligand members of the family include insulin, IGF-1 and IGF-II. There is also a large family of IGF binding proteins, suggesting a high level of regulation within the IGF system. In mammals, the receptors for the IGF family ligands include the insulin receptor (IR) and the IGF-1 receptor (IGF-1 R), which share significant sequence homology, as well as the IGF-II receptor (IGF-IIR) also referred to as the mannose-6-phosphate (M-6-P) receptor, which is of a considerably different structure (Figure 8). The M-6-P receptor appears to be of little consequence to this study; although 48 Figure 7: Trk signalling impacts on both neurite outgrowth and neuronal survival. Dimerized Trk receptors signal via adaptor proteins that bind phospotyrosine. Activation of the MAP Kinase pathway is associated with neurite outgrowth. PI-3K activation has been implicated in neuronal survival. (Modified from Kaplan and Miller, 2000). 49 Trk Receptor Signalling Neurotrophin t t t t t t f f i II II II II II n II n M o o o o o <OQ o n 1 1 1 1 1 1 1 1 1 1 1 D O O O O O O O O O O Neurite Outgrowth Neuron Survival 50 V expressed in the chick, it lacks the high affinity binding state for IGF's present in mammals (Canfield and Kornfeld, 1989; Clairmont and Czech, 1989) and thus is not. considered a functional IGF receptor in the chick model (Duclos and Goddard, 1990). 3.1.5 The IGF-1 Receptor In mammals and other non-mammalian species the IGF-1 receptor is thought to mediate the majority of trophic effects produced by the IGF family (reviewed in Connor and Dragunow, 1998). The insulin receptor and the IGF-1 R are heterotetrameric complexes consisting of an extracellular a-subunit and a transmembrane p-domain with tyrosine kinase activity. The subunits are linked by disulphide bonds into an cc-p-p-a configuration (Figure 8). A single 180 kDa precursor is cleaved to produce an a and a P-subunit. The a-subunits are approximately 135 kDa and radioligand cross-linking studies show them to contain the cysteine rich ligand binding domain (Czech and Massague, 1982; Rosen, 1987). The p-subunits are 95kDa and activation of the intracellular RTK activity permits autophosphorylation of the receptor. The insulin receptor has long been known to play a role in energy regulation in the cell, mediating processes such as glucose transport and metabolism and lipid synthesis. The IGF-1 receptor is believed to play a more active role in cell motility, process extension and cell survival. The phosphorylated IGF-1 receptor is a binding target of the insulin receptor substrate proteins (IRS-1, IRS-2) (De Meyts et al., 1994). The IRS proteins are in turn bound by docking proteins such as growth factor receptor bound-2 (Grb2) protein, the p85ct subunit of phosphatidylnositol-3 kinase (PI-3K) as well as adapter proteins with 51 Figure 8: The Insulin Like Growth Factor Family. Insulin binds to the IR with high affinity but will also activate the IGF-IR at high concentrations. IGF-1 binds with highest affinity to the IGF-IR but also has lesser affinities for the IR and the IGF-IIR. IGF-II preferentially binds the IGF-IIR receptor, but has also been show to weakly activate the IGF-1 R. In the chick, the IGF-IIR is not a functional receptor. 52 The Insulin-like Growth Factor Family Insu l in IGF-1 IGF-II 53 SH2 binding domains (Eck et al., 1996; White, 1994; De Meyts et al., 1994). Grb2 binds the guanine exchange factor (GEF) sos that triggers the activation of the Ras/MAP kinase pathway. The end result of the MAP kinase cascade is activation of the nuclear transcription factors c-fos, c-jun, and c-myc. These factors modify mRNA production and thus de novo protein synthesis in the cell. There is evidence that suggests that the MAP kinase pathway has significant impact on neurite outgrowth (Kim et al., 1997). The P85a subunit of PI-3K activates a larger subunit, which phosphorylates phosphoinositide molecules, which in turn have varied downstream targets. The PI-3K pathway has been implicated in neuronal survival and alterations in cell motility associated with IGF exposure (Singleton et al., 1996; Leventhal et al., 1997). These cascades are pictorially depicted in Figure 9. 3.1.6 GDNF and the GDNF Receptors GDNF and its family of neurotrophic factors neurturin, artemin, and persephin are biologically active molecules known for their support of various neuronal populations (Henderson et al., 1994; Garces et al., 2001; Li et al., 1995; Oppenheim et al., 1995; Houenou et al., 1996). The receptors of this family consist of a Ret receptor tyrosine kinase (RTK) in addition to one of four GPI-linked cell surface proteins, G F R a l , G F R a 2 , G F R a 3 or G F R a 4 . GDNF responds to the Ret-RTK in association with either G F R a l or GFRa2 . In situ hybridization studies have shown high levels of G F R a l and G F R a 2 as well as Ret-RTK in the mouse brainstem (Golden et al., 1999; Golden et al., 1998). 54 Figure 9: IGF-1 impacts on neurite outgrowth and survival. IGF-IR signals via adaptor proteins that bind phospotyrosine. Activation of the MAP Kinase pathway is associated with neurite outgrowth. PI-3K activation has been implicated in neuronal survival. Modified from BioCarta Signalling Pathways, W. O'Dell http://www.biocarta.com/pathfiles/igf1 Pathway.asp 55 IGF-1 Receptor Signalling 56 GDNF activates the MAP kinase pathway (Chen et al., 2001; Hayashi et al., 2000) as well as the phosphatidylinositol-3 kinase (PI-3K) (van Weering and Bos, 1998) signaling cascade. 3.1.7 E G F and the E G F receptor E G F has a family of four receptors ErbBI (EGFR), ErbB2, ErbB3, and ErbB4. These members form homo- and heterodimers and initiate distinctive signaling pathways by recruiting various effector proteins with SH2 domains. Stimulation of the E G F R activates the MAP Kinase pathway, believed to be involved in neurite outgrowth. Indeed, in certain subpopulations of PC-12 cells, E G F has been shown to increase neurite outgrowth (Yamada et al., 1996). This effect is not consistent between sub groups of PC-12 cells. In other PC-12 cells that express the E G F R , no neurite outgrowth is seen (Yasui et al., 2001). This lack of response has been attributed to the duration of MAP kinase activation. These inconsistencies make it difficult to predict the effect of E G F on brainstem-spinal cell populations. The adult rat and human brainstem have been shown to express high levels of E G F R mRNA (Kaser et al., 1992; Ferrer et al., 1996). 3.2 Methods and Materials Egg incubation, retrograde labelling, and brainstem dissections were performed as described in Chapter 2. 57 3.2.1 Explant Culture The brainstems were cut coronally into 300um slices on a Mcllwain Tissue Chopper (Brinkmann, Westbury, NY). Several tissue slices lying in the appropriate anatomical area for the desired nuclei (raphe or vestibular) were examined under rhodamine epifluorescence (Zeiss Axioskop, Germany, 5x magnification) and the slices sorted according to whether the labelled nuclei were present. Using a dissection microscope (Wild, Heerbrugg, Switzerland) the slices containing labelled nuclei were microdissected using a scalpel. Vestibular nuclei were dissected by making a single cut just medial to the nuclei. Raphespinal neurons were separated by making a cut lateral to each side of the raphe nuclei. Explants were plated for 14-16 hours in Dubecco's Modified Eagle Medium solution (DMEM), supplemented with 5% Fetal Calf Serum (FCS, Canadian Life Technologies) to promote adherence, 2mM glutamine, and 100 units/ml penicillin/streptomycin (pen/strep, Gibco-BRL) antibiotic in 48 well culture dishes (Costar, Corning, NY) that had been incubated with 25 ug/ml mouse laminin (Gibco-BRL), for 3 hours. After the allotted time for adherence, the media was carefully siphoned off as not to disturb the explant, and replaced with DMEM (2mM glutamine, pen/strep) along with the treatment or control being tested. Growth factors used were as follows: Recombinant human p-NGF, BDNF, NT-3, NT-4, bFGF ( R & D Systems), GDNF, IGF-1 and EGF(Sigma, St. Louis). Raphespinal cultures were grown for 24 hours in the treatment, for a total of 38-40 hours. The vestibular cultures were grown for a further 24 hours for a total of 62-64 hours. Experiments had between 3-7 replicate wells per treatment and identical trials were performed at least twice. 58 Some cultures were grown on sterile glass coverslips (22mmx22mm, VWR, Westchester, PA) for later immunohistochemistry on fibers. Coverslips were treated the same as plastic culture wells for laminin coating, etc. Cultures were fixed by replacing the media with a solution of 4% formaldehyde or paraformaldehyde in 0.1 M chick PBS with 10% sucrose. These cultures were stored at 4°C for no longer than one month before being photographed. 3.2.2 Ana lys is of Explant Cultures Images were captured using an inverted Nikon fluorescence microscope and a Princeton Micromax cooled CDD camera using the Metaview image software. Due to the insufficient size of the visual field at the smallest magnification, it was often necessary to montage several pictures together to include the length of all neurites. This was accomplished by importing the digital picture files into Photoshop 5.0 (Adobe Systems) and overlapping photographs to create an accurate picture of the culture. The neurites were then manually traced in Photoshop. Quantitative analysis of neurite length was performed using the public domain software ImageJ provided on the NIH website (http://rsb.info.nih.gov/nih-image). The calibration was performed by photographing a um scale and utilizing the object measurement function in ImageJ. Graphical analysis and descriptive statistics were performed on Microsoft Excel and post hoc statistical comparisons (the Scheffe test) were carried out on Statistica. Controls for the experiments included a serum free negative control, a positive control of 5% F C S and/or 10 ng/ml FGF-2. Due to small, uncontrollable variations in the culture conditions, neurite lengths were only compared within the same trial, not across 59 different trials. For cross trial comparison mean neurite lengths were converted to percent of serum free controls and graphed. 3.3 Resul ts Results of experiments to determine the mean neurite length are given as a series of graphs, all of which follow a standardized format. The first bar is invariably that of the serum free control. This represents the baseline of neurite outgrowth with nothing other the basic support required for growth. This is the basis of comparison for all the other treatments. The second and third bars represent a pair of positive controls, 5% Fetal Calf Serum (FCS) and FGF-2 (10ng/ml) respectively. F C S provides a rich source of trophic support, however the mechanism of this support is unknown. FGF-2 has previously been identified as a promoter of neurite outgrowth (Pataky et al., 2000). Support media supplemented with 5% F C S approximately doubles the mean neurite length, while FGF-2 generally appears to have a slightly less potent effect. Interestingly, FGF-2 appears to cause a dense halo of glia around vestibular explants. It is uncertain whether this effect is due to migration or cell division. The remaining bars represent factors being tested. If the application is titered, the lowest concentration is represented by the left most experimental bar (bar 4) and the highest is on the right. Error bars represent the standard error about the mean, and significance is determined by the Scheffe test, the most conservative of the post hoc comparisons. It should also be noted that only values greater that 30um (1 cell body lengths) were used, and that the ten smallest and 10 largest values were eliminated from each pooled treatment group in an attempt to reduce outliers. 60 3.3.1 The Effect of Growth Factors on Mean Neurite Length In order to quickly identify potential trophic factors a screen was performed testing various factors on vestibulospinal neuronal explants. Figure 10a shows a screen for NGF, BDNF, NT-4 and GDNF. The first bar is the mean neurite length of the serum free control. The numerical results of the two trials are summarized in Tables 2 and 3. Table 2: Summary Table for Effects of NGF, BDNF, NT-4 and GDNF on Vestibulospinal Neurite Outgrowth. Treatment applied, mean neurite length, standard error, number of pooled explants and total number of neurites are shown. Bold values are significant: p<10"5. Treatment Mean Neurite Std. Error % of Serum #of # of Pooled Length (urn) (+/-) Free Control Explants Neurites Serum Free 496 17 100 5 330 Control 5% FCS 1130 32 227.76 6 326 FGF-2 10ng/ml 996 23 200.74 5 517 NGF 10ng/ml 569 15 114.71 6 538 BDNF 10ng/ml 838 20 168.98 5 426 NT-4 10ng/ml 614 18 123.87 5 507 GDNF Wng/ml 555 14 111.96 5 459 The serum free control has a mean neurite length of 496 ± 17u.m. Of the four factors screened only BDNF and NT-4 had significant effects (p<.005 and p<.05 respectively) on mean neurite length. BDNF at 10ng/ml increased the neurite length to 169% of the serum free control. NT-4 increased the mean neurite length to 124% of control values. Though NGF and GDNF appeared to slightly increase mean neurite length, neither of these effects was significant. 61 Figure 10: Growth factor screens on vestibulospinal neurons. (A,B) NGF, BDNF; NT-3, NT-4, GDNF, BDNF and IGF-1 were applied to cultured vestibular explants at a concentration of 10 ng/ml. IGF-1, BDNF, EGF and NT-3 treated explants all showed highly significant increases in mean neurite length (p<.005). 62 250 P 200 o 9 150 2 100 9 V) 50 0 Trophic factor screen ** P<QQ5 * p<.05 D M E M 5% F C S FGF - 2 N G F 10ng/ml 10ng/ml 10ng/ml 10ng/ml 10ng/ml B D N F NT-4 G D N F treatment 250 Growth factor screen p<.005 DMEM 5% FCS FGF-2 NT-3 IGF-1 EGF 10ng/ml 10ng/ml 10ng/ml 10ng/ml treatment 53 Table 3: Summary Table for Effects of NT-3, IGF-1 and E G F on Vestibulospinal Neurite Outgrowth. Treatment applied, mean neurite length, standard error, number of pooled explants and total number of neurites are shown. Bold values are significant: p<10"4. Treatment Mean Neurite Std. Error % of Serum # of Explants # of Pooled Length (urn) (+/-) Free Control Neurites Serum Free 417 25 100 5 206 Control 5% FCS 838 34 200.68 5 191 FGF-2 10ng/ml 708 15 169.53 5 308 NT-3 10ng/ml 549 16 131.42 5 264 IGF-1 10ng/ml 791 20 189.39 5 404 EGF 10ng/ml 607 30 145.38 4 234 In Table 3, the mean neurite length of the serum free control was 417 + 25 um. All three growth factors screened in this trial showed a significant increase in neurite outgrowth (Figure 10b). NT-3 increased the neurite length 31% compared to the control, while IGF-1 increased the mean length by 89%. E G F increased the mean neurite length to 145% of control levels. 3.4 D iscuss ion It is important to note that the screen of growth factors was performed on vestibulospinal explants only. Raphespinal populations were not screened with all the trophic factors and may show a response to some of the factors where vestibulospinal populations did not. It is important for future work to investigate this as different neuronal populations may have different trophic requirements. In the trophic factor screens, IGF-1, BDNF, E G F and NT-3, had a significant impact (p<.005) on the elongation of vestibulospinal neurites while NT-4 also appeared 64 to affect neurite length (significant to a level of p<.05). In this section I will discuss the growth factors that did not affect neurite length and potential reasons for their failure to stimulate neurite elongation, and then present a more in depth discussion on the factors that this study focuses on (IGF-1, BDNF) in the following two chapters. 3.4.1 NGF Does not Affect Vest ibulospinal Outgrowth At first glance it seems surprising that NGF does not encourage neurite outgrowth in the vestibular nucleus as trkA has been identified in bulbospinal populations, along with trkB and trkC (Gibbs and Pfaff, 1994; Holtzman et al., 1995; Medio et al., 1992). A more recent study has shown that few bulbospinal neurons actually express trkA in the adult rat (King et al., 1999). This lack of receptor expression, compounded by numerous failures for NGF to induce regeneration associated phenomenon (Tetzlaff et al., 1994; Kobayashi et al., 1997) show that my finding, that NGF does not have an effect on vestibulospinal neurons, is in agreement with the literature. 3.4.2 GDNF Does not Affect Vest ibulospinal Outgrowth When applied to cultured vestibulospinal chick explants there is no increase in mean neurite length. It is possible that receptors are not present in the developing chick vestibular complex, although studies in the adult rat have confirmed that both G F R a l and Ret mRNA are found in the lateral vestibular nucleus (Glazner et al., 1998). No receptor expression studies have been performed on the embryonic chick, however the developing mouse brainstem shows'strong expression of G F R a l and Ret (Golden, 65 1999). This does not however mean that that they are expressed specifically in the vestibular complex. To determine if this is indeed the case, a detailed study of Ret and G F R a l expression in the embryonic chick should be performed in the future. An alternative explanation for the failure of GDNF to influence neurite outgrowth in these experiments is that the human GDNF used in the experiments was too different than chick GDNF. Results of a pairwise BLAST comparison reveal only a 53% identity between human and chicken GDNF. This large variation in sequence may prevent human GDNF from binding to the chick receptors (Accession #XP 031129 (human), AAF26685 (chick)). As GDNF has been observed to possess potent growth effects, it would be interesting to try this experiment again using chick GDNF. 3.4.3 NT-3 Effects Require a More Thorough Examination It has been shown that trkC expression is as high as 84% of brainstem spinal neurons (King et al., 1999) in the adult rat. If embryonic expression of trkC in the chick were similar to these results, it would not be surprising that NT-3 increases neurite outgrowth. The literature suggests otherwise; a study using the same in vitro embryonic explant culture model as this one initially reported that NT-3 was an ineffective agent for stimulating vestibulospinal neurite outgrowth (Pataky et al., 2000). The same study showed NT-3 to be effective for increasing the neurite outgrowth of pontine reticulospinal neurons. Culture conditions were different in this work using a higher laminin concentration (75ug/ml) which may have masked the effect of NT-3. Another study (Ye and Houle, 1997) reports that NT-3 has no significant effect on vestibulospinal neurons. Upon closer examination of their data, it appears there is a 66 trend toward increased numbers of regenerating vestibulospinal neurons, however the high level of statistical variation prevented this from being significant. From these varied results only one thing is clear. Further experimentation is required to determine the potential of NT-3 to promote regenerative behavior. 3.4.4 BDNF and NT-4 Increase Mean Neurite Length The effect of BDNF on vestibulospinal neurite outgrowth and the lesser effect by NT-4 are not surprising. Several studies show expression of TrkB, the receptor for both BDNF and NT-4 to be present in the vestibular complex in both the developing or adult mouse (Klein et al., 1990) and rat (King et al., 1999; Yan et al. 1997). BDNF was selected for further study (Chapter 4), while due to the smaller increase in neurite length, no further experiments were done using NT-4. Investigation of NT-4 effects on bulbospinal neurite outgrowth needs to be pursued further as NT-4 may yet prove a valuable factor in promoting regeneration. 3.4.5 IGF-1 Increases Mean Neurite Length IGF-1 increased the mean neurite length during screening with vestibulospinal explants. For this reason IGF-1 was selected for further investigation in this study. A detailed discussion of IGF-1 effects is provided in Chapter 5. 3.4.6 EGF Does not Affect Vestibulospinal Outgrowth While E G F appeared to increase neurite length in the growth factor screen further investigation revealed that this was a false positive. In the results presented in 67 Table 8 (Appendix), the serum free control has a mean neurite length of 556 ± 1 5 um. Increasing concentrations of E G F appear to have no significant impact on vestibulospinal mean neurite length. At its highest concentration, 10 ng/ml the mean neurite length is 527 ± 13 um. This lack of effect was confirmed in a second trial (data not shown). The fact that E G F produced a false positive result in the neurotrophin screen is difficult to explain. The mean neurite length of the serum free control of the second screen (NT-3, IGF-1, EGF) is the lowest of all the trials. It is possible that a localized infection or mechanical damage to the explants resulted in a lower mean neurite length for the serum free control, making all the other results appear elevated. This could have increased the difference in means, making random variability appear significant. The adult rat and human brainstem have been shown to express high levels of E G F R mRNA (Kasereta l . , 1992; Ferrer et al., 1996), however this does not mean that receptor activation will increase neurite length. Perhaps E G F impacts on factors not observed in this study such as survival, or plasticity. While it is unfortunate that E G F does not promote neurite elongation, it is comforting to know that not every growth factor will increase neurite outgrowth, and increased outgrowth is not due to a change in protein concentration. E G F was not tested on raphespinal populations nor were any immunohistochemical studies performed. 3.5 Conclusions 68 Using this screening method I selected the three factors that appeared to have the greatest impact on neurite outgrowth, IGF-1, BDNF and E G F . Further experimentation resulted in discovery that E G F was a false positive hence it was eliminated from the factors selected for further testing. This fallibility makes it difficult to draw any solid conclusions from the trophic factor screens, however it does provide a starting point for a more in depth investigation of the growth factors. BDNF and IGF were investigated further and the next two chapters are devoted to these experiments. The findings in this section support my first hypothesis that appropriate trophic support encourages neurite outgrowth. 69 Chapter 4: BDNF and TrkB in Bulbospinal Neurite Outgrowth 4.1 Introduction. In the previous chapter several growth factors were screened for positive effects on neurite outgrowth. This chapter focuses on the effects of BDNF on vestibulospinal and raphespinal neurons. A review of the neurotrophic factors (including BDNF) was provided in Chapter 3, and a section of this chapter will provide a further discussion of specific BDNF effects. Due to the large increase in neurite outgrowth affected by BDNF in the vestibulospinal growth factor screen, BDNF was one of three factors selected for further investigation. Experiments performed in this chapter include: a dose-response study of BDNF effects on both vestibulospinal and raphespinal populations. An attempt will be made to delineate potential differential effects on nuclear subpopulations using greater-than-X plots, in which the shift about the X-axis corresponds to whether all or only some subpopulations are responding to the trophic factors. To determine if trophic effects are direct or indirect, immunohistochemical double labelling studies, co-localizing Dil and TrkB or p 7 5 N T R receptors are presented for vestibular and raphe neuronal populations on E9, the first day of growth factor treatment for both vestibular and raphe populations. Similar co-localization results are present for E11 vestibular neurons and E10 raphespinal neurons, the last day of culture in each case. 70 4.2 Methods and Materials Egg incubation, retrograde labelling, and brainstem dissections were performed as described in Chapter 2. Explant culture and neurite length analysis was performed as in Chapter 3. 4.2.1 Immunohistochemistry Chick brainstems were removed as in the explant culture steps between E9 and E11. Brainstems were fixed, sectioned and cut into 20 um sections as described in Chapter 2. The slides were stored at -20°C until use (less than 4 weeks). Slides were thawed, and soaked in 0.1 M chick P B S ( 3 x 5 min) to wash off any excess OCT compound and blocked with 10% normal donkey serum (NDS) and 3% B S A (Fraction V, Sigma, St. Louis) in chick P B S for 1-2 hours. Slides were incubated overnight at room temperature with 0.1 M P B S , .03% Triton X-100/0.3% Tween-20/detergent free (BDH Chemicals, Toronto), with the appropriate antibodies. Antibodies used for TrkB immunohistochemistry were as follows: Ab-1 (Oncogene Research Products), H-181/sc-8316, 794/sc-12 (Santa Cruz Biotechnology Inc.), and anti-TrkB (a generous gift of Dr. F. Lefcort, Montana State University). 794/sc-12 provided the best results. For p 7 5 N T R immunohistochemistry rabbit anti mouse polyclonal antibody (AB1554, Chemicon Int. Inc. Temecula) was used. The sections were washed in 0.1 M P B S ( 3 x 5 min) and then incubated with biotinylated donkey anti-rabbit antibody (1:200, Jackson ImmunoResearch Laboratories, Inc.) for 2 hours to amplify the signal. The slides were 71 washed in 0.1 M P B S (3 x 5min) and incubated in darkness with strep-avidan-conjugated Alexa 488 (1:150, Molecular Probes, Oregon). For both the amplification step and the fluorescence incubation detergent type, concentration or the absence of was kept the same as in the primary incubation step. The slides were soaked in Hoechst 33258 in 0.1 M PBS (1:1000) for one minute to stain the nuclei, rinsed in 0.1 M P B S ( 3 x 5 min) and coverslipped using 0.1 M P B S as a mounting medium. Coverslips were sealed to the slide using clear nail polish, and photodocumented immmediately. Immunofluorescence was visualized under the FITC filter. 4.3 Resul ts To determine how varied concentrations of BDNF affected neurite length, concentrations between 0.2 ng/ml and 10 ng/ml were applied to the cultured explants. As it is impossible to compare the trials directly due to the slight variation in culture conditions during each separate experiment, the values were converted to percent of the serum free control and plotted as paired values on a histogram. Greater-than-X plots are presented for the serum free control and 10 ng/ml BDNF (Figure 11). In these plots the shift about the X-axis corresponds to whether all or only some neuronal subpopulations are responding to BDNF treatment. The results of two representative trials with BDNF on vestibulospinal neurons are summarized in Tables 4a and 4b (Appendix), as are graphs of neurite length vs. treatment for the raw data (Appendix). In an attempt to determine whether the nuclear groups in question expressed receptors forthe trophic factors that stimulated neurite outgrowth, immunohisto-chemistry was performed for trkB and the p 7 5 N T R . Expression was examined at two 72 120% 200 400 600 800 1000 1200 1400 1600 neurite length (um) Figure 11: BDNF increases vestibulospinal neurite length. Main: Projection neurons from the vestibular complex treated with 10 ng/ml BDNF (mean neurite length = 872 ± 28 urn) show a right shift in distribution as compared to the serum free control (DMEM; mean neurite length = 602 ± 17 urn). The shift appears to shift the entire distribution of neurite lengths to the right. From data in table 4a (Appendix). Inset: Projection neurons from the vestibular complex treated with 10 ng/ml BDNF (mean neurite length = 652 ± 20 nm) show a right shift in distribution as compared to the serum free control (DMEM; mean neurite length = 484 ± 17 pirn). The shift appears to shift the entire distribution of neurite lengths to the right. From data in table 4b (Appendix). 73 time points for each nuclear group. Vestibular trkB expression was examined at E9 and E11, the first and last days of growth factor treatment. Raphe trkB expression was examined at E9 and E10, also the first and last day of growth factor treatment. The sections were also stained with Hoechst 33258, a nuclear stain. The nuclear stain was used to visualize the location of all nucleated cells, however it often made the figures cluttered (Figure 12). For this reason the Hoechst staining was omitted from the majority of the figures and only Dil labelling and immunoreactivity to the particular receptor were shown. These experiments were purely qualitative, with no attempt being made to determine the proportion of labelled neurons that expressed receptors. 4.3.1 The Effect of BDNF on Vestibulospinal Neurons In Figure 11 data from the Tables 4a and 4b are represented as greater than X plots. The mean neurite length is shifted from 602 ± 17pm (serum free control) to 871 ± 28um in populations treated with 10ng/ml BDNF (145% of the serum free control). Inset is a similar graph for the data presented in Table 4b as a greater-than-x plot. BDNF (10ng/ml) increased the mean neurite length from 484±14 urn (serum free control) to 652 ± 20|um (135% of serum free control values). Both curves appear to be shifted symmetrically to the right. Figure 13 is the normalized data of all treatments from tables 4a and 4b (Appendix). Dose response for varied concentrations of BDNF looks similar between the two trials. It appears that increasing concentration of BDNF increase the mean neurite length, although the differences are only significant at the higher concentrations. 74 Figure12: A) Cells in the E9 vestibular complex are retrogradely labelled with Dil (red). B) TrkB expression in the E9 vestibular complex (green). Trk B expression C) Hoechst 33258 nuclear stain indicates the position of all cells. D) A composite of A), B), and C). Arrows show cells double labelled with Dil and anti-trkB antibody(yellow). Horizontal chevrons highlight cells that are Dil positive but do not strongly express trkB. Vertical arrowheads indicate cells that express trkB but are not Dil labelled. 75 Figure 13: BDNF increases vestibulospinal neurite outgrowth. Mean neurite lengths of two separate trials were normalized by converting to % of serum free control. BDNF significantly increases vestibulospinal neurite length at concentrations higher than 5ng/ml. Data from tables 4a and 4b. 77 BDNF Increases Vestibulospinal Neurite Outgrowth DMEM 5% FCS FGF-2 0.2ng/rr1 1 ng/ml 5ng/rri 10ng/ml 10ng/mJ BDNF BDNF BDNF BDNF Treatment 78 4.3.2 The Effect of BDNF on Raphespinal Neurons In Figure14 data from the Tables 5a and 5b are represented as greater-than-X plots. In the large graph the mean neurite length (from Table 5a (Appendix)) is shifted from 512+15 (serum free control) to 736 + 18 um in populations treated with 10ng/ml BDNF (144% of the serum free control value). Inset is a similar graph for the data presented in Table 5b (Appendix). BDNF (10ng/ml) increased the mean neurite length from 382 ± 13 um (serum free control) to 573 ± 17pm (150% of the serum free control value). Both curves appear to be shifted symmetrically to the right. Figure 15 is the normalized data of all treatments from tables 5a and 5b (Appendix). Percent increase in growth as compared to the serum free control looks similar between the two trials for varied concentrations of BDNF. 4.3.3 TrkB Immunoreactivity As BDNF was observed to significantly increase neurite outgrowth in vestibulospinal and raphespinal projection neurons, I observed expression of trkB, the receptor through which BDNF is believed to have the majority of its effect on neurite outgrowth. Expression was observed at E9, the first day of treatment and at E10 (raphe) or E11 (vestibular), the final day of treatment. Control sections with primary antibody omitted were also prepared. These were not shown due to the lack of any signal when exposed for the same time as the experimental sections. 79 0 200 400 600 800 1000 1200 1400 1600 neurite length (um) Figure 14: BDNF increases raphespinal neurite length. Main: Raphespinal neurons treated with 10 ng/ml BDNF (mean neurite length = 736 ± 18 urn) show a right shift in distribution as compared to the serum free control (512 ± 15 urn). The shift appears to affect all labelled neuronal populations. From data in Table 5a (Appendix). Inset: Raphespinal neurons treated with 10 ng/ml BDNF (mean neurite length = 573 ± 17 urn) show a right shift in distribution as compared to the serum free control (382 ±13 urn). The shift appears to affect all labelled neuronal populations. From data in Table 5b (Appendix). 80 Figure 15: BDNF increases raphespinal neurite outgrowth. Mean neurite lengths of two separate trials were normalized by converting to % of serum free control. BDNF significantly increases raphespinal neurite length at concentrations higher that 5ng/ml. Data from tables 5a and 5b. 81 BDNF Increases Raphespinal Neurite Outgrowth 200 § 150 o £ fc 100 E 3 l_ V 2 50 o • I I *p<10 -5 Trial 1 Trial 2 DMEM 5% FCS FGF-2 .2ng/ml 1ng/ml 5ng/ml 10ng/ml 10ng/ml BDNF BDNF BDNF BDNF Treatment 82 4.3.1.1 TrkB Immunoreactivity in the Vestibular Complex At E9, the vestibular complex shows expression of trkB in many, but not all of the cell bodies. Figure 12a indicates Dil labelled cells. Dil labelling was photodocumented before immunohistochemical procedures as the detergents used washed out most of the lipophillic tracer from the sections. Figure 12b shows trkB immunoreactivity in the same section. TrkB labelling is spread broadly across the section. Strong labelling is also observed on the large cell bodies of the projection neurons. Figure 12c shows nuclear labelling in the section, effectively identifying the position of all cell nuclei. Figure 12d is a combination of 12a, b, and c. Arrows indicate cells that are labelled with both Dil and trkB. These double labelled cells appear yellow in the figure. Chevrons indicate cells labelled with Dil but lacking strong trkB immunoreactivity. Vertical arrowheads indicate cells that show strong trkB expression but no Dil label. Figure 16 shows trkB immunoreactivity between E9 and E11 tissue sections of the vestibular complex. Both 16a and 16b show strong immunoreactivity in cells labelled with Dil (arrows). Also present are Dil labelled cells that do not show strong immunoreactivity (chevrons) and conversely, immunoreactive cells that are not labelled with Dil (veritcal arrowheads). It is unclear whether non-neuronal cells at either timepoint are trkB labelled due to the high background signal. Figure 17 shows strong trkB immunofluoresence on cultured Dil labelled projection fibers from the vestibular complex, as well as on fibers not labelled with Dil. 83 Figure 16: TrkB expression in Dil labelled vestibulospinal neurons at E9 (A) and E11 (B). Some, but not all cell bodies retrogradely labelled with Dil express trkB (arrows). These appear yellow in the micrographs. Other neurons carry the Dil label, but show no evidence of trkB immunoreactivity (chevrons). TrkB immunoreactive cells with a neuronal morphology that are not Dil labelled are also present (vertical arrowheads). It appears that overall trkB immunoreactivity is decreased at the later time point. 84 8 5 Figure 17: TrkB immunoreactivity on fibers of cultured vestibular explants. Neurites extruded from a cultured explant are strongly immunoreactive for trkB (A), but relatively few carry the Dil label (B). C) a composite of (A) and (B). Similar immunoreactivity is also observed on raphespinal neurites (data not shown). 86 87 4.3.1.2 TrkB Immunoreactivity in the Raphe Nuclei Figure 18 shows trkB immunoreactivity on the raphe nucleus at E9 and E10. TrkB immunoreactivity is apparent on some of the Dil labelled cells at E9 (arrows) and by E10, trkB expression appears on virtually all Dil labelled cells. The high background immunoreactivity makes it difficult to determine if this is true double labelling, or artifactual. It is noteworthy that sections from both time periods also show strong immunoreactivity adjacent to the Dil labelled raphe neurons. In explant cultures this adjacent tissue would be likely also be plated. TrkB negative projection neurons are present (chevrons), as are neurons that are trkB positive but lack Dil labelling (vertical arrowheads). Strong trkB immunoreactivity is not clearly defined on non-neuronal cells at either time point. Raphespinal fibers from cultured explants show a very similar expression pattern to those observed in vestibular projection fibers (not shown). 4.3.2 P75NTR Immunoreactivity The p 7 5 N T R also binds BDNF, and is believed to influence survival response. For this reason I examined p 7 5 N T R expression in the chick vestibular complex at E9 and E11 and in the raphe nuclei at E9 and E10 by immunohistochemistry. 4.3.2.1 p 7 5 N T R Immunoreactivity in the Vestibular Complex Figure 19 shows clear p 7 5 N T R expression in the E9 vestibular complex, on both Dil neurons and non-neuronal cells. P 7 5 N T R is expressed on some Dil labelled cells 88 Figure 18: TrkB expression in Dil labelled raphespinal neurons at E9 (A) and E10 (B). At E9 some, but not all cell bodies retrogradely labelled with Dil that express trkB (arrows). These appear yellow in the micrographs. Other neurons carry the Dil label, but show no evidence of trkB immunoreactivity (chevrons). TrkB immunoreactive cells with a neuronal morphology that are not Dil labelled are also present (vertical arrowheads). By E10 it appears that virtually all labelled cell bodies express trkB. At both time points there is strong trkB immunoreactivity in the regions surrounding the raphespinal cells. TrkB expression in fibers is also apparent. 89 Figure 19: p 7 5 N T R expression in Dil labelled vestibulospinal neurons at E9 (A) and E11 (B). Some, but not all cell bodies retrogradely labelled with Dil express p 7 5 N T R (arrows). These appear yellow in the micrographs. Other neurons carry the Dil label, but show no evidence of p 7 5 N T R immunoreactivity (chevrons). p 7 5 N T R immunoreactive cells with a neuronal morphology that are not Dil labelled are present (vertical arrowheads) as are numerous non-neuronal cells. 91 (arrows), however not all Dil labelled cells express p75 (chevrons), nor are all p75 expressing cells Dil labelled (vertical arrowheads). At E11 p 7 5 N T R expression is similar to that of E9 expression. Double labelled neurons (arrows) are present, as well as Dil positive neurons that appear to lack p 7 5 N T R expression (chevrons). Cell bodies with a neuronal appearance that are not Dil positive also express p 7 5 N T R on both E9 and E11 (vertical arrowheads), as do numerous cells with a non-neuronal morphology. Background immunofluorescence is reduced in comparison to TrkB and IGF-1 R immunohistochemistry suggesting fibers may not be strongly labelled for p 7 5 N T R . This was confirmed on vestibular explant culture fibers that lacked p 7 5 N T R immunofluorescence. 4.3.2.2 p 7 5 N T R Immunoreactivitv in the Raphe Nuclei In Figure 20, it appears that all cells at both time points express p 7 5 N T R , however it is difficult to determine whether these Dil labelled projection neurons are truly double labelled or if the highly immunoreactive fibers at the midline are creating false positives for double labelled cells. Dil labelled projection neurons that appear immunoreactive for p 7 5 N T R are marked with arrows. Dil labelled cells that do not appear immunoreactive are indicated with chevrons, and immunofluorescent cells with a neuronal morphology are also marked (vertical arrowheads). Cells with a non-neuronal morphology also show strong labelling for p75 N T R , and it appears fibers in the raphe nuclei are weakly immunoreactive for p 7 5 N T R . In culture, axons of raphespinal projection neurons were not immunopositive for p 7 5 N T R . 93 Figure 20: p 7 5 N T R expression in Dil labelled raphespinal neurons at E9 (A) and E10 (B). Some, but not all cell bodies retrogradely labelled with Dil appear to express p 7 5 N T R (arrows). It is difficult to determine if these raphespinal neurons are truly expressing p 7 5 N T R a s strong immunoreactivity of fibers at the midline may be producing artifactual double-labelling. Other neurons carry the Dil label, but show no evidence of p 7 5 N T R immunoreactivity (chevrons). p 7 5 N T R immunoreactive cells with a neuronal morphology that are not Dil labelled are present (vertical arrowheads) as are numerous non-neuronal cells. 94 4.4 Discussion BDNF consistently increased mean neurite length in two bulbospinal populations (vestibulospinal and raphespinal) as seen in sections 4.3.1 and 4.3.2. The increase appears to be dose dependant, and at high concentrations (10ng/ml) ranged from 35-45% in vestibulospinal explants and 44-50% in raphespinal populations. This is not at all surprising as the trkB receptor is expressed in the vestibular complex of various vertebrates (Klein et al., 1990; King et al., 1999), as well as in various raphe nuclei (King et al., 1999; Yamuy et al., 2000). It has also been shown that BDNF encourages the sprouting of mature, uninjured serotonergic axons (Mamounas et al., 1995). Potential regeneration of the raphespinal neurons raises serious concerns as aberrant sprouting or growth may result in phantom pain (Sanchez et al., 1995; Apfel, 2000). Other neuronal populations have been observed to express trkB and show a response to BDNF treatment. The red nucleus shows increased regeneration upon in vivo application of BDNF to the cell body (Kobayashi et al., 1997; Liu et al., 1999; Namiki et al., 2000), as do neurons of the reticular formation (Menei et al., 1998). In this study it is clear that trkB is expressed on some but not all labelled projection neurons in the vestibular complex. TrkB is also seen on projecting fibers. This expression is seen both on E9, the first day of treatment and E11, the last day of treatment. It is likely that trkB is also expressed in between these time points. 96 Strong expression of p75 , the other BDNF receptor was observed in the vestibular complex was at both time points. p 7 5 N T R is strongly expressed on both neuronal and non neuronal cells but not on projections of explant cultures. The raphespinal neurons show what appears to be increasing trkB immunoreactivity between E9 and E10, as well as strong p 7 5 N T R immunoreactivity on the first and last day of growth factor treatment. While the literature suggests that trkB is responsible for the changes in neurite growth, it is impossible to discount the p 7 5 N T R a s the signal transducer or a potential co-receptor. Anti-sense knockdown experiments for either receptor might help to decisively determine which receptor is responsible for the effects of BDNF. Alternatively, if similar retrograde labelling could be performed in mice, a complete knockout would prove even more informative. Both trkB and p 7 5 N T R receptors are also expressed on non-neuronal cells making it impossible to determine whether the BDNF acts directly on the neuron or indirectly, via paracrine mechanisms (as observed with fibroblast growth factors (Engele and Bohn 1991)). It should also be noted that these two possibilities are not mutually exclusive and that some combination of the above scenarios may be occurring. Isolation of labelled projection neurons from non-neuronal cells might provide insight into the mechanism of BDNF effects. This could be accomplished by use of a mitioic poison, killing off dividing glial cells while not affecting post-mitotic neurons. If the effect is a direct one, it is would also be interesting to determine whether the growth factor is acting on the cell bodies, or the at the growth cone/axon. This question might be answered using the present assay technique in conjunction with 97 Campenot compartmentalized chambers (Campenot, 1979) to isolate the explant from the growing neurites. BDNF could then be applied to either the growth cones or the cell bodies and the changes in neurite outgrowth quantified and compared. 4.5 Conclusions The experiments performed in this section support the hypothesis that appropriate trophic support will enhance bulbospinal neurite outgrowth. Both trkB and the p 7 5 N T R are present on cell bodies of spinally projecting neurons and trkB is strongly expressed on axons of both populations. This supports my second hypothesis, that receptors for growth factors that increase mean neurite length will be present on projection neurons. While these immunohistochemical studies are informative, it is difficult to draw satisfying conclusions from them due to broad expression of both receptors on cell bodies of both neuronal and non-neuronal cells, as well as the expression of trkB on the neurites. The mechanism of BDNF effects on bulbospinal neurons remains unclear. 98 Chapter 5: IGF-1 and the IGF-1 R in Bulbospinal Neurite Outgrowth 5.1 Introduction In the previous chapter, I focused on the effects of B D N F and the express ion of its receptors on vest ibulospinal and raphespinal cel ls. Similarly, this chapter will focus on the effects of IGF-1 on mean neurite length of bulbospinal neurons, as well as on express ion of the IGF-1 R. Chapter 3 introduced the IGF family of growth factors, their receptors and assoc ia ted signall ing pathways. The d iscuss ion sect ion of this chapter will cons ider how this appl ies to the exper iments performed in this study. Due to the large increase in neurite outgrowth affected by IGF-1 in the vest ibulospinal growth factor sc reen , it was one of three factors se lec ted for further investigation. Exper iments performed in this chapter are similar to those in Chapter 4. A dose response study of IGF-1 effects on both vest ibulospinal and raphespinal populat ions is presented as are greater-than-X plots, in which the shift about the X-ax is cor responds to whether all subpopulat ions are responding to I G F - 1 . Immunohistochemical studies to local ize IGF-1 R express ion are presented for vest ibular and raphe neuronal populat ions on E 9 , the first day of growth factor treatment for both vest ibular and raphe populat ions. Simi lar exper iments are present for E11 vestibular neurons and E10 raphespinal neurons, the last day of culture in each case . 99 5.2 Methods and Materials Egg incubation, retrograde labelling, and brainstem dissections were performed as described in Chapter2. Explant culture and neurite length analysis was performed as in Chapter 3. Immunohistochemistry was done as in Chapter 4, using different primary antibodies. Immunohistochemistry for the IGF-1 R was attempted using IGF-IRp (C-20), IGF-IRa(H-78) affinity purified rabbit polyclonal antibodies(Santa Cruz Biotechnology Inc.), monoclonal GR11 antibody (Oncogene Research Products) as well as M2, M3, and R3 antibodies (generous gifts from Dr. Flora dePablo, Centra de Investigaciones Biologicas, CSIC). R3 provided the best results. 5.3 Results Titered trials were performed using IGF-1 between concentrations of 0.2 ng/ml to 20 ng/ml on vestibulospinal neurons, and 0.2 ng/ml to10 ng/ml on raphespinal neurons. Data was normalized between trials by converting neurite lengths to percent of serum free control, and the two trials were plotted side by side on a single graph. As in the previous chapter, greater-than-X plots have been created to determine if IGF-1 affects all or just specific populations of projection neurons. In addition, representative samples of explant photomontages are displayed for each treatment group (Figure 21) Raw data for these trials is presented in Tables 6 and 7 (Appendix). Data has also been presented as graphs of mean neurite length vs. treatment (Appendix). In an attempt to determine whether the nuclear groups in question expressed receptors for IGF-1, immunohistochemistry was performed for the IGF-1 R. Expression 100 Figure 21: IGF-1 increases neurite outgrowth in vestibulospinal explant cultures. Representitive micrographs of explants in a trial are shown. The serum free control has short neurites (A). Treatment with 5% F C S or FGF-2 increases neurite length (B and C respectively). IGF-1 treatment at 0.2 ng/ml, 1 ng/ml, 5 ng/ml, 10 ng/ml and 20 ng/ml (D, E, F, G, H respectively) increases neurite outgrowth in a dose dependant manner. 101 102 was examined at two time points for each nuclear group. Vestibular IGF-1 R expression was examined at E9 and E11, the first and last day of growth factor treatment, respectively. Raphe IGF-1 R expression was examined at E9 and E10, also the first and last day of growth factor treatment. Hoechst staining was performed but not shown as in the previous chapter. Again, no attempt was made to quantify the proportion of labelled neurons that expressed IGF-1 R. 5.3.1 The Effect of IGF-1 on Vestibulospinal Neurons In Figure 22 data from the tables 6a and 6b are represented as greater-than-X plots. From the data in Table 6a (Appendix 1), the mean neurite length is shifted from 453 + 16 um (serum free control) to 880 + 19 um in populations treated with 20 ng/ml IGF-1, 194% of the serum free control (large plot). The inset figure represents data from Table 6b (Appendix). In this trial the same IGF-1 concentration increased the mean neurite length to 167% of the serum free control (535 ± 14 um to 893 ± 16 um). Both curves appear to be symmetrically shifted to the greater neurite lengths. Figure 23 is the normalized data of all treatments from Tables 6a and 6b (Appendix). Dose response for varied concentrations of IGF-1 looks similar between the two trials. 5.3.2 The Effect of IGF-1 on Raphespinal Neurons In Figure 24 data from the Tables 7a and 7b (Appendix) are represented as greater-than-X plots. From the data in table 7a (Appendix) the mean neurite length is 103 120% 1 neurite length (um) Figure 22: IGF-1 increases vestibulospinal neurite length. Main: Projection neurons from the vestibular complex treated with 20ng/ml IGF-1 (mean neurite length = 880 ± 19 urn) show a right shift in distribution as compared to the serum free control (DMEM; mean neurite length = 453 ± 16 urn). The shift appears to shift the entire distribution of neurite lengths to the right. From data in Table 6a (Appendix). Inset: Projection neurons from the vestibular complex treated with 20ng/ml IGF-1 (mean neurite length = 893 ± 16 urn) show a right shift in distribution as compared to the serum free control (DMEM; mean neurite length = 534 ± 14 urn). The shift appears to shift the entire distribution of neurite lengths to the right. From data in Table 6b (Appendix). 104 Figure 23: IGF-1 increases Vestibulospinal neurite outgrowth. Mean neurite lengths of two separate trials were normalized by converting to % of serum free control. IGF-1 significantly increases vestibulospinal neurite length at concentrations higher than 5ng/ml. Data from tables 6a and 6b. 105 IGF-1 Increases Vestibulospinal Neurite Outgrowth **p<10" 5 250 -, , Serum 5 % F C S F G F - 2 0.2ng/ml 1 ng/ml 5ng/ml 10ng/ml 20ng/ml Free 10ng/ml K3F-1 IGF-1 IGF-1 IGF-1 IGF-1 Control Treatment 106 neurite length (um) Figure 24: IGF-1 increases raphespinal neurite length. Main: Projection neurons from the raphe nuclei treated with 10 ng/ml IGF-1 ( mean neurite length = 721 ± 30um) show a right shift in distribution as compared to the serum free control (512 ± 15um). Inset: IGF-1 increases raphespinal neurite length. Projection neurons from the raphe nuclei treated with 10 ng/ml IGF-1 ( mean neurite length = 543 ± 15um) show a right shift in distribution as compared to the serum free control (382 ± 13um). 107 shifted from 512 ± 15 um (serum free control) to 722 ± 30 um in populations treated with 10 ng/ml IGF-1, 141% of the serum free control (large plot). The inset figure represents data from Table 7b (Appendix). In this trial the same IGF-1 concentration increased the mean neurite length to 142% of the serum free control (382 ± 13 um to 543 ± 15 um). Both curves appear to be symmetrically shifted to the right for all populations. Figure 25 is the normalized data of all treatments from tables 7a and 7b (Appendix). Dose response for varied concentrations of BDNF looks similar between the two trials. 5.3.3 IGF-1 R Immunoreactivity IGF-1 was observed to significantly increase neurite outgrowth in both vestibulospinal and raphespinal neuronal populations. To determine the expression pattern of the IGF-1 R, the high affinity receptor for IGF-1, on these populations, immunohistochemistry was performed. 5.3.3.1 IGF-1 R Immunoreactivitv in the Vestibular Complex Figure 26 shows IGF-1 R expression on vestibular sections on E9 and E11. Despite high background it is possible to see double-labelled projection neurons (arrows) in addition to cells labelled with only Dil (chevrons), or expressing only IGF-1 R (vertical arrows). On E9, IGF-1 R expression is very broad. The background is also high, potentially due to expression of the IGF-1 R on the fibers. 108 Figure 25: IGF-1 increases raphespinal neurite outgrowth. Mean neurite lengths of two separate trials were normalized by converting to % of serum free control. IGF-1 significantly increases raphespinal neurite length at concentrations of 5ng/ml or higher. Data from tables 7a and 7b. 109 IGF-1 Increases Raphespinal Neurite Outgrowth ** p<.10"6 * p<.05 DMEM 5%FCS FGF-2 .2ng/ml 1ng/ml 5ng/ml 10ng/ml 10ng/ml IGF-1 IGF-1 IGF-1 IGF-1 treatment 110 Figure 26: IGF-1 R expression in Dil labelled vestibulospinal neurons at E9 (A) and E11 (B). Some, but not all cell bodies retrogradely labelled with Dil express IGF-1 R (arrows). These appear yellow in the micrographs. Other neurons carry the Dil label, but show no evidence of trkB immunoreactivity (chevrons). IGF-1 R immunoreactive cells with a neuronal morphology that are not Dil labelled are also present (vertical arrowheads). 111 112 By E11, expression appears to be more confined to cell bodies, although high background suggests expression on fibers. While the majority of Dil labelled cells do express the IGF-1 R (arrows) there are also projection neurons that are free of IGF-1 R expression (chevrons). IGF-1 R positive cells with a neuronal morphology but no Dil labelling are also evident on E11 (vertical arrows). Cells that appear morphologically non-neuronal also express IGF-1 R. Neurites from vestibular explants at E11 show strong immunoreactivity (Figure 27). 5.3.3.2 IGFR Immunoreactivitv in the Raphe Nuclei Figure 28 shows IGF-1 R expression in the raphe nucleus on E9 and E10. At E9, expression of IGF-1 R is observed on the majority of labelled cells (arrows). It is difficult to determine whether cells are truly double labelled or if double labelling is an artifact of immunoreactivity of fibers at the midline. Dil labelled cells that are not immunoreactive for IGF-1 R are present (chevrons), while immunoreactive neurons lacking Dil are not clearly visible. By E11 IGF-1 R expression is similar the raphe nucleus although it is still difficult to determine whether Dil labelled cells are truly expressing IGF-1 R it appears that this is the case. Non-Dil labelled immunoreactive cells are visible (vertical arrowheads) and all Dil positive cells appear to double labelled. Strong IGF-1 R labelling is also observed in neurites growing from raphe explants at E10 (not shown). 113 Figure 27: IGF-1 R immunoreactivity on fibers of cultured vestibular explants. Neurites extruded from a cultured explant are strongly immunoreactive for IGF-1 R (A), but relatively few carry the Dil label (B). C) a composite of (A) and (B). Similar immunoreactivity is also observed on raphespinal neurites (data not shown). 114 115 Figure 28: IGF-1 R expression in Dil labelled raphespinal neurons at E9 (A) and E10 (B). At E9 some, but not all cell bodies retrogradely labelled with Dil that express IGF-1 R (arrows). These appear yellow in the micrographs. It is difficult to determine if these raphespinal neurons are truly expressing IGF-1 R a s strong immunoreactivity of fibers at the midline may be producing artifactual double-labelling. Other neurons carry the Dil label, but show no evidence of IGF-1 R immunoreactivity (chevrons). IGF-1 R immunoreactive cells with a neuronal morphology that are not Dil labelled are also present (vertical arrowheads). By E10 it appears that virtually all labelled cell bodies express IGF-1 R . 116 5.4 Discussion The increased neurite length observed after treatment with IGF-1 is in agreement with results presented in the literature. IGF-1 has been shown to promote neurite outgrowth in various neuronal populations both in vitro and in vivo, across a range of organisms (Bondy, 1991; Fernyhough et al., 1993; Zackenfels eta l . , 1995; Kim etal . , 1997; Leventhal et al., 1997; Kurihara et al., 2000). IGF-1 R mRNA expression has been observed in the brainstem (Holzenberger and Lapointe, 2000; Lobie et al., 1993; Gehrmann et al., 1994) as has IGF-1 mRNA (Holzenberger and Lapointe, 2000) and related binding proteins (Bondy and Lee, 1993). The symmetrical shift in the greater-than-X plots indicates that IGF-1 increases neurite outgrowth in all axonal populations in both vestibulospinal and raphespinal populations. In an attempt to determine whether these effects were direct or indirect, immunohistochemistry was performed. While no commercial antibodies were successful in revealing chick IGF-1 R expression, the R3 antibody developed by Dr. Flora dePablo's lab appeared to label specific cells quite well. Unfortunately, this antibody has not been fully characterized and until this is done IGF-1 R immunohistochemical studies must regarded with some caution. At E9, sections through the vestibular complex showed strong labelling of both neuronal and non-neuronal cell bodies. What appears to be high background is more likely heavy labelling of the fibers as seen in Figures 17 and 27. 118 It is impossible to determine whether the IGF-1 effects are direct or indirect due to strong labelling at both time points on both neuronal and non-neuronal cells. If the effect is assumed to be direct, it is also impossible to determine whether IGF-1 is acting on the cell body or the axon and growth cone with the present studies. Segregation of the growing axons and cell bodies by use of Campenot chambers would again be a powerful tool in determining if growth effects are mediated by the axon and growth cone, the cell body, or both. 5.5 Conclusions The experiments performed in this section support the hypothesis that appropriate trophic support will enhance bulbospinal neurite outgrowth. IGF-1 R is present on cell bodies of spinally projecting neurons and trkB is strongly expressed on axons of both populations, supporting the second hypothesis. IGF-1 R also appears to be expressed by non-neuronal cells making it impossible to determine if its effects are direct or indirect. 119 Chapter 6: General Discussion In the previous chapters, evidence was presented to support the hypotheses that were put forth in Chapter 1. This chapter will discuss ideas that have not yet been considered while examining specific aspects of the study. In it, I will attempt to try to tie together the various aspects of the experiments that merit further discussion. 6.1 Drawbacks to the Model While this assay is a powerful tool, it does have some drawbacks. In this section these failures will be discussed, and suggestions made for how experiments might be modified or supplemented to ameliorate future studies. 6.1.1 Use of a Developmental System to Study Spinal Cord Regenerat ion One of the first questions that arises from this study is: how valid is the use of an in vitro, embryonic model in studies that pertain to in vivo, adult conditions in the clinical situation? Gene expression can be radically different between developing and adult organisms. Data produced in this model may not be relevant to adult systems. The model also utilizes an animal (chick) that is further removed from the target organism (humans) than the ideal. Use of similar techniques in a mammalian model may be looked upon more favorably when extrapolating results to humans. While the research performed here may not have direct impact on the clinical aspect of spinal cord injury, it provides essential information about axonal regrowth after injury. This information may lead to future experiments that would more directly impact the clinical situation. The in vitro brainstem explant culture model is ideal for the 120 assessment of growth factor effects on immature chick bulbospinal neurons. The model is simple, yet has the power to observe the effect of growth factors on individual populations of projection neurons, for which there are no reliable, specific molecular markers. This is particularly important, as different bulbospinal populations may have varied trophic requirements. Identification of particularly responsive projection neurons may prove to be an important factor in a multifaceted therapy for SCI. Many studies have shown that young animals show a much greater ability to recover from SCI, especially during embryonic development (Chick: Hasan et al., 1993; Keirstead et al., 1992), opossum (Nicholls et al., 1994; Saunders et al., 1995)). C N S regeneration may depend on reverting adult neurons and/or associated glial cells to an earlier stage in development. Re-capitulation of the developmental state may allow us to utilize information from this study, and other studies of developmental processes, in order to facilitate axonal regeneration. While the chick model has many advantages, it is unlikely to gain popular support until it is transferred into a mammalian model. Similar retrograde labelling and explant culture studies might be performed in the mouse whose corticospinal tract develops postnatally (Gianino et al., 1999), or in the precociously born opossum in which many spinal pathways are incomplete neonatally (Martin et al., 1994). 6.1.2 Use of a Single Growth Factor Another question that should be addressed is the use of a lone growth factor to stimulate neurite outgrowth. In this in vitro study we have, in each case, used a single growth factor in an attempt encourage neurite outgrowth. This, however, is not 121 necessarily the situation in vivo. It is much more likely that several growth factors will be present in the environment, albeit potentially regulated by binding proteins, truncated receptors or various other mechanisms. Future studies must address the impact multiple growth factors on these bulbospinal populations. It has been seen that various factors interact synergistically promoting somite myogenesis (Pirskanen et al., 2000). The same might be true with neurite elongation. While beyond the scope of this work, efforts must be made to identify potential synergistic effects of various growth factors. There is also potential for increased complexity in the systems due to multiple receptor isoforms. The antibody used in the immunohistochemical studies is only sensitive to the full-length trkB isoform. It would be interesting to study expression of the TK- (gp95) isoform, which while having some signaling effects (Baxter et al., 1997; Mamounas et al., 2000; Yacoubian and Lo, 2000), is also believed to prevent BDNF diffusion (Anderson etal . , 1995; Biffo et al.,1995). In addition, the IGF-1 R has been seen to form hybrid receptors with components of the insulin receptor (Siddle et al., 1994). The role and frequency of these hybrid receptors is unknown in the present culture system, nor is information available on whether the R3 antibody has affinity for the hybrid IGF-1 R/IR. The variability in the receptors must be further explored if we are to understand what downstream signaling cascades are involved in the growth promoting effects of BDNF and IGF-1. We must also keep our mind open to the possibility of previously unidentified receptors for the growth factors. There is still a multitude of orphan receptors that have yet to be associated with a specific ligand. 122 6.1.3 Mixed Subpopulations It is also unfortunate that subpopulations of nuclear groups cannot be cultured separately due to mixing within the populations and the difficulties in manipulating such miniscule pieces of tissue. This failing makes it impossible to determine whether there are differences in reaction to growth factors between the subpopulations of projection neurons. Greater-than-X plots can determine if all subpopulations are impacted by a growth factor, but if growth does not affect all projection neurons there is no way of determining which are affected. These distinctions would be made simpler by identification of cellular markers distinct to each subpopulation. 6.1.4 Bias While the analysis of the assay was performed blind, it is important to consider potential bias in the system. For instance, after analyzing several trials, it was relatively easy to pick out vestibulospinal explants treated with FGF-2 due to the halo of non-neuronal cells that would generally surround the explant. While it is hoped that this knowledge would not affect the results of the assay, it is possible that it did. A potential solution to this dilemma would be creation of image software that could trace the neurites accurately and without such bias. 6.1.5 Fasiculation or Collateralizaton? Quantification of the model was achieved by tracing Dil labelled axons projecting from the explant. It does not take into account axons that may produce collateral 123 branches (which would be counted as two separate axons) nor does it account for axons that have fasiculated together. It would not be difficult to utilize techniques such as confocal microscopy to determine whether an axon with a branched appearance was two separate axons growing along each other, or true collateral branching, however to do this for entire data sets would be extraordinarily labor intensive. If a defasiculating agent were added to either the substrate or the media it may eliminate axons growing together allowing for more accurate quantification, as well as studies on growth factor effects on axonal branching. 6.1.6 Increased Survival or Enhanced Outgrowth Throughout the course of these experiments we assume that these growth factors are increasing the neurite outgrowth, that is the rate of process elongation. Alternatively these factors might support neuronal survival. A robust cell body is more likely to put forth a healthy projection than one that is deprived of growth molecules. Despite these drawbacks, the advantages of the model are also numerous. It is a simple, cost-effective and quick way to test potential growth factors. It could just as easily be put to used in determining the effects of various substrates, both permissive and inhibitory. It could also be used in conjunction with Campenot chambers to delineate axon/soma effects. 6.2 Regarding the Cell Populations Used The nuclear populations that this study focuses on are the vestibulospinal and raphespinal groups. These populations were chosen for two reasons: 124 1) they play a significant role locomotor activities and 2) consistant results were obtained from them. Pontine reticular explants were tried in a number of experiments, however results were highly inconsistent. This is unfortunate as the reticular formation plays a major role in locomotion. It is important that further attempts are made to examine growth factors that will stimulate neurite outgrowth in the reticular formation. Other labelled populations of neurons such as the medullary reticulospinal and the noradrenergic coeruleospinal neurons were not attempted for culture. Future studies must explore the trophic requirements of these populations. 6.3 Unmasking Potential Growth Factor Effects by Minimizing Baseline Growth In the experiments performed to determine growth factor effects on neurite length, an attempt was made to keep the serum free control growing at a minimal rate. This was done to ensure that changes caused by the growth factors were not masked by an overly-permissive environment. I used a lower concentration of laminin (25ug/ml) than used in previous studies (75 ug/ml; Pataky et al., 2000), in an attempt to unmask effects that may have been previously hidden. Laminin is found in the C N S developmental^ and is also associated with olfactory bulbs in the mammalian C N S that experience regeneration. Laminin is also expressed on astrocytes in the optic nerves of frogs and goldfish which experience regeneration after injury. It has been suggested that the neurite outgrowth is the sum of positive and negative cues (Rose and Chiba, 1999; Stoeckli, 1997). If too many positive cues are present they might mask potential effects by growth factors. By reducing the level of positive cues it was hoped to reveal any effects of growth factors that have previously gone unobserved. This logic appears 125 to have been effective, as increases in neurite outgrowth have been observed that had previously gone unnoticed. Along similar lines of thought, molecules determined to be inhibitory to neurite outgrowth, fibronectin and the C S P G aggrecan were used as substrates in addition to laminin. Unfortunately, the results (data not shown) of these experiments proved to be inconsistent, and this aspect of the investigation was abandoned early in the research. By reducing permissive cues or addition of actively inhibitory molecules, it may be possible to observe growth factor effects that are unnoticed in highly permissive environments. 6.4 Posi t ive Controls Addition of fetal calf serum to the culture media resulted in increased mean neurite length implying an increased rate of outgrowth. This change in the mean could have resulted from increasing outgrowth in certain subpopulations of neurons, however greater-than-X plots reveal a shift of all neuronal populations to greater length (data not shown). The increases in outgrowth produced are likely the result of various trophic factors present in the serum. The exact mechanism of the agents involved and whether they directly or indirectly impact neurons is yet unknown. FGF-2 at a concentration of 10ng/ml also results in large increases in neurite length. This is in agreement with the effect of FGF-2 in the literature. It is interesting to observe the 'halo' of glia surrounding vestibular explants, but not raphe explants. It is tempting to accredit the glial migration/multiplication for the increased neurite outgrowth, however, this may not be the case as we see that in explants conditioned with F C S , 126 BDNF, or IGF-1 there are significant increases in outgrowth, but no obvious changes in the glia cells. The increase in neurite length with FGF-2 is also observed in raphespinal cells however the halo effect does not occur. This implies that glial proliferation/migration is not a requirement for increased neurite outgrowth, and may just be a secondary effect of FGF-2 treatment. mRNA for FGF-2 receptors was found in glial cells (Pataky et al., 2000) so it is no surprise that non-neuronal cells are effected by FGF-2 exposure. The same study suggests that FGF-2 stimulation of glial cells may be indirectly responsible for increased neurite outgrowth via paracrine interactions. 6.5 Variation in Neurite Outgrowth The phenomenon of some explants not growing as well as others is difficult to explain. Localized infection may be one cause of variation in growth, as might physical damage incurred during the microdissection and plating procedure. Alternatively, it might be caused by the orientation of the explant in culture; that is whether the rostral side is plated up or down. It seems likely that neurons will project caudally as that is their natural pathway. If the explant is plated with its rostral side up, newly extruded neurites will immediately contact the laminin substrate as they emerge from the explant. If the explant is plated with its caudal side up, the neurites may grow upward and finding nothing, have to redirect themselves to locate points of adhesion. The rostral/caudal level of the explant relative to where it is taken from the brainstem is also likely to cause variation between explants in the same treatment group. 127 6.6 Trophic Factor Therapy: A Potential Contributor to SCI Treatment The evidence of trophic factor involvement in both neurodevelopment and neuronal survival and regenerative behavior (i.e. sprouting, axon elongation) certainly suggests that trophic factor replacement therapy may be a candidate for SCI treatment. There are, however, many inherent problems with such therapies. Many trophic factors are peptides that are too large to cross the blood-brain barrier. This leads to invasive intraventricular application methods (cannulas, osmotic pumps), that are often nonspecific, and potentially hazardous. It seems unlikely that such methods will lead to successful clinical improvement, however, as scientific techniques evolve, solutions to such difficulties may arise. Adenoviral vectors, and selectively inducible gene promoters allow for the possibility of gene therapy on specific neuronal groups. Over-expression of trophic factors in desired cell populations for a controlled duration may indeed prove part of a multifaceted therapy for SCI. The study of these factors also leads to an awareness of the intracellular signaling processes that mediate their effects. If these cascades could be effectively manipulated, we would be provided with a powerful tool in the pursuit of an effective treatment for spinal cord injury. 6.7 Conclusions In the first chapter of this study two main hypothesis were put forth: 1. Neurite outgrowth in bulbospinal neurons will be enhanced by appropriate trophic support. 2. Neurons and/or surrounding non-neuronal cells will express receptors for trophic molecules that enhance neurite outgrowth. 128 With regards to the first hypothesis I determined that E G F , GDNF and NGF did not significantly increase vestibulospinal neurite outgrowth. NT-3 and NT-4 did appear to promote significant increases in vestibulospinal neurite outgrowth, but require further assessment to confirm this. IGF-1 and BDNF treatment resulted in significant increases of mean neurite length in both vestibulospinal and raphespinal populations. IGF-1 (20ug/ml) increased mean vestibulospinal neurite length to between 167-193% of the serum free control. IGF-1 (10ug/ml) increased mean raphespinal neurite length to 141-142% of the serum free control. BDNF (10ng/ml) increased mean vestibulospinal neurite to 135-145% of the serum free control, and raphespinal neurite length to between 144-150%of the serum free control. In all cases, the increase in mean neurite length appears to affect the length of all projecting neurites, as opposed to a specific population. With regard to the second hypothesis, it was determined that some, but not all labelled vestibulospinal projection neurons express trkB, p 7 5 N T R , and IGF-1 R, at two time points , E9 and E11. Expression was not confined to neuronal cells, as cell with non-neuronal morphologies were also seen to express the three receptor molecules at both time points. Fibers (E11) were also shown to be highly immunoreactive for trkB and IGF-1 R, with little immunoreactivity for the p 7 5 N T R . Receptor expression on raphespinal projection neurons was more difficult to judge as their localization near the highly immunoreactive fibers at the midline may have produced artifactual double labelling. While it remains unclear if Dil labelled raphespinal soma express these receptors, it was clear that fibers (E10), both Dil labelled and non-129 labelled, appeared highly immunoreactive for trkB and IGF-1 R. These fibers did not show immunoreactivity the p 7 5 N T R . Non-neuronal cells were strongly immunoreactive for all three receptors. 6.8 Future Experiments While the experiments conducted in this study have yielded interesting results, this work is in no way exhaustive, either in regards to the potential of this model system, or to how the selected growth factors affect regenerative behaviors. This study focused on two major populations of bulbospinal neurons, those from the vestibular complex and the raphe nuclei. It is essential that further work be done on neurons of the pontine and medullary reticular formation, as these also play a key role in locomotion. 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Bold values are significant: p<10"4 Treatment Mean Neurite Std. Error % of Serum #of # of Pooled Length (um) (+/-) Free Control Explants Neurites Serum Free 602 17 100 5 427 Control 5% FCS 1285 29 213.29 5 316 FGF-2 10ng/ml 956 20 158.70 5 269 0.2ng/ml BDNF 595 16 98.79 5 293 1 ng/ml BDNF 801 19 132.91 5 486 5ng/ml BDNF 769 23 127.57 5 277 10ng/ml BDNF 872 28 144.69 5 284 Table 4b: Summary Table for Effects Titred BDNF on Vestibulospinal Neurite Outgrowth. Treatment, mean neurite length, standard error, number of pooled explants and total number of neurites are shown. Bold values are significant: p<10"5 Treatment Mean Neurite Std. Error % of Serum #of # of Pooled Length (um) (+/-) Free Control Explants Neurites Serum Free 484 14 100 6 538 Control 5% FCS 809 24 167.29 6 402 FGF-2 10ng/ml 749 15 154.82 4 431 0.2ng/ml BDNF 403 14 83.24 4 329 1 ng/ml BDNF 426 14 87.95 5 314 5ng/ml BDNF 595 15 122.88 6 518 10ng/ml BDNF 652 20 134.79 6 452 149 Figure 29: BDNF increases vestibulospinal neurite outgrowth. A) Graph of Mean Neurite Length (um) vs. Treatment for data in Table 4a. B) Graph of Mean Neurite Length (um) vs. Treatment for data in Table 4b. 150 1400 £)200 3^ £1000 Ui c ® 800 © • C 600 3 9 ~~ 400 BDNF Increases Vestibulospinal Neurite Outgrowth ,*p<1° p<10-c ra o 200 Serum Free 5% FCS FGF-2 0.2ng/ml 1 ng/ml 5ng/ml 10ng/ml Control 10ng/ml BDNF BDNF BDNF BDNF Treatment 900 I 800 E 700 -3 600 -.C O) c 500 -_ l 400 -1 • "C 300 -(1) z 200 -100 -BDNF Increases Vestibulospinal Neurite Outgrowth y<w DMEM 5% FCS FGF-2 10ng/ml .2 ng/ml BDNF Treatment 1 ng/ml BDNF 5 ng/ml BDNF p<10" 10 ng/ml BDNF 151 Table 5a: Summary Table for Effects of Titred BDNF on Raphespinal Neurite Outgrowth. Treatment, mean neurite length, standard error, number of pooled explants and total number of neurites are shown. Bold values are significant: p<10"5 Treatment Mean Neurite Std. Error % of Serum #of # of Pooled Length (um) (+/-) Free Control Explants Neurites Serum Free 512 15 100 4 385 Control 5% FCS 648 30 126.63 3 155 FGF-2 10ng/ml 658 25 128.52 4 175 0.2ng/ml BDNF 525 16 102.54 5 492 1 ng/ml BDNF 607 17 118.51 4 312 5ng/ml BDNF 693 19 135.43 5 312 10ng/ml BDNF 737 18 143.92 4 379 Table 5b: Summary Table for Effects of Titred BDNF on Raphespinal Neurite Outgrowth. Treatment, mean neurite length, standard error, number of pooled explants and total number of neurites are shown. Bold values are significant: p<10"5 Treatment Mean Neurite Std. Error % of Serum #of # of Pooled Length (urn) (+/-) Free Control Explants Neurites Serum Free 382 13 100 7 677 Control 5% FCS 449 25 117.45 7 186 FGF-2 10ng/ml 522 19 136.62 5 347 0.2ng/ml BDNF 248 11 64.94 3 141 1 ng/ml BDNF 446 46 116.58 5 194 5ng/ml BDNF 551 22 144.17 6 316 10ng/ml BDNF 573 17 149.92 5 566 152 Figure 30: BDNF increases raphespinal neurite outgrowth. A) Graph of Mean Neurite Length (um) vs. Treatment for data in Table 5a. B) Graph of Mean Neurite Length (um) vs. Treatment for data in Table 5b. 153 7 0 0 BDNF Increases Raphespinal Neurite Outgrowth p<10" BDNF Increases Raphespinal Neurite Outgrowth c _J 0) 3 800 700 600 500 400 300 200 100 0 D M E M 5 % F C S F G F - 2 10ng/ml .2ng/ml B D N F Treatment 1 ng/ml B D N F 5ng/ml B D N F p<10-** 10ng/ml B D N F 154 Table 6a: Summary Table for Effects of Titred IGF-1 on Vestibulospinal Neurite Outgrowth. Treatment, mean neurite length, standard error, number of pooled explants and total number of neurites are shown. Bold values are significant: p<10"5 Treatment Mean Neurite Std. Error % of Serum #of # of Pooled Length (urn) (+/-) Free Control Explants Neurites Serum Free 453 16 100 6 335 Control 5% FCS 984 33 216.97 7 334 FGF-2 10ng/ml 821 22 181.08 6 346 0.2ng/ml IGF-1 554 16 122.23 7 431 1 ng/ml IGF-1 597 17 131.57 7 397 5ng/ml IGF-1 775 19 170.89 7 507 10ng/ml IGF-1 689 21 151.88 6 403 20ng/ml IGF-1 880 19 193.98 6 478 Table 6b: Summary Table for Effects of Titred IGF-1 on Vestibulospinal Neurite Outgrowth. Treatment, mean neurite length, standard error, number of pooled explants and total number of neurites are shown. Bold values are significant: p<10"5 Treatment Mean Neurite Std. Error % of Serum #of # of Pooled Length (um) (+/-) Free Control Explants Neurites Serum Free 535 14 100 5 427 Control 5% FCS 931 30 174.13 5 213 FGF-2 10ng/ml 897 15 167.82 5 311 0.2ng/ml IGF-1 446 12 83.39 5 194 1 ng/ml IGF-1 564 13 105.46 5 288 5ng/ml IGF-1 727 15 135.94 5 365 10ng/ml IGF-1 791 14 147.97 5 358 20ng/ml IGF-1 893 16 167.13 5 377 155 Figure 31: IGF-1 increases Vestibulospinal neurite outgrowth. A) Graph of Mean Neurite Length (um) vs. Treatment for data in Table 6a. B) Graph of Mean Neurite Length (um) vs. Treatment for data in Table 6b. 156 1200 ^ 1000 E r 800 600 cn c c 0) •Z 400 C 200 4-IGF-1 Increases Vestibulospinal Neurite „ p < 1 c r i Outgrowth S e r u m Free Contro l 5 % F C S F G F - 2 10ng/ml .2ng/ml IGF-1 1 ng/ml IGF-1 5ng/ml 10ng/ml 20ng /ml IGF-1 IGF-1 IGF-1 treatment E 1 2 0 0 3 1 0 0 0 SZ O) c 8 0 0 6 0 0 3 4 0 0 a> c c 2 0 0 tz ffl 0 IGF-1 Increases Vestibulospinal Neurite Outgrowth p<10-S e r u m 5 % F C S F G F - 2 0 .2ng /m l 1 ng /m l 5 n g / m l 1 0 n g / m l 2 0 n g / m l F ree 1 0 n g / m l IGF-1 IGF-1 IGF-1 IGF-1 IGF-1 Cont ro l treatment 157 Table 7a: Summary Table for Effects Titred IGF-1 on Raphespinal Neurite Outgrowth. Treatment, mean neurite length, standard error, number of pooled explants and total number of neurites are shown. Bold values are significant: p<10"5. IGF Raphe Treatment Mean Neurite Std. Error % of Serum #of # of Pooled Length (urn) (+/-) Free Control Explants Neurites Serum Free 511.79 14.54 100 4 385 Control 5% FCS 648 30 126.63 3 155 FGF-2 10ng/ml 658 25 128.52 4 175 0.2ng/ml IGF-1 510 19 99.60 5 281 1 ng/ml IGF-1 474 22 92.68 4 167 5ng/ml IGF-1 605 16 118.14 5 403 10ng/ml IGF-1 722 30 140.99 3 170 Table 7b: Summary Table for Effects Titred IGF-1 on Raphespinal Neurite Outgrowth. Treatment, mean neurite length, standard error, number of pooled explants and total number of neurites are shown. Bold values are significant: p<10"5. Treatment Mean Neurite Std. Error % of Serum #of # of Pooled Length (urn) (+/-) Free Control Explants Neurites Serum Free 382 13 100 7 677 Control 5% FCS 449 25 117.45 7 186 FGF-2 10ng/ml 522 19 136.62 5 347 0.2ng/ml IGF-1 397 15 103.85 6 406 1 ng/ml IGF-1 385 19 100.69 5 255 5ng/ml IGF-1 559 14 146.35 5 688 10ng/ml IGF-1 543 15 142.16 7 665 158 Figure 32: IGF-1 increases raphespinal neurite outgrowth. A) Graph of Neurite Length (um) vs. Treatment for data in Table 7a. B) Graph of Neurite Length (um) vs. Treatment for data in Table 7b. 159 IGF-1 Increases Raphespinal Neurite Outgrowth. p<.05 ' D<10"5 800 700 3, 600 °> 500 <D 400 i 300 c ro 200 C) 100 ** r l n r -E-l - i -DMEM 5%FCS FGF-2 .2ng/ml 1 ng/ml 5ng/ml 10ng/ml 10ng/ml IGF-1 IGF-1 IGF-1 IGF-1 Treatment IGF-1 Increases Raphespinal Neurite Outgrowth 700 600 g 500 3 £ 400 ra c 0) O 300 3 « 200 100 0 DMEM •p<10" 5% FCS FGF-2 10ng/ml .2ng/ml IGF-1 1ng/ml IGF-1 5ng/ml IGF-1 10ng/ml IGF-1 treatment 160 Table 8: S u m m a r y Tab le for Effects Titred E G F on Vest ibu lospinal Neurite Outgrowth. Treatment, mean neurite length, standard error, number of pooled explants and total number of neurites are shown. Treatment M e a n Neurite Std. Error % of Se rum #o f # of Poo led Length (urn) (+/-) Free Control Explants Neurites Serum Free 556 15 100 6 399 Control 5% FCS 627 34 112.70 6 98 10ng/ml FGF-2 892 14 160.40 5 371 0.16 ng/ml EGF 494 14 88.84 6 349 0.64 ng/ml EGF 432 12 77.77 6 285 2.5ng/ml EGF 425 10 76.41 5 243 10ng/ml EGF 528 12 94.95 6 349 161 

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