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Immunological suppression of central nervous system (CNS) myelin and the effect of myelin suppression… Keirstead, Hans Stegmann 1994

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IMMUNOLOGICAL SUPPRESSION OF CENTRAL NERVOUS SYSTEM (CNS) MYELIN AND THE EFFECT OF MYELIN SUPPRESSION ON CNS REPAIR AFTER INJURY By HANS STEGMANN KEIRSTEAD B.Sc., University of British Columbia, 1990  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (ZOOLOGY) We accept this thesis as conforming to he required standard  THE UNIVERSITY OF BRITISH COLUMBIA September 1994 ©Hans Stegmann Keirstead, 1994  In presenting this thesis in partial fulfillment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission.  Department of The University of British Columbia Vancouver, Canada Date S  II  ABSTRACT In higher vertebrates, axons within the differentiated central nervous system (CNS) possess a very limited capacity for repair after injury. The following experiments were designed to determine the contributions of CNS myelin to the lack of regeneration observed following transection of the late embryonic and hatchling chick spinal cord. The developmental onset of myelination in the chick begins at embryonic day (E) 13 of the 21 day developmental period. Spinal cord transections after the developmental onset of myelination result in little or no neuroanatomical repair or functional recovery. However, intraspinal injection of complement-binding galactocerebroside (GalC) antibodies or 04 antibodies (which react with sulfatide, seminolipid and an unidentified antigen on oligodendrocyte progenitors) plus complement between E9-E12 results in a delay in the onset of myelination until E17 (developmental myelin-suppression). A subsequent transection of the spinal cord as late as El 5 (i.e. during the normal restrictive period for repair) results in complete neuroanatomical regeneration and functional recovery. Spinal cord transections on El5 in a normally-myelinated embryo result in no neuroanatomical regeneration or functional recovery. These findings indicate that CNS myelin is inhibitory to the functional regeneration of transected spinal cord in embryonic chick (Keirstead et al. 1992). These studies also suggest that myelin suppression might also facilitate regeneration after adult spinal cord injury. Hatchling chickens are precocial and  III  their brainstem and spinal cord can be considered in all respects adult-like. Administration of complement-binding GaIC antibodies or 04 antibodies plus complement to the hatchling spinal cord results in the transient removal of spinal cord myelin (immunological demyelination). The thoracic cord of posthatching day (P)2-P10 chickens were completely transected and immunological demyelination was simultaneously initiated. Fourteen to 28 days later, retrograde tract tracing, including double-labeling studies, indicated that approximately 5-15% of the brainstem-spinal projections had regenerated across the transection site to lumbar levels. Even though voluntary locomotion was not observed after recovery, focal electrical stimulation of identified brainstem locomotor regions evoked either stepping movements or ‘fictive’ stepping in paralysed animals (collaborative studies, see chapter 5). This indicates that the transient demyelination of injured hatchling (i.e. mature) chick spinal cord facilitated axonal regeneration resulting in some functional synaptogenesis with spinal neurons.  iv TABLE OF CONTENTS ABSTRACT TABLE OF CONTENTS  iv  LIST OF TABLES  vi  LIST OF FIGURES  vii  LIST OF ABBREVIATIONS  ix  ACKNOWLEDGEMENTS  xi  CHAPTER 1 General introduction  1  CHAPTER 2 Myelin development in the embryonic chick spinal cord  18  INTRODUCTION  19  MATERIALS AND METHODS  29  RESULTS  34  DISCUSSION  50  CHAPTER 3 Developmental myelin-suppression in the embryonic  55  chick spinal cord INTRODUCTION  56  MATERIALS AND METHODS  65  RESULTS  67  DISCUSSION  90  V  CHAPTER 4 Neuroanatomical repair and functional recovery of  104  transected spinal cord in embryonic chick INTRODUCTION  105  MATERIALS AND METHODS  111  RESULTS  114  DISCUSSION  129  CHAPTER 5 Neuroanatomical repair and physiological recovery  139  following transection and immunological demyelination of the hatchling chick spinal cord INTRODUCTION MATERIALS AND METHODS  140 -  147  RESULTS  150  DISCUSSION  180  CHAPTER 6 General discussion  196  REFERENCES  213  vi LIST OF TABLES  Table 5-1  Summary of immunological demyelination with single injection  156  vii LIST OF FIGURES  Chapter 2 Figure 2-1  GaIC during development  35  Figure 2-2  CNP during development  37  Figure 2-3  MBP during development  40  Figure 2-4  MAG during development  42  Figure 2-5  Toluidine blue E15 spinal cord  45  Figure 2-6  GFAP during development  47  Figure 3-1  Developmental myelin-suppression (MBP, MAG, CNP)  68  Figure 3-2  Developmental myelin-suppression (toluidine blue)  73  Figure 3-3  Onset of myelination following developmental  76  Chapter 3  myelin-suppression Figure 3-4  Hatchling levels of myelination following developmental  79  myelin-suppression Figure 3-5  Immunological control injections  82  Figure 3-6  GFAP during and after developmental myelin-suppression  85  Figure 3-7  MAP-2 during developmental myelin-suppression  88  Figure 3-8  Schematic representation of mechanism of developmental  91  myelin-suppression Figure 3-9  MBP mRNA during developmental myelin-suppression  96  VIII  Chapter 4 Figure 4-1  Spinal cord transection control  115  Figure 4-2  Confirmation of developmental myelin-suppression  117  Figure 4-3  Neuroanatomical regeneration in the embryo  120  Figure 4-4  Neuroanatomical regeneration with transection control  1 26  Figure 4-5  Physiological recovery  131  Figure 5-1  Immunological demyelination  151  Figure 5-2  Immunological control injections  154  Figure 5-3  Time course of immunological demyelination  157  Figure 5-4  14-day immunological demyelination and remyelination  160  Figure 5-5  GFAP during immunological demyelination  163  Figure 5-6  Transection site 20 days post-transection  166  Figure 5-7  Spinal cord transection control  168  Figure 5-8  Neuroanatomical regeneration in the hatchling  172  Figure 5-9  Neuroanatomical regeneration in hatchling- double labelling  178  Chapter 5  Figure 5-10 Physiological recovery- EMG  185  Figure 5-1 1 Physiological recovery- ENG  187  Figure 5-12 Immunological demyelination in mice  191  Chapter 6 Figure 6-1  Astrogliosis following hatchling spinal cord transection  205  ix LIST OF ABBREVIATIONS bFGF  Basic fibroblast growth factor  CAM  Cell adhesion molecule  CBDA  Cascade blue labelled dextran amine  CNP  2’,3’-cyclic nucleotide 3’-phosphodiesterase  CNS  Central nervous system  E  Embryonic day  EAE  Experimental allergic encephalomyelitis  ECM  Extracellular matrix  EGF  Epidermal growth factor  EMG  Electromyogram  ENG  Electroneurogram  FGF  Fibroblast growth factor  GalC  Galactocerebroside  GFAP  Glial fibrillary acidic protein  hr  Hour  HSPG  Herarin sulfate proteoglycan  MAP-2  Microtubule-associated protein-2  MBP  Myelin basic protein  MAG  Myelin-associated glycoprotein  mRNA  Messenger ribonucleic acid  NCAM  Neural cell adhesion molecule  x NGF  Nerve growth factor  NTF  Neurotrophic factor  02A  Oligodendrocyte, Type-2 Astrocyte  P  Posthatching day  PDGF  Platelet derived growth factor  PECT  Pectoralis  PN  Postnatal day  PNS  Peripheral nervous system  RDA  Rhodamine labelled dextran amine  SART  Sartorius  TGF  Transforming growth factor  pm  Micron  xi ACKNOWLEDGEMENTS  I will begin by acknowledging three people whose influence led me to embark on the academic road that I find myself on. My father Kenneth Eugene Keirstead has shown me by example that a diligent work ethic unfailingly yeilds rewards. I have learnt to identify the price to pay for every action, and so doing am able to make new beginnings with an informed and realistic perspective. My mother Sandra Marlene Keirstead has enforced in me a ‘geshtalt’ method of viewing existing and foreseen situations. She has also provided unfailing emotional support for which I am eternally grateful. David John Roberts has been a friend and inspiration to me from a very young age. I recognized many years ago that it would take more than a Ph.D. to equal his creative and active mind. I thank my wife Melanie Gaye ter Borg for all of the love, balance and support that she has provided. Melanie, you have shown me that it is possible to enjoy a rich and diverse lifestyle, even during times of chaos and confusion at the workplace. Thank-you for making my life so much fun. John Douglas Steeves has taught me far more than research skills during my graduate years. You have been both mentor and friend. Thank-you for your wisdom and perspective. I would also like to thank Gillian Dawn Muir for her help with aspects of my thesis, but more importantly for making me smile so much at work. Gillian, it  xl’  has been nothing but fun working with you. Thanks also to Diane Henshel, David M. Pataky and Gerald Sholomenko for their advice and collaborative efforts. This thesis would not have been possible without the technical support of Karen Goh, John McGraw, Karin Mathias and Ania B. Wisniewska. I cannot thank these individuals enough for their commitment, interest and hard work. Finally, I would like to thank the Natural Sciences and Engineering Research Council of Canada and the Network of Centres of Excellence for Neural Regeneration and Functional Recovery for their scholarship support.  1  CHAPTER 1  GENERAL INTRODUCTION  2 Traumatic injury to the central nervous system (CNS) sets into motion a myriad of intracellular and intercellular events involved in the immune response, cellular proliferation, axonal degeneration and, in few cases, neuronal regeneration. Successful neuronal regeneration requires the re-extension of severed axons, axonal pathfinding, synapse formation, and ultimately the restoration of physiological functions. Different types of neurons within and between species differ in their ability to regenerate following injury. For example, CNS neurons of invertebrates and lower vertebrates are able to regenerate axonal processes. Severe injury to the tectum of goldfish (Grant and Keating 1986), the spinal cord of goldfish (Bernstein 1964) and axolotl (Grimm 1971; Holder et al. 1982), the retinotectal projections of fish and amphibians (Easter 1983) or the VIlIth cranial nerve of Rana pip/ens (Zakon and Capranica 1 981) results in successful neuronal regeneration and restoration of function. The ability of CNS neurons to regenerate has been gradually lost over the course of evolution, such that regeneration is generally restricted to anamniotes  in a state of continuous growth. Perhaps the best studied example in this context is the regenerative ability of the optic nerve and tectal efferents of Rana pip/ens (Easter 1983). The retinotectal system of post-metamorphic Rana pip/ens undergoes continuous neurogenesis of retinal ganglion cells, followed by axonal projection and functional synapse formation (Easter 1983). In contrast to the dramatic regenerative ability of the Rana pip/ens optic nerve, tectal efferents fail to regenerate following injury (Lyon and Stelznêr 1987). Neurogenesis in the  3 frog tectum stops at metamorphosis (Grant and Keating 1986). Thus, Rana pipiens may exist at an intermediate stage in the phylogenetic trend towards a loss of regenerative capabilities. The adult CNS of higher vertebrates has a very limited capacity for regeneration following injury. CNS regeneration in higher vertebrates is restricted to primary olfactory axons (Barber 1981), monoaminergic fibres (Bjorklund et al. 1971), unmyelinated cholinergic axons (Bjorklund et al. 1971) and neurosecretory fibres (Dellman 1973). Axotomy in any other region of the adult CNS results in traumatic degeneration of both the proximal and distal stumps of the severed axons (Ramon y Cajal 1959). Proximal stumps undergo an initial stage of retrograde degeneration (usually back to the nearest collateral branch) followed by an aborted attempt at regeneration (growth cone formation and elongation of approximately 1-2mm; Cajal 1928). Due to a lack of trophic support, distal axonal stumps undergo Wallerian degeneration and are ultimately resorbed by glial cells (WaIler 1852; Ranvier 1871, 1873; Ramon y Cajal 1959). The lack of regeneration exhibited by adult CNS neurons of higher vertebrates does not imply that these neurons lack the intrinsic capability to regenerate following injury, If peripheral nerve segments containing Schwann cells are grafted to a site of CNS injury, severed CNS axons are able to grow out and make functional synaptic connections with their targets (Ramon y Cajal 1959; David and Aguayo 1981; Aguayo et al. 1991). Similarly, axotomized CNS neurons will extend axons in vivo through implants of fetal CNS tissue  4 (Bjorklund and Stenevi 1979; Kromer et al. 1981; Bjorklund 1991), or implants consisting of fibroblasts genetically modified to express growth factors (Fisher and Gage 1993; Tuszynski et al. 1994). In addition, motor neurons residing within the ventral horn of the spinal cord are capable of regenerating their peripheral projecting axon if the axotomy took place within the PNS (Ramon y Cajal 1959). These findings indicate that CNS neurons retain intrinsic growth programs which enable long-distance axonal regeneration in the presence of a favorable extraneuronal environment. The factors contributing to the poor regenerative capacity of the mature CNS of higher vertebrates can be divided into two general categories: 1) growth promoting factors present in the developing system which are either absent or aberrently expressed following adult injury, and 2) growth inhibitory factors which are chronically expressed in the adult CNS or are upregulated following adult injury.  Growth Promoting Factors Growing, regenerating or mature axons in the adult state are constantly exposed to an extremely complex molecular environment. Extracellular matrix (ECM) molecules facilitate cell attachment, cell migration and process extension during development and are in a position to intimately influence regenerating axons. Fibronectin is a widely distributed ECM component which is predominantly expressed by radial glia during a limited period of neuritic growth  5 in development (Sheppard et al. 1991). Astrocytes, however, fail to upregulate fibronectin following injury to the adult CNS (Egan et al. 1991). Laminin is also present in the developing ECM of virtually all higher vertebrates, providing a substrate for cell migration and anchorage (Carbonetto 1984). However, like fibronectin, laminin does not appear to be detectably upregulated following CNS injury (Sosale et al. 1988). Laminin upregulation has been observed following optic nerve transection, however, injury-induced sprouting was confined to laminin(-) areas (Giftochristos and David 1988). Finally, heparin sulfate proteoglycan (HSPG; one of several proteoglycans which form the major constituents of CNS ECM) has been shown to promote cell attachment and speading. Again, HSPG is only seen transiently in development during the period of pattern formation and connectivity (Steindler et al. 1990). Cell adhesion molecules (CAMs) constitute another set of adhesive proteins that are fundamental to the motility of the growth cone. CAMs are divided into two groups, the Ca - independent immunoglobulin superfamily and 2 the Ca - dependent, homophilically-interacting cadherin family. Molecules 2 belonging to the immunoglobulin superfamily share structural motifs referred to as immunoglobulin domains, each consisting of approximately 100 amino acids looped by a disulfide bridge (Cunningham et al. 1987). Members of the immunoglobulin superfamily include Li, Ng-CAM, contactin, Fl 1 /F3, Tag 1/axonin-1, MAG, P (Grumet 1991), Nr-CAM (Grumet et al. 1991), SC1/DM0 GRASP (Tanaka et al. 1991) as well as the phylogenetically oldest and best-  6 characterized member, neural cell adhesion molecule (N-CAM; reviewed in Carbonetto and David 1993). N-CAM isoforms are expressed by oligodendrocytes (Bhat and Silberbeg 1986), astrocytes (Noble et al. 1985) and neurons (Nybroe et al. 1989) and bind in a homophilic or heterophilic manner to components of the ECM including heparin sulfate proteoglycan (Cole and Akeson 1989). A threshold level of N-CAM expression is required before neurite outgrowth can occur; small increases in N-CAM levels beyond this threshold result in great increases in neurite outgrowth-promoting ability (Doherty et al. 1990a). During development, however, the neurite outgrowth-promoting ability of N-CAM is lost (Doherty et al. 199Ob) N-CAM becomes less sialyated (Rothbard et al. 1982), less sulfated and phosphorylated with development (Linnemann et al. 1985). The loss in neurite outgrowth-promoting ability can be attributed to the reduction in polysialic acid content, which increases the avidity of N-CAM-N-CAM interactions (Doherty et al. 1990b). An advancing filopodium must make contacts that are adhesive enough to support the contractile forces that lead a growth cone, but not so adhesive as to inhibit further advance. Cadherins are a superfamily of CAMs that include CNS-specific neural (N), retinal (R), brain (B) and truncated (T) cadherins. Cadherins interact intracellularly with actin microfilaments and cytoplasmic proteins called catenins (McGee and Buxton 1991) and share several common structural features including Ca - binding sites (Takeichi 1991). All of the various types of 2 cadherins interact homophilically with like cadherins on another cell to generate  7 adhesion (Takeichi 1991). Cadherins are expressed early in the developing nervous system (Takeichi 1991). CNS regeneration- or degeneration-induced changes in cadherin expression have yet to be documented. Integrins are a superfamily of cell-surface receptors for ECM molecules that are found in abundance on growth cones (Carbonetto 1984). lntegrins are heterodimers that share a common beta subunit and are classified into families according to their alpha subunit. The presence of appropriate ECM receptors is clearly a prerequisite for the advancement of developing or regenerating neurites. Although integrins are found in many developing systems (Reichardt and Tomaselli 1991), they are functionally down-regulated during development, which may partially account for the reduced axonal regeneration seen in vivo as the CNS matures. In order to facilitate axonal advancement, ECM degradation may be initiated by proteases such as plasminogen activators (Pittman 1 985), protease nexins (Rovelli et al. 1992) and metalloproteases (Machida 1991). Although proteases have been associated with axonal growth in many CNS areas (Sumi et al. 1992), an in vivo role can not be firmly established since studies of proteases have generally been limited to culture assays. Nonetheless, it has been suggested that proteases may facilitate axonal regeneration by degrading or altering the ECM in advance of the growth cone (Brodkey et al. 1993). Perhaps the most important group of molecules influencing CNS development and regeneration are neurotrophic factors (NTFs), or growth  8 factors. Over 40 years ago Hamburger and Levi-Montalcini proposed that neurons are overproduced during development and, during maturation of the nervous system, compete for a target-derived factor or factors. Due to a limited supply of these NTFs, only a portion of the original population of neurons survive. Thus, it was postulated that specific NTFs promote the survival, differentiation and maturation of different populations of neurons in development (Hamilton and Levi-Montalcini 1949). These early experiments facilitated the discovery and characterization of nerve growth factor (NGF), which has been shown to regulate the size of specific neuronal populations during periods of naturally-occurring cell death (Harper and Thoenen 1981). NGF has subsequently been shown to belong to a family of related molecules, collectively known as the neurotrophins. Basal forebrain cholinergic neurons, sympathetic neurons and a subpopulation of sensory neurons are sensitive to NGF, while the other members of the neurotrophin family, brain-derived neurotrophic factor (BDNF), neurotrophin (NT)-3 and NT-4/5 have more widespread effects on central and peripheral neurons (Thoenen 1991). For example, NT-3 has been shown to prevent the death of noradrenergic neurons of the locus coeruleus (Arenas and Persson 1994), enhance sprouting in corticospinal tract axons during development and after spinal cord injury (Schnell et al. 1 994), and play a role in the survival of spinal proprioceptive afferents and their peripheral sense organs (muscle spindles and Golgi tendon organs) in development (Ernfors et al. 1994).  9 Other NTFs not belonging to the neurotrophin family also influence neuronal survival during development and after CNS injury. Transforming growth factor (TGF)-beta, platelet-derived growth factor (PDGF), fibroblast growth factor (FGF) and epidermal growth factor (EGF) have all been shown to have direct or indirect effects on CNS neurons (reviewed in Brodkey et al. 1993). Many of these NTFs also have dramatic effects on the glial population of the CNS as well, such as gliogenesis, the induction of macrophage motility, stimulation of angiogenic growth, or facilitation of a new glial limitans formation around a site of CNS injury (Brodkey et al. 1993). NTF receptor distribution is also an important consideration in determining the functions of specific NTFs in the developing and regenerating CNS. Members of the neurotrophin family bind with low affinity to a common receptor (p75), but additionally require specific high-affinity receptors encoded by trk proto-oncogenes for signal transduction (Meakin and Shooter 1992). Clearly, attempts to manipulate the NTF environment of regenerating neurons must also consider the expression of appropriate NTF receptors by the neuronal population of interest.  Growth Inhibitory Factors The growth of neurites to their targets during nervous system development clearly requires the appropriate spacial and temporal expression of adhesion molecules. While the adhesion molecules described above direct  10 neurite growth by enhancing cellular adhesion, several adhesion molecules have been described that direct neurite growth by providing a non-permissive substrate. Anti-adhesive molecules may increase the efficiency of surface recognition processes for directed neurite outgrowth. Injury to the CNS results in the local release of cytokines and NTFs from injured neurons and glial cells (Hugh Perry et al. 1993; Nakajima and Kohsaka 1993). These factors stimulate astrocytes directly or indirectly (via the stimulation of microglia) to assume a reactive state, characterized by the upregulation of glial fibrillary acidic protein and expression of several cell-surface molecules that are normally not expressed, or are expressed at low levels (Hertz et al. 1990; Reier and Houle 1988). Tenascin is one such molecule that is upregulated by astrocytes after injury (Brodkey et al. 1993). Tenascin is expressed in the CNS during development (Chiquet 1989) and, as a result of its anti-adhesive properties, may guide growing neurites by growth cone diversion (Martini and Schachner 1991). In stripe culture assays, neurons tend to avoid areas containing tenascin, preferring to grow on laminin, fibronectin or tenascin free polyornithine (Faissner and Kruse 1990). Developing astrocytes also express several anti-adhesive proteoglycans including chondroitin sulfate, dermatan sulfate and keratan sulfate, which promote cell detachment and inhibition of attachment (Herndon et al. 1990). The neurite-inhibitory properties of chondroitin sulfate proteoglycan define functional boundaries during development in the rodent somatosensory barrel field (Sheppard et al. 1991)  11 and the roof plate of the spinal cord (Snow et al. 1990). Lesions to the adult CNS result in an upregulation of proteoglycans (McKeon et al. 1991). Thus, injury to the adult CNS appears to induce astrocytes to re-express several developmentally-regulated anti-adhesive molecules including tenascin and various proteoglycans. Reactive astrocytes may also inhibit neurite growth through a CNS lesion site by providing a physical barrier to growing neurites. Astrocytes undergo extensive proliferation and hypertrophy in and around a site of CNS injury (Hertz et al. 1990; Reier and Houle 1988). It has been proposed that the dense network of astocytes which forms at the lesion site provides a barrier through which growing neurites are unable to navigate (reviewed in Reier and Houle 1988). The complexly interdigitating astrocytic processes often exhibit an increased number of junctional complexes, which may contribute to the formation of an impenetrable physical barrier to growing neurites. In the hibernating ground squirrel, however, axonal regeneration fails to occur despite the lack of glial scar formation following spinal cord transection (Guth et al. 1981). Oligodendrocytes and oligodendrocyte-produced myelin also express neurite-inhibitory molecules. Myelination within the developing CNS generally takes place after neuronal projections have reached their targets (Schreyer and Jones 1982; Okado and Oppenheim 1985; Schneider et al. 1990; Keirstead et al. 1992; Jhaveri et al. 1992). It has thus been hypothesized that one of the  12 functions of myelin within the CNS is to stabilize newly-formed neural connections (Kapfhammer and Schwab 1994). One such molecule expressed by developing and mature CNS oligodendrocytes is janusin, originally referred to as J1-160/180 (Morganti et al. 1990). Binding of janusin to its putative receptor, the F3/1 ladhesion molecule, inhibits neurite outgrowth (Pesheva et al. 1993). Schwab and collegues have identified two proteins, termed Nl-35 and NI250 that account for much of the neurite-inhibitory activity in CNS myelin (Caroni and Schwab 1988). The identification of these inhibitors began with the observation that neurons, astrocytes and fibroblasts in culture preferred to grow on substrates other than oligodendrocytes and their radial, highly-branched process networks (Schwab and Caroni 1988). In contrast, frozen sections from neonatal rat spinal cords treated with mitotic inhibitors or X-irradiated (to prevent myelin formation) proved to be permissive substrates for growing neuroblastoma cells (Savio and Schwab 1989; Savio and Schwab 1990). The CNS myelin inhibitory activity was largely recovered after protein separation in SDS-PAGE and reconstitution into liposomes, and was subsequently identified as 35 kDa (Nl-35) and 250 kDa (Nl-250) proteins (Bandtlow et al. 1990). When added to favorable substates, these proteins inhibited neurite growth. Functionally-blocking monoclonal antibodies to these proteins (IN-35 and IN 250) were then generated (Caroni and Schwab 1988). Application of these antibodies (by implantation of antibody-secreting hybridoma cells near the lateral ventrical) in rats with transections of the corticospinal tract resulted in limited  13 anatomical regrowth of axons approximately 5mm caudal to the lesion site 2-3 weeks after injury (Schnell and Schwab 1990). Despite the limited regeneration obtained with this approach, these studies identified specific proteins in CNS myelin that contribute to the inhibition of neuronal repair after injury. Finally, it has been proposed that myelin-associated glycoprotein (MAG) may contribute to the neurite-inhibitory activity of CNS myelin (L. McKerracher et al. 1994). Experiments involving protein fractionation of bovine CNS myelin and in vitro testing of growth inhibitory activity have identified MAG as an inhibitor of process extention for differentiated PC12 cells. Further experimentation, including the generation and use of functionally-blocking antibodies, is necessary to elucidate the relative contribution of MAG to the neurite-inhibitory activity of CNS myelin. Strategies to evoke substantial regeneration following injury to the CNS must take into consideration the many growth-promoting and growth-inhibiting factors that may influence a particular population of injured neurons. Precluding the formulation of such strategies, however, is information regarding the relative contribution of each of these factors to the ability of injured neurons to regenerate. Although neurite-inhibitory proteins have been identified in CNS myelin, any in vivo attempts to neutralize the inhibitory activity have resulted in very limited regeneration (Schnell and Schwab 1990), possibly due to limitations  in the neutralization technique itself. The experiments outlined in this thesis were designed to address the question: to what extent does CNS myelin  14 contribute to the lack of regeneration observed following spinal cord injury to the late embryonic and hatchling chick? The chick offers several advantages as an experimental model. The developmental stages of the chicken Gallus domesticus have been thoroughly documented (Hamburger and Hamilton 1951). Embryonic surgery in vivo is difficult to perform on mammalian embryos due to maternal complications, however, the avian egg provides for easy and uncomplicated access to the embryonic chick at any stage of development. In addition, the structure and function of the vertebrate brainstem and spinal cord has remained virtually unchanged throughout evolution (Sarnat and Netsky 1981). In particular, the brainstem-spinal control of locomotion in birds very similar to that of other vertebrates, including mammals (Bekoff 1976; Okado and Oppenheim 1985; Sholomenko and Steeves 1987; Steeves et al. 1987; Webster and Steeves 1988, 1991; Sholomenko et al. 1991a,b,c; Steeves and Jordan 1980; Armstrong 1986). Following injury to the adult spinal cord, birds suffer the same motor deficits as a mammal (Eidelberg et al. 1981; Zemlan et al. 1983; Sholomenko and Steeves 1987; Webster and Steeves 1991). Previous studies of developing chick embryos have indicated that the ability to recover from spinal cord injury is lost in development (Okado and Oppenheim 1985; Hasan et al. 1991). Transections of the developing spinal cord early in embryonic development result in complete functional recovery assessed after hatching. Transections of the spinal cord after embryonic day (E)  15 13 of the 21 day developmental period result in no anatomical or physiological repair, rendering such an animal completely incapable of voluntary locomotion after hatching. Thus, there appears to be a transition during development of the embryonic chick, from a permissive to a restrictive period for spinal cord repair. Investigations of extraneuronal changes during development that are coincident with the transition from permissive to restrictive repair periods may provide insights into the lack of spinal cord repair following injury to the adult spinal cord. I began the studies outlined in this thesis with an investigation of myelin development within the spinal cord of the chick. Histological and immunohistochemical analysis of the developing chick spinal cord revealed that the developmental onset of myelination is coincident with the transition from permissive to restrictive repair periods (chapter 2). This finding, taken together with other studies outlined above indicating the neurite-inhibitory properties of CNS myelin, suggested that myelin may inhibit the regeneration of transected spinal cord in embryonic chick. In order to test this hypothesis, a method of delaying the developmental onset of myelination in vivo was developed (chapter 3). Due to the presence of several different putative neurite-inhibitors in CNS myelin (see above) a method of complete myelin ablation was desirable. The studies outlined in chapter 3 indicated that direct spinal cord injections of complement proteins plus complement-fixing antibodies to oligodendrocyte cell surface-specific antigens  16 resulted in a delay of the developmental onset of myelination until El 7. This technique was also shown to be effective in suppressing the developmental onset of myelination in the neonatal mouse spinal cord. Transections of the spinal cord during developmental myelin-suppression then serve as a direct test of whether myelin inhibits the regeneration of transected spinal cord in embryonic chick. Complete spinal cord transections as late as E15 in myelin-suppressed embryonic chicks resulted in complete neuroanatomical and physiological repair (Chapter 4). Normally-myelinated embryos transected on E15 exhibited no anatomical or physiological repair after hatching. These studies indicated that myelin inhibits the functional regeneration of transected spinal cord in  -  embryonic chicks and suggested that spinal cord myelin may likewise inhibit regeneration following injury to the adult spinal cord. In order to test the inhibitory properties of adult CNS myelin, a method of transiently demyelinating the adult spinal cord was developed. The procedure developed for myelin-suppression in the embryo was modified so as to transiently demyelinate the adult chick spinal cord over a 2-3 week period. Immunological demyelination was also shown to be effective in removing myelin from the mouse spinal cord, indicating that this procedure is not species restricted. Complete transection of the hatchling chick spinal cord, followed by demyelination over a 2-3 week period resulted in partial neuroanatomical regeneration of axotomized fibres (chapter 5). Collaborative studies outlined in  17 Chapter 5 demonstrate that this neuroanatomical regeneration is accompanied by functional synaptogenesis within the spinal cord. Brainstem stimulation of transected and demyelinated hatchling chicks 2-4 weeks after surgery elicited electrical activity within the muscles of the legs. These studies indicate that adult spinal cord myelin inhibits the regeneration of transected spinal cord in the hatchling chick. Furthermore, these findings constitute the first demonstration in higher vertebrates of functional regeneration following spinal cord injury. In contrast to the complete regeneration observed in the myelin suppressed embryo, transection and demyelination of the hatchling chick spinal cord elicited only partial regeneration. This observation suggests that other factors affecting regeneration, that may be present in the embryo, may be absent or below threshold levels of effectiveness in the hatchling chick. Nevertheless, myelin appears to be a major factor affecting the ability of the embryonic and hatchling chick spinal cord to regenerate following injury.  18  CHAPTER 2  MYELIN DEVELOPMENT IN THE EMBRYONIC CHICK SPINAL CORD Ñ  19 INTRODUCTION Myelin is a highly differentiated membrane structure produced by oligodendrocytes in the central nervous system (CNS). Although the chief role of myelin is to allow rapid axonal conduction in the fibre systems of the CNS (Ritchie 1984), it has also been implicated in the formation of proper ion channel distribution in axons (Joe and Angellides 1992), restriction of target area innervation by developing neuronal projections (Schwab and Schnell 1991) and several CNS diseases including multiple sclerosis, optic neuritis and experimental allergic encephalomyelitis (Quarles et al. 1989). Recently, CNS myelin has been shown to inhibit the regeneration of transected spinal cord axons in rat (Schnell and Schwab 1990) and embryonic chick (Keirstead et al. 1992). Developmental studies of myelination have proven to be a useful means of elucidating the various functions of myelin in the adult animal. Oligodendrocyte precursors originate from the ventricular zone of the neural tube, and express mRNA encoding the larger of the two isoforms of 2’,3’-cyclic nucleotide 3’-phosphodiesterase (CNP; Yu et al. 1994; Schrerer et al. 1994). Oligodendrocyte precursors isolated from the optic nerve express antigens labelled by GD3 and A2B5 antibodies (Raff 1989: Reynolds and Wilkin 1988). Culture studies of oligodendrocyte progenitors isolated from the rat optic nerve have defined these cells as oligodendrocyte-type-2 astrocyte (02A) progenitor cells which differentiate in vitro into oligodendrocytes or type-2 astrocytes, depending on the culture conditions (Raff 1989). When no serum is  20 provided in the culture medium, all 02A progenitor cells promptly differentiate into oligodendrocytes. This constitutes the ‘default’ pathway of 02A progenitor differentiation. When serum is included in the culture medium, the majority of 02A progenitor cells differentiate into astrocytes. Ciliary neurotrophic factor has been identified as the factor in astrocyte-conditioned medium that is responsible for inducing 02A progenitor cells to differentiate along the astrocyte lineage. Stable differentiation of astrocytes also requires an unidentified component of the extracellular matrix (reviewed in Williams and Price 1992). It should be noted, however, that several investigators have found no evidence of astrocyte differentiation from freshly-dissociated 02A progenitor cells, or from an immortalized line of 02A progenitor cells originally isolated from the optic nerve (Espinosa de los Monteros et al. 1993; Groves et al. 1993). Groves et al. (1993) transgenically modified 02A progenitor cells to express $-galactosidase, the protein product of the LacZ gene. After injecting this cell line into demyelinated lesions of the rat spinal cord, blue X-gal staining of the spinal cord at various time points after injection revealed only ,8-galactosidase-positive oligodendrocytes. No fl-galactosidase-positive astrocytes were identified. These findings suggest that the differentiation of 02A progenitor cells into astrocytes is a phenomenon unique to in vitro experiments. Oligodendrocyte progenitors undergo a set number of cell divisions (about 8) before differentiating into oligodendrocytes (Raff et al. 1985; Raff 1989). Platelet derived growth factor (PDGF) and basic fibroblast growth factor (bFGF)  21 have been identified as factors involved in the division of oligodendrocyte progenitors prior to differentiation (reviewed in Goldman 1992). PDGF upregulates the expression of bFGF receptors on oligodendrocyte progenitors, inducing their responsiveness to the mitogenic effects of bFGF. It is theorized that an internal clock controls the timely differentiation of oligodendrocytes from their progenitors in the presence of bFGF and PDGF (Raff et al. 1985). The timing mechanism of the internal clock may have two separate components; a counting component driven by mitogens (like bFGF), and an effector component driven by hydrophobic signalling molecules (like thyroid hormone, retinoic acid and glucocorticoids) that induce oligodendrocytes to withdraw from the cell cycle (Barres et al. 1994). Receptors for thyroid hormone, retinoic acid and glucocorticoids are able to inhibit the activity of AP-1 transcription factors, which are formed by the heterodimerization of Jun and Fos proteins and mediate the proliferative response to growth factors (Barres et al. 1994; Ransone and Verma 1990). As one might imagine, hyperthyroidism accelerates the deposition of myelin, whereas hypothyroidism delays it (Walters and Morell 1981; Legrand 1986; Dussault and Ruel 1987). It is thus hypothesized that oligodendrocyte mitogens induce cellular division, which decreases the AP-1 activity with each division. When AP-1 activity drops below a threshold level, hydrophobic signals are then able to further reduce the activity below a level required to keep the cell dividing, and division ceases. Subsequent to cessation of cellular proliferation, cellular differentiation  22 takes place (Barres et al. 1994). PDGF and bFGF are detectable in the rat, mouse and chick around the time of oligodendrocyte differentiation (Richardson et al. 1988; Wanaka et al. 1990; Kalcheim and Neufeld 1990; Weise et al. 1993). In the chick spinal cord, bFGF levels peak just prior to the developmental onset of oligodendrocyte differentiation (Kalcheim and Neufeld 1990). Furthermore, the rat thyroid gland becomes active around birth, and begins secreting thyroid hormone at a time when oligodendrocytes first appear in the CNS (Samel 1968; Puymirat 1992). CNS myelination in the human also takes place shortly after the thyroid gland becomes active, at about the 1 2th week of gestation (Friede 1989). The ganglioside GM3 may also play a role in oligodendrocyte differentiation. Differentiation of control cells in vitro is associated with increased metabolic labelling of endogenous GM3, suggesting a role for GM3 as precursors differentiate towards the more mature stages of myelin production (Vim et al. 1994). Furthermore, addition of exogenous GM3 to cultures enriched for 02A cells promotes differentiation through a pre-myelinating stage (labelled by 04 antibodies which react with sulfatide, seminolipid and an unidentified antigen on oligodendrocyte progenitors; Sommer and Schachner 1982) to a myelinating stage, characterized by process formation and expression of myelin related constituents such as GaIC, sulfatide and myelin-associated glycoprotein (Yim et al. 1994). Gangliosides have been shown to promote or inhibit the phosphorylation of many neural proteins including myelin basic protein  23 (Goldenring et al. 1985; Kim et al. 1986). Because these myelin-related constituents are expressed in the later stages of 02A differentiation, the precise stage(s) at which GM3 may exert its effects are unknown. Concurrent with the differentiation of oligodendrocytes from oligodendrocyte progenitors is the expression of galactocerebroside (GaIC) by newly differentiated oligodendrocytes (Benjamins et al. 1987). Newly differentiated pre-myelinating oligodendrocytes also begin to express mRNA encoding the smaller of the two isoforms of CNP, as well as the CNP protein (Scherer et al. 1994; Sprinkle et al. 1978). The cellular functions of GaIC and CNP have not been elucidated. CNP immunoreactivity is found in the cytoplasmic compartments of the oligodendrocyte, mainly in the soma and paranodal loops of the myelin sheaths (Braun et al. 1988). Although extracted CNP fractions have been shown to catalyze 2’,3’-cyclic nucleotide to 2’nucleotide, 2’,3’-cyclic nucleotide has not been found within oligodendrocytes (Sprinkle et al. 1978). GaIC is a highly abundant sphingolipid located within the lipid membranes of the oligodendrocyte (Benjamins et al. 1987). GalC is linked to the cytoskeleton and may play a role in transmembrane signalling (Benjamins and Dyer 1990). Pre-myelinating oligodendrocytes and their progenitors are highly migratory (Small et al. 1987). Early in development these cells migrate from their place of origin in the subventricular zone and infiltrate the fasciculated axonal tracts of the CNS where they differentiate into oligodendrocytes (Warf et  24 al. 1991; NolI and Miller 1993; Yu et al. 1994). The signal for these newlydifferentiated oligodendrocytes to begin myelination of axons has not been determined, although axonal contact has been implicated (Doyle and Colman 1993). Cyclic AMP has been implicated in the activation of myelin genes in both oligodendrocytes and Schwann cells (Goda et al. 1989; Monuki et al. 1989). The O4, GalC oligodendrocyte begins myelination by sending out a process that contacts the axon then begins wrapping around the axon. Myelin associated glycoprotein (MAG) may be involved in the adhesive interactions between the oligodendrocyte process and the axonal membrane (reviewed in Quarles 1988). MAG levels are high during the initial stages of myelination and decrease considerably thereafter. In the mature CNS, MAG immunoreactivity is restricted to the internal mesaxon adjacent to the axonal membrane.  -  After the initial stages of wrapping, myelin undergoes compaction (reviewed in Morell et al. 1989). At this stage of myelin development, two major myelin proteins are expressed that play a significant role in the compaction process. Myelin basic protein (MBP) mRNA is translated within the cell body and oligodendrocyte processes and incorporated into the myelin membranes on the cytoplasmic surface (reviewed in Mikoshiba et al. 1991). During the compaction process, homophilic interactions of MBP bring the cytoplasmic surfaces of the myelin membranes together, resulting in the extrusion of the cytoplasm and the formation of the major dense line. In cross section, the major dense line appears under electron microscopy as a dark spiral  25 originating at the axonal surface. Proteolipid protein (PLP) appears just prior to compaction as a transmembrane protein with a large extracellular domain on the surface of the myelin membrane (reviewed in Mikoshiba et al. 1991). During compaction, the extracellular surfaces are brought together as facing PLP extracellular domains homophilically interact. This results in the formation of the intraperiod line which is seen under electron microscopy as a thin line spiralling between the major dense lines. The mature myelin sheath is interrupted at approximately 1-2 mm intervals along the length of the axon at nodes of Ranvier. A single oligodendrocyte may send out myelinating processes to 30 or more internodal segments on neighboring axons. The compacted myelin sheath increases the membrane resistance and decreases the membrane capacitance of the axon (reviewed in Morell et al. 1989). These two features of myelinated axons result in less decremental decay of the electrical charge as it travels between the Nodes of Ranvier. The myelin sheath may also play a role in determining the distribution of voltage-gated sodium channels along the length of the axon. Although direct causation has not been convincingly demonstrated, myelinated axons consistantly display a characteristic sodium channel distribution quite different from that of an unmyelinated axon. Voltage-gated sodium channels are clustered around the nodes of Ranvier and the adjacent regions under the paranodal loops of the myelin sheath. Voltage-gated sodium channels are absent or very sparse in the internodal segments of the myelinated axon. These  26 properties of myelinated axons result in super-threshold stimulation of the action potential only at the nodes of Ranvier, and highly efficient conduction of the resulting electrical signal within the myelinated internodal segment. This phenomenon is known as ‘saltatory conduction’. Saltatory conduction is disturbed in nervous system diseases which involve demyelination of fiber tracts (Quarles et al. 1989). Multiple Sclerosis (MS) is one such disorder that is relatively prominent in the human population. MS is characterized by transient demyelinating lesions of the CNS displaying abnormally large numbers of inflammatory cells (T-cells, B-cells, monocytes and macrophages/microglia). Physiological deficits during periods of exacerbations of the disease are related to the CNS area displaying the demyelinated lesion. Analysis of the cerebrospinal fluid (CSF) of MS patients also reveal ‘oligoclonal banding’, which indicates an abnormally high concentration of antibodies to one or very few unidentified antigens. Although an immunological regulatory malfunction is clearly present in MS, it has not been determined whether the autoimmunity is causitive or correlative. Both genetic and environmental factors have been implicated in triggering the disease (reviewed in livanainen 1981). A popular animal model of MS is the experimental allergic encephalomyelitis (EAE) mouse (reviewed in Quarles et al. 1989). This pathogenic condition is inducible in several species by the injection of CNS tissue or MBP into the circulatory system. The presence of myelin antigens in the bloodstream induces an autoimmune reaction which results in the formation of demyelinated lesions  27 within the CNS. Interestingly, neonatal exposure to MBP protects mice from developing EAE when challenged as an adult (Adorini et at. 1990). In such an animal, the developing thymus of the neonatal mouse generates T-cells which recognize the MBP antigen as ‘self’. More recently, myelin has been shown to display an activity which inhibits the growth and regeneration of neurites. Growing neurites in culture will avoid white matter when an alternative substrate is available (Schwab and Caroni 1988). When plated directly onto CNS white matter, cells fail to extend processes beyond a few millimeters. This inhibitory activity has been isolated and found to reside in 35kD and 250kD proteins isolated from rat CNS white matter (Bandtlow et al. 1990). Antibodies to these inhibitory proteins have been generated and, when administered to the corticospinal lesioned rat, facilitate partial anatomical regeneration of corticospinal axons (Schnell and Schwab 1990). Although no functional repair follows such an intervention, this study clearly demonstrates that CNS myelin contains a component that inhibits the regeneration of axotomized corticospinal fibres. Furthermore, myelin-associated inhibitors have been suggested to play a developmental role in axonal pathfinding (Schwab and Schnell 1991). The following chapters of this thesis also describe the inhibitory properties of myelin for the regeneration of axotomized brainstem-spinal projections (also see Keirstead et al. 1992). Recognizing that myelin may be inhibitory to the growth and regeneration of spinal cord axons, I asked the question; when does the process of  28 oligodendrocyte differentiation and myelin development occur relative to axonal development in the chick spinal cord? Neurogenesis of brainstem-spinal projecting neurons is complete by embryonic day (E)3- E5 (McConnell and Sechrist 1980), and brainstem-spinal projections controlling locomotion are complete to all levels of the spinal cord by E12 (Okado and Oppenheim 1985). I hypothesized that myelination of the spinal cord would begin after neuronal development and axonal projections were complete. In order to determine the developmental onset of spinal cord myelination, I conducted a series of immunohistochemical investigations using antibodies to various myelin-specific antigens including GalC, CNP, MBP and MAG (described above).  29 MATERIALS AND METHODS Fertilized White Leghorn eggs were incubated at 37°C in an automatic rotating incubator. Prior to surgery or sacrifice, all eggs were developmentally staged using accepted protocols (Hamburger and Hamilton 1951). Paraffin Embedding Animals were perfused intracardially at the appropriate developmental stage (see results) with O.1M phosphate buffered saline (PBS) containing 2500 USP units of heparin per 50 mIs PBS, pH 7.4 (at 37°C) followed by perfusion with 4% paraformaldehyde in 0.1M phosphate buffer, pH 7.4 (at 4-10°C). The dissected spinal cords were then immersed in the same fixative for 24hrs at 4°C and subsequently returned to PBS pH 7.4 (at 4-10°C) until further processing. In preparation for paraffin embedding, the spinal cords were immersed in 50% ethanol for two hours then transferred to 70% ethanol where they remained overnight. The following morning the spinal cords were transferred to embedding cartridges and placed in an automated histomatic tissue processor. The histomatic tissue processor was adjusted so as to sequentially expose the tissue to 30 minutes 80% ethanol, 30 minutes 90% ethanol, 60 minutes 100% ethanol, 60 minutes fresh 100% ethanol, 60 minutes paraffin at 50-60°C under vacuum, and a final 60 minutes paraffin at 50-60°C under vacuum. The tissue was then transferred from embedding cartridges to embedding blocks and surrounded by hot paraffin which was then allowed to cool and harden. The paraffin blocked tissue was cut on a Leitz microtome into 10pm parasagittal  30 sections and mounted on gelatin-coated slides for immunohistochemical processing. Immunohistochemistry Antigens were localized using indirect immunofluorescence. Galactocerebroside immunohistochemistry was performed  Ofl  Gyrostat sectioned  tissue that was perfused as outlined above. All other antigens were localized on paraffin-embedded tissue sections. The paraffin-embedded tissue sections were sequentially immersed in two, 60 second xylene baths to remove the paraffin then brought up to 100% hydration with two minute immersions in 100% ethanol, 95% ethanol, 70% ethanol, 50% ethanol and a five minute immersion in PBS pH 7.4. In order to block non-specific background staining, excess PBS was removed and the slides were incubated with 200-400 p1 per slide of 10% goat serum in PBS containing 3% Triton-X 100. After 45 minutes, excess fluid was removed from the slides and the sections were circled with rubber cement. The primary antibody (diluted in 1 % goat serum in PBS containing 3% Triton-X 100) was applied (200-400 p1 per slide) and incubated for 3 days at 4 degrees Celcius. After this 3 day incubation period, excess primary antibody was removed with 3 washes of 5 minutes each in 1 % goat serum in PBS containing 3% Triton-X 100. The secondary antibody (diluted in 1% goat serum in PBS containing 3% Triton-X 100) was applied (200-400 p1 per slide) and incubated for one hour at room temperature. Slides were rinsed in PBS and fitted with microscope cover glass in 1:2 PBS, glycerol prior to microscopic analysis. For  31 long-term storage, the cover glass was sealed with a small amount of nail polish around the perimeter. The rabbit anti-human myelin basic protein antibody (MBP; Accurate Chemical Scientific Corp., #AXL746), the rabbit anti-cow glial fibrillary acidic protein (GFAP; Dakopatts Corp., #Z334) and the mouse anti-bovine microtubule associated protein- 2 (MAP-2; Amersham #RPN1194) were all used at a dilution of 1:100 in 1% goat serum in PBS containing 3% Triton-X 100. The mouse anti-bovine Myelin Associated Glycoprotein (MAG; Boehringer Mannheim #1 450 972) and the rabbit anti-bovine 2’,3’- Cyclic Nucleotide 3’- Phosphodiesterase (CNP; a gift from Peter Braun, McGill University, Canada) were used at a dilution of 1:500 in 1% goat serum in PBS containing 3% Triton-X 100. The rabbit anti-bovine galactocerebroside antibody (Chemicon AB 142) was used at a dilution of 1:10. The secondary antibodies were either goat anti-rabbit FITC conjugated immunoglobulin (Caltag Laboratories, #L42001) diluted 1:100 in 1% goat serum in PBS containing 3% Triton-X 100 or a goat anti- mouse FITC conjugated immunoglobulin (Caltag Laboratories, #L42001) diluted 1:100 in 1% goat serum in PBS containing 3% Triton-X 100. Standard immunohistochemical controls (eg. omission of primary and/or secondary antibodies) were processed alongside tissue sections from experimental and control animals. Pre-absorption controls were conducted by the respective supplier of each antibody. Photomicrographs were taken on a Zeiss Axiophot using epifluorescent illumination with the appropriate filters.  32 Histological Staining Animals designated for toluidine blue staining were perfused intracardially at the appropriate developmental stage (see results) with 0.1M phosphate buffered saline (PBS) containing 2500 USP units of heparin per 50 mIs PBS, pH 7.4 (at 37°C) followed by perfusion with 2.5% glutaraldehyde in O.1M phosphate buffer, pH 7.4 (at 4-10°C). The dissected tissue was rinsed and stored in 0.1M Na-cacodylate buffer (pH 7.4) overnight. The next morning the tissue was transferred to 1 % osmium tetroxide in 0.1 M Na-cacodylate buffer (pH 7.4) and placed on a rotator for one hour. The tissue was rinsed with distilled water and sequentially exposed to 70% ethanol, 85% ethanol, 95% ethanol, 100% ethanol and a final 100% ethanol for 10 minute intervals on a rotator. After a 30 minute exposure to propylene oxide on a rotator, the tissue placed on the rotator for one hour while exposed to 1:1 propylene oxide and Spurr resin. The tissue was exposed to 100% Spurr resin on a rotator for twenty-four hours, then placed in labelled block moulds with fresh Spurr resin and incubated at 60 degrees Celcius for 16 hours. The hardened blocks were cut into 1pm transverse sections on an ultra microtome and placed on glass slides for toluidine blue staining. 0.1% toluidine blue stain was filtered and dropped onto sections on a hot plate. The toluidine blue was left on the sections for one or two minutes, long enough for metallic rings to form around the drying toluidine blue drops. The slides were then rinsed with hot tap water followed by distilled water, then placed on a hot plate to dry.  33 Photomicrographs were taken on a Zeiss Axiophot microscope.  34 RESULTS Galactocerebroside (GaIC) immunoreactivity was not detected at any level of the spinal cord in animals sacrificed on embryonic day (E) 8 (n  =  3) or E9  (n=2; Fig. 2-lA). lmmunoreactivity for GaIC was first detected in 4 of 6 animals sacrificed on ElO (Fig. 2-1B). GaIC immunoreactivity in these animals was punctate, and was most often located in the lateral-most regions of the white matter tracts. In 3 of 4 animals analysed on El 1, GalC immunoreactivity was similarly organized. GaIC immunoreactivity was consistently more abundant and present throughout the spinal cord white matter in animals analysed on E12 (n=3), E13 (n=4), E14 (n=3) and E15 (n=5). GaIC immunostaining of P1, P3, P7 and PlO spinal cords were quantitatively similar, suggesting that myelination is complete by the time of hatching (n=6 for each day; Fig. 2-iC) 2’,3’- cyclic nucleotide 3’- phosphodiesterase (CNP) immunoreactivity was not detected at any level of the spinal cord in animals sacrificed on ElO (n=3) or Eli (n=3; Fig. 2-2A). CNP immunoreactivity was first detected in the spinal cord on Ei2.5 in 6 animals (Fig. 2-28). CNP immunoreactivity was always more abundant throughout the spinal cord white matter on E13 (n=4), E14 (n=8), and Ei5 (n=8). CNP immunostaining of P1, P3, P5 and PlO spinal cords were quantitatively similar, suggesting that myelination is complete by the time of hatching (n=6 for each day; Fig. 2-2C). Myelin basic protein (MBP) or myelin-associated glycoprotein (MAG) immunoreactivity was not detected at any level of the spinal cord on E9 (6  35 Figure 2-1. Developmental pattern of galactocerebroside (GaIC) immunoreactivity in the chick spinal cord. A: GaIC immunofluorescence staining of unoperated control embryonic day (E) 9 thoracic spinal cord in parasaggital section showing no GaIC immunoreactivity. B: GalC immunofluorescence staining of unoperated control ElO thoracic spinal cord in parasaggital section showing the developmental onset of GaIC immunoreactivity. C: GalC immunofluorescence staining of unoperated control posthatching day (P) 7 thoracic spinal cord in parasaggital section showing the hatchling pattern of GaIC immunoreactivity. In all photographs the outer edge of the spinal cord lies on the right hand side, with the white matter adjacent. (Bars  =  lOOum).  (J%J  37 Figure 2-2. Developmental pattern of 2’,3’-cyclic nucleotide 3’-. phosphodiesterase (CNP) immunoreactivity in the chick spinal cord. A: CNP immunofluorescence staining of unoperated control embryonic day (E) 11 thoracic spinal cord in parasaggital section showing no CNP immunoreactivity. B: CNP immunofluorescence staining of unoperated control E12.5 thoracic spinal cord in parasaggital section showing the developmental onset of CNP immunoreactivity. C: CNP immunofluorescence staining of unoperated control posthatching day (P) 5 thoracic spinal cord in parasaggital section showing the hatchling pattern of CNP immunoreactivity. In all photographs the outer edge of the spinal cord lies on the right hand side, with the white matter adjacent. (Bars =  lOOum).  GD  CL)  39 animals tested with MBP antibody), ElO (6 animals tested with MBP antibody, 4animals tested with MAG antibody), Eli (12 animals tested with MBP antibody, 4 animals tested with MAG antibody) or Ei2 (12 animals tested with MBP antibody, 4 animals tested with MAG antibody; Fig. 2-3A; Fig. 2-4A). MBP and MAG immunoreactivity first appeared in the ventrolateral funiculi of the cervical spinal cord on E13 and appeared to proceed in a ventro-dorsal and rostral-caudal direction with development (14 animals tested with MBP antibody and 6 animals tested with MAG antibody; Fig.2-3B; Fig. 2-4B). Comparison of age-mached El 3 embryos revealed that CNP immunoreactivity was consistently more abundant than MBP or MAG immunoreactivity. On E14 all levels of the spinal cord displayed MBP, MAG and CNP immunoreactivity with the ventral funiculi markedly more dense than other areas (18 animals tested with MBP antibody, 12 animals tested with MAG antibody and 8 animals tested with CNP antibody). By E15 a dense network of immunoreactivity was observed within all spinal cord white matter (28 animals tested with MBP antibody, 8 animals tested with MAG antibody and 8 animals tested with CNP antibody). A dramatic increase in MBP and MAG immunoreactivity was observed in animals sacrificed on E17 and El8, suggesting a second wave of myelination at this stage of development (18 animals tested with MBP antibody for each day, 6 animals tested with MAG antibody for each day). MBP and MAG immunostaining of P1, P3, P5, P7 and PlO spinal cords were qualitatively similar, suggesting that myelination is complete by the time of hatching (n=6 for each day, for each of  40 Figure 2-3. Developmental pattern of myelin basic protein (MBP) immunoreactivity in the chick spinal cord. A: MBP immunofluorescence staining of unoperated control embryonic day (E) 1 2 thoracic spinal cord in parasaggital section showing no MBP immunoreactivity. B: MBP immunofluorescence staining of unoperated control E13 thoracic spinal cord in parasaggital section showing the developmental onset of MBP immunoreactivity. C: MBP immunofluorescence staining of unoperated control posthatching day (P) 7 thoracic spinal cord in parasaggital section showing the hatchling pattern of MBP immunoreactivity. In all photographs the outer edge of the spinal cord lies on the right hand side, with the white matter adjacent. (Bars  =  lOOum).  42 Figure 2-4. Developmental pattern of myelin-associated glycoprotein (MAG) immunoreactivity in the chick spinal cord. A: MAG immunofluorescence staining of unoperated control embryonic day (E) 12 thoracic spinal cord in parasaggital section showing no MAG immunoreactivity. B: MAG immunofluorescence staining of unoperated control E13 thoracic spinal cord in parasaggital section showing the developmental onset of MAG immunoreactivity. C: MAG immunofluorescence staining of unoperated control posthatching day (P) 7 thoracic spinal cord in parasaggital section showing the hatchling pattern of MAG immunoreactivity. In all photographs the outer edge of the spinal cord lies on the right hand side, with the white matter adjacent. (Bars  =  lOOum).  44 MBP and MAG; Fig. 2-3C; Fig. 2-4C). Toluidine Blue staining of plastic embedded tissue on E15 revealed dense areas of myelination in all regions of the spinal cord white matter (n=4). At this stage of development the toluidine blue stained myelin profiles appeared to be tightly compacted (Fig. 2-5). In all of the immunohistochemical analyses, myelin-specific immunoreactivity (for CNP, MBP and MAG) within the spinal cord gray matter was dramatically reduced compared to myelin-specific immunoreactivity within the spinal cord white matter. Only a few sparse fibres were detected in embryos analysed prior to E15. After E15, all animals consistently displayed myelin specific immunoreactivity within their spinal cord gray matter. Myelin-specific immunoreactivity in the adult spinal cord gray matter was consistently denser than in the embryonic spinal cord gray matter. Glial fibrillary acidic protein (GFAP) immunoreactivity was not detected at any level of the spinal cord in animals sacrificed on embryonic day (E) 9 (n=4), ElO (n=6) or Eli (n=6; Fig. 2-6A). Immunoreactivity for GFAP was first detected in 4 of 6 animals sacrificed on E12 and was almost exclusively localized in the white matter (Fig. 2-6B). On E13 and E14 GFAP immunoreactivity was evident in both the white and gray matter (n =6 for each), however, the white matter was far more immunoreactive. GFAP immunoreactivity increased noticably in both the white and gray matter on El 5, relative to the levels of immunoreactivity in younger animals (n=6). After  45 Figure 2-5. Toluidine Blue staining of unoperated control embryonic day (E) 15 chick spinal cord white matter in transverse section. Notice the densely compacted profiles of myelinated axons (dark rings; Bars  =  25um).  47 Figure 2-6. Developmental pattern of glial fibrillary acidic protein (GFAP) immunoreactivity in the chick spinal cord. A: GFAP immunofluorescence staining of unoperated control embryonic day (E) 11 thoracic spinal cord in parasaggital section showing no GFAP immunoreactivity. B: GFAP immunofluorescence staining of unoperated control E12 thoracic spinal cord in parasaggital section showing the developmental onset of GFAP immunoreactivity. C: GFAP immunofluorescence staining of unoperated control posthatching day (P) 5 thoracic spinal cord in parasaggital section showing the hatchling pattern of GFAP immunoreactivity. In all photographs the outer edge of the spinal cord lies on the right hand side, with the white matter adjacent. (Bars  =  lOOum).  -  49 hatching, GFAP immunoreactivity increased dramatically. Spinal cords from hatchlings sacrificed on posthatching day (P) 2 (n=4), P4 (n=6), P5 (n=6), P8 (n=6), PlO (n=7), P12 (n=6) and P15 (n=3) consistently displayed intense GFAP immunoreactivity in both the white and gray matter (Fig. 2-6C). Again, the level of GFAP immunoreactivity was greater in the white matter than the gray matter.  50 DISCUSSION Oligodendrocytes differentiate 2-3 days prior to the developmental onset of myelination in the embryonic chick spinal cord (Benstead et al. 1957). In vitro experiments have indicated that the oligodendrocyte-specific expression of galactocerebroside (GaIC) is concurrent with the differentiation of oligodendrocytes from oligodendrocyte progenitors (Benjamins et al. 1987). The developmental expression of GalC observed in the present in vivo study supports these findings. GaIC immunoreactivity was first detected in the embryonic chick spinal cord on embryonic day (E) 10, 3 days prior to the developmental onset of spinal cord myelination (described below; Fig. 2-1). This suggests that pre-myelinating oligodendrocytes are present within the embryonic chick spinal cord white matter approximately 3 days prior to the developmental onset of myelination. Oligodendrocyte-specific 2’,3’- cyclic nucleotide 3’- phosphodiesterase (CNP) has also been reported to be expressed by pre-myelinating oligodendrocytes and/or oligodendrocyte progenitors (Braun et al. 1988). It is interesting in this regard that I observed the developmental onset of CNP immunoreactivity in the embryonic chick spinal cord on E12.5 (Fig. 2-2), just prior to the onset of myelin basic protein (MBP) and myelin-associated glycoprotein (MAG) immunoreactivity on E13 (described below). These findings do not concur with previous histological studies which indicate that pre myelinating oligodendrocytes or oligodendrocyte progenitors appear 2-3 days  51 prior to myelination (Benstead et al. 1957), nor do they concur with the GaIC developmental study outlined above, which indicates that pre-myelinating oligodendrocytes are present as early as ElO. This discrepency may reflect a low sensitivity of the CNP antibody used in this study. Paraffin processing may have additionally contributed to a decreased immunoreactivity of the CNP antigen. Alternatively, oligodendroglial development may be characterized by a GaIC, CNP stage, followed by a GaIC, CNP stage which briefly precedes myelination. Myelination in the developing chick spinal cord begins in the ventrolateral funiculi of the spinal cord on E13 and proceeds in a rostral-caudal direction with development. During the early stages of myelination, MBP, MAG or CNP immunoreactivity was consistently more abundant in the ventrolateral funiculi than other areas of the myelinating spinal cord (Fig. 2-3; Fig. 2-4). By El 5, toluidine blue stained transverse sections of the spinal cord revealed tightly compacted myelinated axonal profiles (Fig. 2-5). These results are in agreement with observations of myelin development in the rat and mouse spinal cord, although myelination in these animals begins much later in embryonic development (Foran and Peterson 1992). By E17, all levels of the myelinating chick spinal cord showed similar levels of MBP, MAG or CNP immunoreactivity. On E17 a dramatic increase in MBP and MAG immunoreactivity occured in all levels of the myelinating spinal cord. This second wave of myelination may be due to an increase in MBP and MAG incorporation into the myelin processes,  52 extention of myelin processes on or around this stage of development or to an ongoing increase in oligodendrocyte number in the early myelinating spinal cord and a corresponding increase in the number of myelin processes at this stage of myelination. The onset of myelination in the developing chick spinal cord begins following the completion of axonal projections from the brainstem to the lumbar levels of the spinal cord. Brainstem-spinal projections are complete to the cervical spinal cord by E8 and are complete to the lumbar spinal cord by El 2 (Okado and Oppenheim 1985). Myelination of the spinal cord begins in the ventrolateral funiculi shortly afterwards on E13. The ventrolateral funiculi surround the ventral horns, which are the site of spinal cord motorneurons. Interestingly, E13 also marks the onset of kicking movements of developing embryos. Thus, the developmental onset of spinal cord myelination occurs shortly after brainstem-spinal neurons have completed their projections and have made functional synaptic connections with their targets. The development of the corticospinal tract of the rat also follows this temporal sequence. The rat corticospinal tract is a late-developing tract that begins descending from the brainstem only after the other tracts of the spinal cord have completed their development and have been myelinated (Schreyer and Jones 1982). Myelination of the corticospinal tract begins only after axonal projections are complete and functional connections have been formed. The onset of myelination in the developing chick spinal cord may be  53 under the control of astrocytes within the spinal cord. In vitro experiments have demonstrated that oligodendrocyte differentiation is under the control of at least two growth factors, basic fibrillary growth factor (bFGF) and platelet-derived growth factor (PDGF), which are found in both astrocytes and neurons (reviewed in Goldmanl992). In the presence of bFGF and PDGF, oligodendrocyte progenitors undergo a set number of cell divisions before differentiating into oligodendrocytes (Raff et al. 1985; Raff 1989). bFGF alone is a potent mitogen for dividing oligodendrocyte progenitors. PDGF upregulates the expression of bFGF receptors on oligodendrocyte progenitors, increasing their sensitivity to this potent mitogen. It is theorized that an internal clock within the oligodend rocyte progenitor controls their timely differentiation into oligodendrocytes (Raff et al. 1985). Oligodendrocyte progenitors may receive an additional astrocyte-derived signal which over-rides the mitogenic effects of bFGF and PDGF and prompts oligodendrocyte progenitors to differentiate (Sam David, McGill University, Canada; unpublished observations). Astrocyte conditioned medium has recently been shown to overcome the anti differentiative effects of bFGF and PDGF when applied to dividing oligodendrocyte progenitors in culture. When astrocyte-conditioned medium is applied to bFGF- and PDGF-supplemented cultures of dividing oligodendrocyte progenitors, the cells promptly exit the cell cycle and differentiate into oligodendrocytes. It is interesting in this regard that I observed that the onset of astrocyte-specific glial fibrillary acidic protein (GFAP) immunoreactivity occurs  54 on El 2, just prior to the onset of myelination. Given the results of these two studies, it is conceivable that astrocytes may directly induce oligodendrocyte differentiation in the developing chick spinal cord. In the rodent spinal cord, astrocytes appear several days prior to the appearance of oligodendrocytes (Raff et al. 1984). Perhaps the putative astrocyte-derived factor influencing oligodendrocyte differentiation in the rat is present, or reaches threshold levels of effectiveness, around the time of oligodendrocyte differentiation. The onset of myelination in the developing chick spinal cord is coincident with the loss of regenerative capacity of spinal cord axons (Keirstead et al. 1992). Complete transection of the embryonic chick spinal cord prior to the developmental onset of myelination at E13, results in complete anatomical and physiological repair resulting in 100% functional recovery of locomotor activity (Shimizu et al. 1990; Hasan et al. 1991). Double-labelling studies have illustrated that this repair is due in part to true regeneration of axotomized fibres (Hasan et al. 1993). Transections on or after E13 result in limited or no repair, leaving the transected animal completely incapable of voluntary locomotion (Shimizu et al. 1990; Hasan et al. 1991). This restricted period for repair extends for the duration of CNS maturation and adult life (Shimizu et al. 1990). The transition at E13 from a permissive to a restrictive period for spinal cord repair is coincident with the developmental onset of myelination within the embryonic chick spinal cord (Benstead et al. 1957; Hartman et al. 1979; MackIm  and Weill 1985; Keirstead et al. 1992). These findings suggest that  55 myelin may be inhibitory to the regeneration of transected spinal cord axons, and prompted the studies outlined in the remainder of this thesis.  55o  CHAPTER 3  DEVELOPMENTAL MYELIN-SUPPRESSION IN THE EMBRYONIC CHICK SPINAL CORD  56 INTRODUCTION  Studies of the regenerative capacity of the developing chick spinal cord have determined that the spinal cord looses the ability to functionally regenerate following complete transection on or about embryonic day (E) 13 of the 21 day developmental period prior to hatching (Hasan et al. 1991). Transections of the spinal cord after E13 result in little or no neuroanatomical regeneration or functional recovery, rendering the animal completely incapable of voluntary locomotion after hatching (Shimizu et al. 1990; Hasan et al. 1991). Chapter 2 of this thesis outlines a series of experiments which led to the observation that the developmental onset of myelination in the embryonic chick spinal cord begins on E13, coincident with the transition from a permissive to a restrictive period for spinal cord repair. This observation suggested that myelin may be inhibitory to the functional repair of transected spinal cord in embryonic chick. In order to test this hypothesis, it was necessary to devise a method of delaying the onset of myelination (myelin-suppression) until later stages of development (well into the restrictive period for repair). A subsequent transection of the spinal cord during the normally restrictive period for repair (eg. E14 or E15) in such a myelin-suppressed embryo would then serve as a direct test of whether myelin is inhibitory to the functional repair of transected spinal cord in embryonic chick. This chapter discusses the development and characterization of a  57 protocol effective in delaying the developmental onset of myelination in the embryonic chick spinal cord. Several mouse models have been developed that show characteristics of abnormal myelin development. The jimpy mouse is a neurological mutant characterized by a deficiency of oligodendrocytes and myelin in the CNS (Sidman et al. 1964). All major components of myelin are reduced in jimpy mice including myelin basic protein (MBP; Matthieu et al. 1973), galactolipids (Nussbaum et al. 1969), myelin-associated glycoprotein (MAG; Yanagisawa and Quarles 1986), 2’,3’- cyclic nucleotide 3’- phosphodiesterase (CNP; Sarlieve 1976) and proteolipid protein (PLP; Yanagisawa and Quarles 1986). Mutation of the PLP gene has subsequently been shown to be responsible for the jimpy mutation (Spreyer et al. 1993). The number of oligodendrocytes generated in jimpy mice increases over the first two weeks postnatally, and is comparable to the number of oligodendrocytes generated by normal mice (Ghandour and Skoff 1988). However, this population of jimpy oli.godendrocytes is not stable, showing high levels of cell death early in development (Knapp et al. 1986). Thus, the adult population of jimpy oligodendrocytes is dramatically reduced compared to normal mice and, additionally, cannot be maintained in culture as long as oligodendrocytes from normal mouse brains (Privat et al. 1972). Besides showing abnormally high levels of cell death, young jimpy oligodendrocytes are generally more highly branched than normal oligodendrocytes (Ghandour and Skoff 1988). This stellate characteristic of young jimpy oligodendrocytes may  58 adversely affect early oligodendrocyte-neuron interactions leading to decreased myelination. In the neonatal jimpy brain, oligodendrocytes often display a polarized morphology, with thicker and stubbier processes as compared to normal oligodendrocytes (Ghandour and Skoff 1988). Astrocytes are also abnormal in jimpy mice, displaying characteristics of a chronic reactive state (Dupouey et al. 1080). Jimpy mice usually survive for a maximum of 2 weeks postnatally (Sidman et al. 1964). Due to these various complications, the jimpy phenotype is an unsuitable model for studies of CNS injury and repair. A dysmyelinated transgenic mouse line termed ‘wonky’ has also been developed, resulting from expression of class 1 histocompatibility molecules in oligodendrocytes (Turnley et al. 1991). Oligodendrocytes do not normally express class 1 histocompatibility molecules (Traugott 1 987; Bartlett et al. 1989). Although the mechanism of cellular destruction associated with aberrant MHC molecule expression is unclear, overexpression of class 1 histocompatibility molecules in other cell types (eg. pancreatic B cells) leads to nonimmune destruction on the cells (Allison et al. 1988). DNA consisting of the murine class 1 H2Kb protein-coding sequences of the genomic clone linked to the Hind 1 11 fragment of the murine MBP promoter, when microinjected into fertilized eggs, resulted in the ‘wonky’ phenotype (Turnley et al. 1991). Wonky mice are characterized by hypomyelination in the CNS, and exhibit neurological symptoms at 11-14 days of age, just after the onset of myelination in normal mice. Homozygous transgenic mice have a shivering phenotype during  59 locomoter activity and develop tonic seizures which lead to death at 15-22 days. The number of astrocytes is also increased in wonky mice, which may indicate reactive gliosis. Due to the short life span of the wonky phenotype, this model is not suitable for studies of CNS injury and repair. Experimental methods of myelin-suppression in vivo include X-ray irradiation (Blakemore 1977), drugs (Blakemore 1978: Ludwin 1978: Eames et al. 1977), viruses (DalCanto and Lipton 1980; Herndon et al. 1977), nerve compression (Bunge et al. 1961; Clifford-Jones et al. 1980) and cell-mediated immunological reactions (Lampert 1968; Raine and Bornstein 1970). Although effective in suppressing myelin, none of these methods have demonstrated the cell selectivity and reparative benefits observed in the use of developmental myelin suppression. Several in vitro studies have described inhibition of myelin formation mediated by anti-galactocerebroside antibodies or sera from rabbits innoculated with whole CNS tissue (Bornstein and Raine 1970; Dorfman et aI.1979; Ranscht et al. 1987). Bornstein and Raine report myelin inhibition via complement mediated cytotoxicity. In this study, anti-CNS anti-serum inhibited oligodendrocyte differentiation and myelin formation in rat spinal cord cultures (Bornstein and Raine 1970). Exposure of neonatal rat cerebellum cultures to anti-galactocerebroside anti-serum has also been reported to inhibit myelin formation, although this study suggests that complement proteins are not required for the inhibition of primary myelination (Dorfman et aI. 1979).  60 Inhibition of in vitro peripheral myelin formation has also been demonstrated by exposing cultured rat sensory neurons and Schwann cells to monoclonal anti galactocerebroside immunoglobulin (Ranscht 1987). Anti-galactocerebroside immunoglobulin prevented elongation of the mesaxon, inhibiting myelin formation by more than 99% as compared to control cultures. This study suggests that the mechanism of myelin inhibition is the removal of galactocerebroside (GaIC) from Schwann cell surfaces by internalization of the GaIC-anti-GaIC antigen-antibody complex. Antisera against GaIC have also been shown to demyelinate guinea pig optic nerve (Sergott et al. 1984; Ozawa et al. 1989), cat optic nerve (Carrol et al. 1984; Carrol et al. 1985) and rat spinal cord in vivo (Mastaglia et al. 1989). Although GaIC is the major sphingolipid produced by oligodendrocytes in the CNS, its function has not yet been elucidated (Ransht et al. 1982; Dyer and Benjamins 1990). However, several experiments suggest that GalC may be involved in transmembrane signalling (Dyer and Benjamins 1990; Benjamins and Dyer 1990). GaIC immunostaining of oligodendrocytes in culture reveals confluent GalC labelling of the oligodendrocyte cell body and membrane sheets (Ghandour and Skoff 1988). In vitro studies have demonstrated that GaIC antibodies, when applied to oligodendrocyte cultures, cause patching of GaIC over internal domains of MBP (Dyer and Benjamins 1988). GaIC patching is then followed by microtubule disruption in oligodendroglial membrane sheets and the fusion of MBP domains. The depolymerization of microtubules is mediated by an  61 influx of calcium through plasma membrane channels (Dyer and Benjamins 1990). Extracellular EGTA (a calcium chelator) blocks anti-GaIC induced microtubule dissassembly, suggesting that an extracellular source of calcium mediates this effect. Microtubule depolymerization is believed to occur by phosphorylation of tubulin and its associated proteins by calcium, calmodulin dependent kinases. Actin monomers, normally colocalized with microtubules, reorganize and form filaments in the absence of microtubular structures. As a result of these cellular alterations, blebbing of the membrane surface and process contraction eventually lead to elimination of the oligodendrocyte myelin membrane sheets. These studies indicate that anti-GaIC antibodies are an efficient means of suppressing myelin in oligodendrocyte cultures and prompted an investigation of their effectiveness in suppressing myelin development in the embryonic chick spinal cord in vivo. The efficiency of GalC antibodies in removing myelin (outlined above) and inhibiting myelin formation suggests that other oligodendrocyte surface-specific, complement binding antibodies may be equally effective for this purpose. In order to bind and activate the first component of complement (Cl q), two juxtaposed surface-bound lgG molecules are required (Law and Reid 1989). This necessitates a high concentration of antigen on the surface of the oligodendrocyte. The efficiency of GaIC antibodies in evoking myelin suppression may be attributed to the abundance of GaIC on the surface of oligodendrocyte myelin (GaIC is the major sphingolipid in the myelin membrane).  62 Pentameric 1gM molecules, on the other hand, are able to bind the first component of complement and activate the complement cascade when singly bound to a cell surface (Law and Reid 1989). For this reason 1gM-mediated, complement-dependent cellular attack can be initiated with an antibody to a relatively less abundant cell surface antigen and is generally more robust than an lgG-mediated cellular attack. These advantages prompted a study of the effectiveness of myelin-suppression initiated by the 04 antibody (developed by Melitta Schachner). The 04 antibody is an 1gM antibody specific for the myelin membrane (Sommer and Schachner 1981) recognizing an unidentified antigen on the oligodendrocyte progenitor cell surface as well as an antigen present on the mature myelin sheath (Bansal et al. 1989). The 04 antibody has been demonstrated to effectively bind complement in vitro (Sommer and Schachner 1982). The classical pathway of the complement system is activated by the interaction of the first component of complement (Cl complex) with the Fc region of an lgG or 1gM antibody (reviewed in Law and Reid 1989). Different lgG isotypes vary in their ability to bind and activate the Cl complex. IgGi and lgG3 isotypes are very active whereas lgG2 is less active and lgG4 is inactive. Although monomeric lgG is capable of activating the Cl complex, the strength of binding is less than that of multiple Fc regions presented by aggregated lgG in immune complexes. Tight 1gM-Cl complex binding takes place following binding of the 1gM antibody to antigen and the concomitant exposure of binding  63 sites in the many Fc regions of the pentameric antibody. Upon activation, the Cl complex (Clq + Cir + Cis) undergoes a conformational change, allowing sequential autoactivation of the Cir and Cis components. The activated Cis molecule then splits the C4 pro-enzyme, liberating the C4a anaphylatoxin and the large C4b fragment. The activated C4b enzyme then binds and splits the C2 pro-enzyme in a Mg -dependent fashion, producing the C4b2a complex and 2 the non-catalytic C2b molecule. The C4b2a complex activates C3 by a proteolytic cleavage of C3 into the C3a anaphylatoxin and the highly reactive C3b molecule. It is at this point in the complement enzymatic cascade that the complement system may be irreversibly deposited on the target surface. The C3 molecule is a unique design in molecular architecture. Upon activation, the removal of C3a results in a conformational change in the C3b molecule which exposes an internal thiolester. The exposed thiolester is extremely reactive and has the ability to form covalently linked complexes with any nucleophile (a hydroxl or amino group). This implies that C3b can be deposited on any biological surface. The ability of C3b to bind to any foreign life form is clearly an immunological advantage, but protective measures must be present to ensure that host cell binding does not take place. The extremely reactive nature of the thiolester provides its own control. C3b will use water as a nucleophile, limiting the effective range of the activated C3b. Thus C3b is restricted to binding to the surface of the same cell which triggered its activation, or is inactivated by water.  64 The activated C3b molecule complexes with C4b2a and induces a conformational change in C5, liberating the C5a anaphylatoxin and the C5b molecule, responsible for initiating the assembly of the membrane attack complex (MAC). The formation of the MAC constitutes the terminal steps of complement activity. The activated C5b molecule binds C6 and exposes a C6 binding site for C7. In this way C7, C8 and C9 are sequentially bound to the C5b molecule. Binding of C9 initiates a high affinity C9-C9 interaction resulting in the formation of the MAC with a composition of C5b-8(C9), where n may range from 1-18. The MAC presumably disrupts the ionic homeostasis of the . As 2 oligodendrocyte membrane leading to an increase in intracellular Ca 2 results in a disruption of discussed above, the increase in intracellular Ca microtubules resulting in retraction of oligodendrocyte processes. This chapter outlines a protocol for, and demonstrates the effectiveness of, complement proteins plus monoclonal GaIC, polyclonal GaIC or 04 antibodies to delay the developmental onset of myelination in the embryonic chick spinal cord (termed developmental myelin-suppression; Keirstead et al. 1992). Developmental myelin-suppression is documented with MBP, MAG or CNP immunohistochemistry, toluidine blue staining or a combination of these techniques. Furthermore, developmental myelin-suppression is shown to be specific for oligodendrocyte myelin, leaving the neuronal and astrocytic populations of the spinal cord unperturbed.  65 MATERIALS AND METHODS Developmental Myelln-Suppression Thoracic spinal cord injections were performed at embryonic day (E) 9E12 in the chick embryo using a glass micropipette (tip diameter  =  30-40 urn;  A-M Systems, Everett, Washington #6045) connected to a Picospritzer II pressure injection system (General Valve Corp., Fairfield, New Jersey). Myelin 3 mouse suppression in the chick embryo was evoked by injecting either an lgG galactocerebroside (GaIC) antibody (a gift from B. Ranscht, La Jolla Cancer Research Institute, U.S.A.) at a dilution of 1:25 with 20% homologous serum (as a source of complement) in 0.1M phosphate buffered saline (PBS), pH 7.4 or 1 rabbit GaIC antibody (Chemicon International Inc., Ternecula, California an IgG #AB142) at a dilution of 1:25 with 20% guinea pig complement (Gibco BRL, Burlington, Ontario #19195-01 5) in 0.1M PBS pH 7.4 or an 1gM polyclonal 04 antibody (a gift from Melitta Schachner, Neurobiology ETH-Honggerberg HPM E38 CH-8093 Zurich, Switzerland) at a dilution of 1:25 with 20% guinea pig complement in 0.1M PBS pH 7.4. Each animal received a total volume of 2-3 ul, over 1-4 penetrations, injected directly into the mid-to-high thoracic spinal cord. The mouse GaIC antibody was supplied as a hybridoma supernatant (2.67 mg/mi) which was then diluted 1:25, providing an effective concentration of 63.0 ng of GalC hybridoma supernatant injected per gram body weight. To control for non-specific binding of the GaIC antibody effecting our results, control embryos were similarly injected with 20% homologous  66 complement plus a human antibody that does not cross-react with chicken. We chose a monoclonal antibody to glial fibrillary acidic protein (GFAP), a major constituent of astrocytes within the central nervous system (CNS). Other immunological control embryos received injections of either: 1) GalC antibody only, 2) homologous complement proteins only, 3) guinea pig complement only, 4) vehicle only (0.1M PBS, pH 7.4) or, 5) the mouse GaIC antibody plus homologous complement, following heat-inactivation of the complement by exposure at 50°C for 30 mm. Those embryos not undergoing a subsequent thoracic spinal cord transection were perfused intracardially at the appropriate developmental stage (see results) with O.1M PBS containing 2500 USP units of heparin per 50 mIs PBS, pH 7.4 (at 37°C) followed by perfusion with 4% paraformaldehyde in O.1M phosphate buffer, pH 7.4 (at 4-10°C). The dissected brains and/or spinal cords were then immersed in the same fixative for 24hr at 4°C and subsequently embedded in paraffin using standard protocols. Paraffin Embedding See page 29. Immunohistochemistry See page 30. Histological Staining See page 32.  67 RESU LTS The developmental onset of myelination in the embryonic chick spinal cord was delayed by pressure injection of complement proteins plus either monoclonal galactocerebroside (GalC) antibodies, polyclonal GaIC antibodies or polyclonal 04 antibodies directly into the thoracic spinal cord, 1-4 days prior to the normal developmental onset of myelination (embryonic day 13; see chapter 2). Monoclonal galactocerebroside (GalC) antibodies, polyclonal GaIC antibodies and polyclonal 04 antibodies were utilized due to their abilities to recognize oligodendrocyte cell-surface antigens and fix complement. Thoracic spinal cord injections of monoclonal GalC antibodies, plus either chick serum or guinea pig complement, delayed the developmental onset of spinal cord myelination until E17 (Fig. 3-1). Immunohistochemical assessments of myelin-suppressed spinal cords (previously injected on El 1) sacrificed on El 2 (n=6), El3 (n=6), E14 (n=6), El5 (n=12) and E16 (n=6) revealed a complete lack of myelin basic protein (MBP) immunoreactivity throughout the spinal cord excluding the most rostral 1-4 cervical segments. Immunohistochemical assessments of unoperated control El 5 spinal cords revealed a dense network of MBP immunoreactivity (Fig. 3-lA; see chapter 2). The extent and degree of developmental myelin-suppression in animals sacrificed on El 5 was similar when the complement proteins and monoclonal GaIC antibodies were administered on either E9, ElO, Ell or El2 (n=3 for each day).  68 Figure 3-1. Developmental myelin-suppression in the thoracic spinal cord of the embryonic chick in parasaggital section. A: Unoperated (normally-myelinated) control embryonic day (E) 15 spinal cord showing extensive myelin basic protein (MBP) immunoreactivity within white matter. B: MBP immunofluorescence staining of an El 5 spinal cord from a myelin-suppressed animal that received a single injection of polyclonal galactocerebroside (GaIC) antibodies plus complement on El 1; note the absence of myelin. C: Unoperated (normally myelinated) control El 5 spinal cord showing extensive myelin-associated glycoprotein (MAG) immunoreactivity within white matter. D: MAG immunofluorescence staining of an El 5 spinal cord from a myelin-suppressed animal that received a single injection of GaIC antibodies plus complement on El 1; note the absence of myelin. E: Unoperated (normally-myelinated) control El 5 spinal cord showing extensive 2’,3’-cyclic nucleotide 3’-phosphodiesterase (CNP) immunoreactivity within white matter. F: CNP immunofluorescence staining of an El 5 spinal cord from a myelin-suppressed animal that received a single injection of GaIC antibodies plus complement on El 1; note the absence of myelin. Developmental myelin-suppression is also observed with monoclonal GaIC or 04 antibodies plus complement. The previous injection site is undetectable in B, D and F, indicating that the injection does not cause substantial damage. In all photographs the outer edge of the spinal cord lies on the right hand side, with the white matter adjacent. (Bars  =  lOOum).  01:  72 Thoracic spinal cord injections of polyclonal GaIC antibodies plus guinea pig complement on El 1, also delayed the developmental onset of spinal cord myelination until E17 (Fig. 3-1). lmmunohistochemical assessments of myetin suppressed spinal cords on E12 (n=4), E13 (n=4), E14 (n=4), E15 (n=5; Fig. 3-1B) and E16 (n=4) showed a complete tack of myelin basic protein (MBP) immunoreactivity throughout the spinal cord excluding the most rostrat 1-4 cervical segments. Immunohistochemical assessments of myelin-suppressed spinal cords on E13 (n=3), E14 (n=3) and E15 (n=3; Fig. 3-iD) also showed a complete lack of myelin-associated glycoprotein (MAG) immunoreactivity throughout the spinal cord excluding the most rostral 1-4 cervical segments. lmmunohistochemical assessments of myetin-suppressed spinal cords on E13 (n=3), E14 (n=3) and El5 (n=3; Fig. 3-iF) also showed a complete lack of 2’,3’-cyclic nucleotide 3’-phosphodiesterase (CNP) immunoreactivity throughout the spinal cord excluding the most rostral 1-4 cervical segments. Immunohistochemical assessments of unoperated control El 5 spinal cords (n =3 for each) revealed a dense network of MBP, MAG and CNP immunoreactivity (Fig. 3-lA, C and E; also see chapter 2). Five spinal cords injected on El 1 with polyclonal GalC antibodies plus guinea pig complement were sacrificed on E15, and their thoracic spinal cords were embedded in Spurr resin and transversely microsectioned for totuidine blue staining (Fig. 3-2). Light microscopic analysis of toluidine blue stained spinal cord tissue revealed a lack of myelin in all regions (Fig. 3-28). In all instances  73 Figure 3-2. Toluidine Blue staining of chick spinal cord white matter in transverse section. A: Unoperated (normally-myelinated) embryonic day (E) 15 spinal cord. Notice the densely compacted profiles of myelinated axons (dark rings). B: E15 spinal cord from a myelin-suppressed animal that received a single injection of polyclonal galactocerebroside (GaIC) antibodies plus complement on El 1; note the virtual absence of myelinated axons. (Bars 25um).  =  75 only a very few myelinated axons scattered throughout the spinal cord white matter were visible. Unoperated El 5 control tissue (n =5) processed in a similar manner contained an abundance of tightly compacted myelin characteristically viewed as rings surrounding the axon profiles (Fig. 3-2A; also see chapter 2). Thoracic spinal cord injections of polyclonal 04 antibodies plus guinea pig complement on El 1, also delayed the developmental onset of spinal cord myelination until El7 (Fig. 3-1). Immunohistochemical assessments of myelin suppressed spinal cords on E13 (n=3), E14 (n=3), E15 (n=3) and E16 (n=3) showed a complete lack of myelin basic protein (MBP) immunoreactivity throughout the spinal cord excluding the most rostral 1-4 cervical segments. lmmunohistochemical assessments of unoperated control El 5 spinal cords revealed a dense network of MBP immunoreactivity (Fig. 3-lA; see chapter 2). The 04 1gM antibody consistently produced larger and more complete areas of myelin inhibition compared to the monoclonal and polyclonal GaIC IgG antibodies, which were equally effective compared to each other. On or about E17 a period of robust myelination took place in both unoperated control spinal cords (see chapter 2) and experimental spinal cords subjected to developmental myelin-suppression (Fig. 3-3). MBP immunohistochemical analysis of E17 and E18 spinal cords, previously injected with monoclonal GaIC antibodies plus guinea pig complement on El 1, revealed immunoreactivity throughout all levels of the spinal cord (Fig. 3-3B; n  6 for  each day). Similarly, MBP immunohistochemical analysis of E17 and El8 spinal  76 Figure 3-3. Developmental onset of myelination following developmental myelin suppression. A: Unoperated (normally-myelinated) control embryonic day (E) 17 chick spinal cord showing extensive myelin basic protein (MBP) immunoreactivity within white matter in parasaggital section. B: MBP immunofluorescence staining of an E17 spinal cord in parasaggital section from a myelin-suppressed chick that received a single injection of polyclonal galactocerebroside (GaIC) antibodies plus complement on El 1; note that MBP immunoreactivity is comparable to, or less than, normally myelinated levels indicated in A. In all photographs the outer edge of the spinal cord lies on the right hand side, with the white matter adjacent. (Bars  =  lOOum).  78 cords, previously injected with polyclonal GaIC antibodies plus guinea pig complement on El 1, revealed immunoreactivity throughout all levels of the spinal cord (n =4 for each day). Although all myelin-suppressed spinal cords analysed on E17 showed obvious MBP immunoreactivity, they appeared to be less intensely stained than unoperated control E17 spinal cord tissue (Fig. 33A). MBP immunohistochemical analysis of P2, P4 and P6 spinal cords (n=4 for each day), previously injected with monoclonal GaIC antibodies plus guinea pig complement on El 1, showed levels of MBP immunofluoresence comparable to unoperated control tissue (Fig. 3-4A and B; n=25). MBP immunohistochemical analysis of P2, P4 and P6 spinal cords (n=4 for each day), previously injected with polyclonal GaIC antibodies plus guinea pig complement on El 1, also showed levels of MBP immunoreactivity similar to the unoperated control tissue. These results indicate that myelination following developmental myelin-suppression is fully compensatory. After hatching, animals that had previously been subjected to developmental myelin-suppression demonstrated locomotor behavior that was comparable to unoperated control hatchlings of the same age. Locomotor behavior of previously myelin-suppressed hatchlings was assessed on P2, P4 and P6 (n =8 for each day), prior to sacrifice for immunohistochemical analysis (see above). Visual assessments of postural adjustments, walking, running and righting responses suggested that neuronal control of locomotion was not  79 Figure 3-4. Hatchling pattern of myelination following developmental myelin suppression. A: Unoperated (normally-myelinated) control posthatchling day (P) 2 chick spinal cord showing extensive myelin basic protein (M8P) immunoreactivity within white matter in parasaggital section. B: MBP immunofluorescence staining of an P2 spinal cord in parasaggital section from a myelin-suppressed chick that received a single injection of polyclonal galactocerebroside (GalC) antibodies plus complement on El 1; note that MBP immunoreactivity is comparable to normally myelinated levels indicated in A. In all photographs the outer edge of the spinal cord lies on the right hand side, with the white matter adjacent. (Bars  =  lOOum).  oç\ 0  81 altered in hatchlings that had been subjected to myelin-suppression during development. As a control for the possible influence of nonspecific binding of the GaIC antibody affecting our results, 5 control embyos were injected in the thoracic spinal cord at El 1 with an antibody to glial fibrillary acidic protein (GFAP; an astrocyte marker) plus guinea pig complement. MBP immunohistochemistry on El 5 revealed no suppression of myelin development. Other immunological control embryos received injections of monoclonal GalC antibody only (Fig. 35A; n=6), guinea pig complement only (Fig. 3-5B; n=8), PBS vehicle only (n=4) or monoclonal GaIC antibodies plus heat-inactivated serum (heating serum prior to use denatures and inactivates the complement proteins; n=8). In no case was myelin development suppressed or detectably altered, nor was there any evidence of anatomical repair or functional recovery after an E15 spinal cord transection (see chapter 4). Direct pressure injection of control and experimental solutions into the thoracic spinal cord did not result in significant damage to the spinal cord tissue. The injected solution did not detectably displace spinal cord tissue or result in an area of necrosis at the injection site at any of the ages examined (see above). A thin needle tract in the thoracic region was occasionally visible in E12-E14 spinal cords previously injected on E9-E12. After E14, the injection site was undetectable. The developing and mature state of the spinal cord astrocyte population  82 Figure 3-5. Pattern of myelin basic protein (MBP) immunoreactivity in immunological control thoracic spinal cords in parasaggital section. A: MBP immunofluorescence staining of an embryonic day (E) 15 spinal cord from an animal that received a single injection of GaIC antibody only on El 1; note that myelin is unperturbed. B: MBP immunofluorescence staining of an El 5 spinal cord from an animal that received a single injection of complement only on El 1; note that myelin is unperturbed. In all photographs the outer edge of the spinal cord lies on the right hand side, with the white matter adjacent. (Bars 1 OOum).  =  84 did not appear to be disturbed by developmental myelin-suppression, as assessed by GFAP immunohistochemistry (Fig. 3-6). Immunohistochemical assessments of myelin-suppressed and unoperated control E14 and E15 (Fig. 36A and B) spinal cords (n  =  4 for each) showed that the astrocyte number and  distribution were similar for each developmental stage. P6 spinal cord tissue from myelin-suppressed animals (n=6) also showed similar astrocyte numbers and distribution as compared to unoperated control P6 spinal cord tissue (Fig. 36C and D; n=8). Additionally, individual astrocytes in myelin-suppressed spinal cords did not appear to express higher levels of GFAP than individual astrocytes from unoperated controls at any of the developmental stages examined. The neuronal population of the spinal cord did not appear to be disturbed as a result of developmental myelin-suppression (Fig. 3-7). Neuronal development in the embryonic spinal cord was assessed with microtubule associated protein 2 (MAP-2) immunohistochemistry to identify the dendritic morphology, and thionin staining to identify the neuronal and axonal morphology. Myelin-suppressed E15 spinal cords analysed with MAP-2 antibodies (Fig. 3-78; n  =  4) or thioinin staining (n =3) was indistinguishably  different from unoperated control El 5 spinal cord analysed with MAP-2 antibodies (Fig. 3-7A; n=4) or thionin staining (n=3).  85 Figure 3-6. Pattern of glial fibrillary acidic protein (GFAP) immunoreactivity in the myelin-suppressed chick spinal cord in parasaggital section. A: Unoperated (norrnally-myelinated) control embryonic day (E) 15 spinal cord showing extensive GFAP immunoreactivity within the white matter. B: GFAP immunofluorescence staining of an El 5 spinal cord from a myelin-suppressed animal that received a single injection of polyclonal galactocerebroside (GaIC) antibodies plus complement on El 1; note that GFAP immunoreactivity is comparable to A. C: Unoperated (normally-myelinated) control posthatching day (P) 6 spinal cord showing extensive GFAP immunoreactivity within the white matter. D: GFAP immunofluorescence staining of an P6 spinal cord from a myelin-suppressed animal that received a single injection of polyclonal GaIC antibodies plus complement on El 1; note that GFAP immunoreactivity is comparable to C. In all photographs the outer edge of the spinal cord lies on the right hand side, with the white matter adjacent. (Bars urn for C and D.).  =  lOOum for A and B; 50  c4  88 Figure 3-7. Pattern of microtubule-associated protein-2 (MAP-2) immunoreactivity in the myelin-suppressed chick in parasaggital section. A: Unoperated (normally-myelinated) control embryonic day (E) 1 5 spinal cord showing extensive MAP-2 immunoreactivity within the white matter. B: MAP-2 immunofluorescence staining of an E15 spinal cord from a myelin-suppressed animal that received a single injection of polyclonal galactocerebroside (GaIC) antibodies plus complement on El 1; note that MAP-2 immunoreactivity is comparable to A. In all photographs the outer edge of the spinal cord lies on the right hand side, with the white matter adjacent. (Bars  =  lOOum).  4,  4 -&  :  .,  :.-  ‘r’ ‘4  :1  ‘4 M  90 DISCUSSION Although the neural factors controlling the differentiation and development of oligodendrocytes within the CNS have not been fully characterized, it is nonetheless possible to alter the developmental onset of myelination in vivo (Keirstead et al. 1992). By specifically targeting the highly catalytic and toxic activity of a series of blood enzymes (complement), it is possible to destroy a cell population of interest (Law and Reid 1989). Targeting oligodendrocytes in this manner necessitates an oligodendrocyte cell surfacespecific antibody that is capable of readily binding complement via the Fc receptor portion of the antibody (Fig. 3-8). The monoclonal galactocerebroside (GaIC), polyclonal GaIC and polyclonal 04 antibodies described above meet both of these requirements (Ranscht et al. 1982; Sommer and Schachner 1982; Sergott et al. 1984; Ghandour and Skoff 1988; Ozawa et al. 1989; Mastaglia et al. 1989). Direct injection of complement proteins plus monoclonal GaIC, polyclonal GaIC or 04 antibodies into the thoracic spinal cord of an embryonic day (E) 9El 2 chick results in a delay of the developmental onset of myelination until El 7 (developmental myelin-suppression; Fig. 3-1; Fig. 3-2; Fig. 3-3). Myelination of the embryonic chick spinal cord normally begins on E13 (Benstead et al. 1957; Hartman et al. 1979; MackIm  and Weill 1985; Keirstead et al. 1992; see  chapter 2). Developmental myelin-suppression was confirmed immunohistochemically using three myelin-specific antibodies (to myelin basic  91 Figure 3-8. Schematic representation of the postulated mechanism of developmental myelin-suppression. Oligodendrocyte cell surface-specific antibodies bind to the oligodendrocyte surface. Once bound, they fix the first component of complement, which in turn activates the complement cascade. The concommitant formation of the membrane attack complex results in a disruption of the ionic homeostasis of the cell.  X)I(III1()  )  )1II’.1(It[I)I\  IILS)1OI(I JL L)L I LJI( Ii L1O’ ) 1)  ‘1 )1)S1 ) ! 11l1IA/1I’I  93 protein, MBP, Fig. 3-lA and B; myelin-associated glycoprotein, MAG, Fig. 2-iC and D; and 2’,3’-cyclic nucteotide 3’-phosphodiesterase, CNP; Fig. 3-1E and F) as well as histologically by toluidine blue staining of microthin transverse sections of the developing spinal cord (Fig. 3-2). Developmental myelin suppression also inhibited the expression of several proteins that normally appear at the developmental onset of myelination in the chicken embryo (results not shown; D.W. Ethell, H.S. Keirstead and J.D. Steeves, unpublished work). Although GaIC antibodies have been shown to destroy mature myelin in vitro (Dorfman et al. 1979; Dyer and Benjamins 1990) and in vivo (Carroll et at. 1984; Sergott et al. 1984; Carroll et at. 1985; Ozawa et al. 1989; Mastaglia et al. 1989), this is the first demonstration of an anti-GaIC induced developmental suppression of CNS myelin formation. Additionally, this is the first demonstration of myelin-suppression in vivo mediated by 04 antibodies. The antigens recognized by the GaIC and 04 antibodies were selected due to their oligodendrocyte specificity, the availability of complement-binding antibodies as well as the temporal appearance of these molecules in development (Ranscht et al. 1982; Sommer and Schachner 1982; chapter 2 of this thesis). Although the antigen recognized by the 04 antibody is present on mature oligodendrocytes, a like or related antigen recognized by the 04 antibody is also expressed by otigodendrocyte precursors just prior to their differentiation into oligodendrocytes (Sommer and Schachner 1982). GaIC is one of the first markers expressed by oligodendrocytes upon differentiation from their  94 precursors (Ranscht et al. 1982). Histological studies have demonstrated that oligodendrocytes differentiate 2-3 days prior to myelination (Benstead et al. 1957). In support of these findings, a GaIC immunohistochemical investigation of the developing chick spinal cord indicated that GaIC immunoreactivity was first detected on ElO (see chapter 2 of this thesis). For these reasons, the complement proteins plus monoclonal GaIC, polyclonal GaIC or 04 antibodies were injected on E9-E12. The immunological protocol outlined above may elicit developmental myelin inhibition via destruction of all newly-differentiating oligodendrocytes. If this is the case, it follows that the E17 onset on myelination following developmental myelin-suppression is due to the novel differentiation of  -  oligodendrocyte progenitors on or about E17. On the other hand, it is conceivable that the immunological protocol outlined above operates via selective destruction of the myelin sheaths, effectively sparing the oligodendrocyte cell bodies. The E17 onset of myelination following developmental myelin inhibition would then be a result of the re-extention of myelinating processes from the surviving parental cell bodies. These two possibilities are, of course, not mutually exclusive. Several findings support the latter scenario. Firstly, in vitro studies of oligodendrocyte attack mediated by GaIC-antisera (Dorfman et al. 1979) or 04 antibodies and complement (Sommer and Schachner 1981), indicate that the addition of these composite solutions to cultured oligodendrocytes results in withdrawl of the myelinating processes and  95 survival of the oligodendrocyte cell bodies. The subsequent removal of the composite solutions from the culture media resulted in the re-extention of myelinating processes. Secondly, collaborative efforts undertaken with David Pataky (a Ph.D. candidate under the supervision of Dr. John D. Steeves, University of British Columbia, Department of Zoology, Vancouver, B.C., Canada) indicate that E15 myelin-suppressed spinal cords continue to express MBP mRNA despite the lack of myelin (Fig. 3-9; Keirstead et al. 1994). Because mRNA is chiefly located in cell bodies, these findings suggest that oligodendrocyte cell bodies survive developmental myelin-suppression. Lastly, myelination following developmental myelin-suppression takes place in a speedy and fully compensatory manner (see Results section) suggesting that surviving oligodendrocyte cell bodies contribute to myelination following developmental myelin-suppression. MBP mRNA expression in myelin-suppressed spinal cords suggests that surviving oligodendrocyte cell bodies continue to express MBP mRNA despite the loss of myelin processes and sheaths. However, MBP mRNA is found in oligodendrocyte processes as well as oligodendrocyte cell bodies. The translocation of MBP mRNA to oligodendrocyte processes is a reflection of the high turnover of MBP within the myelin sheaths (DesJardins and Morell 1983). Therefore, it is arguable that developmental myelin-suppression results in the  96 Figure 3-9. Representative Northern blots demonstrating myelin basic protein (MBP) mRNA expression in myelin-suppressed animals. A: Unoperated (normally-myelinated) control spinal cords express MBP mRNA. “El 5” means sacrificed on El 5. B: MBP mRNA expression in El 5 spinal cords four days after injection of galactocerebroside (GaIC) antibodies plus complement on El 1. “Ii 1 El 5” means injected on El 1 and sacrificed on El 5. Sizes indicated are in kilobases.  q7  B  A  E15  Ii 1E15 I  28S’ 2.7k 18S’  I  98 loss of both the oligodendrocyte cell body and the myelin sheaths; the MBP mRNA detected in myelin-suppressed spinal cords may reside within the oligodendrocyte processes spared by the immunological intervention. Although no direct evidence exists to dispute this possibility, it is unlikely that myelin processes separated from their parental cell bodies would persist in the spinal cord for four to eight days before being degraded. Therefore it is highly probable that the MBP mRNA detected within myelin-suppressed spinal cords is located within oligodendrocyte cell bodies which have survived developmental myelin suppression. It is also possible that de-differentiation of astrocytes or astrocyte precursors may contribute to spinal cord myelination following developmental myelin—suppression. In demyelinating lesions of the cat optic nerve, reactive glial cells which have begun to differentiate along the astrocyte lineage (GFAP) may reverse their direction of differentiation, evidenced by the coexpression of both astrocyte- and oligodendrocyte-specfic markers (GFAP, GaIC; Kim 1985; Carroll et al. 1987). Switching glial cultures from one type of culture media to another favoring an alternative direction of differentiation results in the transient coexpression of both astrocyte and oligodendrocyte phenotypes (Ingraham and McCarthy 1989). Cells of the 02A lineage may actually exhibit greater plasticity in demyelinating/remyelinating conditions (Kim 1985). Cells coexpressing astrocyte and oligodendrocyte phenotypes can be isolated from the spinal cords of young adult mice demyelinated by corona virus infection (Armstrong et al.  99 1990). In addition, cells coexpressing astrocyte and oligodendrocyte phenotypes have been reported in vivo in a number of demyelinating situations (Bunge et al. 1961; Carroll et al. 1987; Godfraind et a!. 1989). Pending double labelling studies of developmentally myelin-suppressed spinal cords using both astrocyte- and oligodendrocyte-specific antibodies, the possibility of astrocyte dedifferentiation and redifferentiation into myelinating oligodendrocytes can not be excluded. Both normal and myelin-suppressed spinal cords display a ‘wave’ of myelination on or about E17 in development (Fig. 3-3; also see chapter 2 of this thesis). This period of robust myelination is evidenced by a dramatic increase in the MBP, CNP and MAG immunoreactivity detected within the spinal cord. It is unclear whether this period of intense myelination is due to an increase in myelin production by existing oligodendrocytes, or to novel differentiation of oligodendrocyte precursors and a concomitant extention of myelinating processes. Again, these two possibilities are not mutually exclusive. After hatching (E21), levels of MBP, CNP and MAG immunoreactivity in myelin suppressed spinal cords appear to be equivalent to that of unoperated control spinal cords (Fig. 3-4). This indicates that myelination following myelin suppression is fully compensatory, with the E17 ‘wave’ of myelination contributing significantly to myelin recovery within the myelin-suppressed spinal cord. Developmental myelin inhibition in vivo requires both complement  100 proteins and GaIC or 04 antibodies (Fig. 3-5). Immunological control embryos receiving injections of GalC antibody only, complement proteins only, PBS vehicle only or GaIC antibody plus heat-inactivated serum showed no indications of myelin-suppression. In no case was myelin development suppressed or detectably altered, nor was there any evidence of anatomical repair or functional recovery after an E15 spinal cord transection (see chapter 4). This finding is supported by other in vivo studies of anti-GaIC induced demyelination which were also shown to be complement-dependent (Sergott et al. 1984; Ozawa et al. 1989). It is interesting that several in vitro studies have demonstrated anti GaIC induced demyelination in the absence of complement (Dorfman et al. 1979; Ranscht et al. 1987). The susceptibility of cultured myelin to antibodymediated attack in the absence of complement may be somewhat analagous to the susceptibility of myelin proteins to proteolysis. Lysophilized or frozen myelin proteins are rapidly degraded by proteases, whereas freshly isolated myelin proteins are relatively resistant to proteases (Cammer et al. 1986). Thus, the method of myelin isolation and cultivation in the in vitro studies may alter the native state of myelin, increasing suscepibility to antibody-mediated attack. Alternatively, the in vivo mechanism of myelin-suppression may differ significantly from the in vitro mechanism of myelin-suppression. Delaying the developmental onset of myelination does not appear to alter the development and mature state of the astrocyte population of the spinal cord (Fig. 3-6). Immunohistochemical analysis of E15 myelin-suppressed spinal cords  101 indicate that glial fibrillary acidic protein (GFAP) immunoreactivity is similar to unoperated control E15 tissue. Developmental myelin-suppression did not induce detectable changes in astrocyte distribution or individual size. No evidence of astrogliosis was observed in any of the myelin-suppressed spinal cords examined. Additionally, myelin-suppressed spinal cords examined after hatching revealed no signs of abnormal GFAP immunoreactivity as compared to unoperated control spinal cords processed in a similar manner. The neuronal population of the spinal cord did not appear to be disturbed as a result of developmental myelin-suppression (Fig. 3-7). Myelin-suppressed El 5 spinal cords analysed with MAP-2 antibodies to identify dendritic morphology, and thionin staining to identify neuronal and axonal morphology (results not shown), were indistinguishably different from unoperated control E15 spinal cords analysed in a similar manner. After hatching, animals that had previously been subjected to developmental myelin-suppression demonstrated locomotor behavior that was comparable to unoperated control hatchlings of the same age. Visual assessments of postural adjustments, walking, running and righting responses suggested that neuronal control of locomotion was not altered in hatchlings that had been subjected to myelin-suppression during development. Myelination has been shown to affect axonal cytoarchitecture in other developing systems. In the peripheral nervous system (PNS), Schwann cell contact influences axonal morphogenesis, including the segregation of Na  102 channels to the nodes of Ranvier (Joe and Angelides 1992) and regulation of axonal caliber (de Waegh et al. 1992). The trembler mouse is a peripheral myelin-deficient mutant that is characterized by marked hypomyelination as a result of Schwann cell dysfunction. Myelin enwrapment and compaction do not occur, and axonal caliber, phosphorylation of neurofilaments and slow axonal transport are significantly decreased (de Waegh et al. 1992). de Waegh et al. (1992) propose a model in which Schwann cells locally regulate neurofilament phosphorylation via cell-cell contact, mediated perhaps by myelin-associated glycoprotein (MAG). MAG is a member of the immunoglobulin superfamily of recognition/adhesion molecules and is concentrated in the periaxonal space and nodes of Ranvier. MAG may stimulate a neuronal kinase or inhibit a phosphatase, resulting in changes in neurofilament phosphorylation, and therefore axonal caliber. This relationship is reflected in the adult nerve, where regions of the axon not in contact with compact myelin, such as the nodes of Ranvier and the Schmidt-Lanterman incisures in the PNS, have smaller axonal caliber and greater neurofilament packing density than myelinated regions of the same axon (Price et al. 1990). The apparent insensitivity of chick spinal cord axons to myelin-suppression may reflect the immature state of the axons at this stage of development; in a normally-myelinating embryo, myelin-induced alterations of axonal cytoarchitecture may not take place until later stages of embryonic development. Thus, analyses of unoperated control and myelin suppressed El 5 embryos would reveal similar axonal cytoarchitecture.  103 Alternatively, thionin staining may be insensitive to any alterations produced by developmental myelin-suppression. Perhaps a longer period of chick spinal cord myelin-suppression or a more detailed analysis of axonal cytoarchitecture may reveal axonal alterations as a result of the immunological myelin-suppression procedure described here. The ability to delay the developmental onset of myelination in the chick embyo may prove to be a useful tool for investigations of myelin development as well as interactions of myelin with other cell populations within the developing spinal cord. Perhaps more importantly, developmental myelin suppression provides a means of assessing the effects of myelin on the ability of the spinal cord to functionally regenerate following injury. Transections of the embryonic spinal cord on or after the developmental onset of myelination (E13) result in no repair whatsoever (Shimizu et al. 1990; Hasan et al. 1991; Keirstead et al. 1992). Such animals are completely incapable of voluntary locomotion after hatching, suggesting that myelin may be inhibitory to the regeneration of transected axons in the developing embryo. Developmental myelin-suppression provides a means of delaying the onset of myelination until later stages of development; a subsequent transection of the spinal cord during the period of myelin-suppression then serves as a direct test of whether myelin is inhibitory to the regeneration of transected spinal cord.  104  CHAPTER 4  NEUROANATOMICAL REPAIR AND FUNCTIONAL RECOVERY OF TRANSECTED SPINAL CORD IN EMBRYONIC CHICK  105 INTRODUCTION  Transection of the embryonic chick spinal cord prior to the developmental onset of myelination at embryonic day (E) 13 results in complete anatomical and physiological recovery (Shimizu et al. 1990; Hasan et al. 1991). Embryonic spinal cord transections after the developmental onset of myelination result in no recovery, rendering such an animal incapable of voluntary locomotion after hatching (Shimizu et al. 1990; Hasan et al. 1991; Keirstead et al. 1992). These studies suggest that myelin may be inhibitory to the functional regeneration of transected spinal cord in embryonic chick. Chapter 3 of this thesis outlines an immunological method of delaying the developmental onset of myelination within the embryonic chick spinal cord until later stages of development. Developmental myelin-suppression then serves as a tool with which to investigate the effects of myelin on the ability of the spinal cord to regenerate following injury in later stages of embryonic development. This chapter concerns itself with the regenerative capacity of the myelin-suppressed chick spinal cord following late embryonic transection in vivo. There are three mechanisms by which an embryonic spinal cord could effect repair following complete transection in vivo. Repair of brainstem-spinal projections following injury to the developing spinal cord may be due to neogenesis of brainstem-spinal projecting neurons within locomotor nuclei of the brainstem. Although possible following very early embryonic spinal cord  106 transections, neurogenesis is not likely to play a role in spinal cord repair following late-embryonic transection. Neogenesis within the developing chick brainstem-spinal projecting nuclei is complete by E5 (McConnell and Sechrist, 1980; Sechrist and Bronner-Fraser, 1991). Neurogenesis within the central nervous system (CNS) during post-development is limited to the retina, spinal cord and tectum of fish and amphibia, the olfactory epithelium and hippocampus of the rodent and the telencephalic vocal control centers of songbirds (Anderson and Waxman, 1985; Holder and Clarke, 1988). Although it is conceivable that spinal cord injury itself may induce neogenesis of neurons within brainstem locomotor nuclei, it is unlikely that these cells could contribute to any functional recovery of locomotion in hatchlings transected during late embryonic development. Axons grow at a rate of 1-2 mm/day in vivo (Schnell and Schwab 1990). It is unreasonable to assume that late embryonic transection could result in neogenesis, differentiation and axonal outgrowth exceeding a distance of 400 mm within the final 7 days of late embryonic development. Neuroanatomical repair and physiological recovery following injury to the developing spinal cord may alternatively be due to subsequent development of brainstem-spinal projections. Brainstem-spinal projections first descend to the spinal cord by embryonic day (E) 3.5 (2) and complete their axonal projections to lumbar levels by E10-E12 (Okado and Oppenheim 1985). By E12, brainstem spinal projections throughout the spinal cord are equivalent in number and distribution to those in a hatchling chick (Okado and Oppenheim 1985).  107 Therefore, it is reasonable to assume that repair of embryonic spinal cord transected prior to E13 is in part due to late developing brainstem-spinal projections. Because brainstem-spinal projections are complete to all levels of the spinal cord by E12, however, it is unlikely that subsequent development could contribute to recovery from spinal cord transections during the final week of embryonic development. Thirdly, repair of brainstem-spinal projections following injury to the developing spinal cord may be due to true axonal regeneration; that is, the extention of a viable neurite from the proximal end of a severed axon. Although the peripheral nervous system (PNS) is capable of true regeneration following injury, the damaged CNS typically shows little or no ability to effect anatomical or functional regeneration (Sholomenko and Steeves 1987: Eidelberg 1981). Several lines of evidence indicate that the poor regenerative capacity of the CNS is due, not to a lack of intrinsic growth programs, but to environmental influences including an absence of trophic factors and the presence of axonal growth inhibitors (Ramon y Cajal, 1914; David and Aguayo 1981; Schwab and Caroni 1988). Axons of CNS neurons are able to grow out and make functional connections with their targets if peripheral nerve segments containing Schwann cells are grafted to the site of injury (Ramon y Cajal 1914; David and Aguayo 1981; Aguayo et al. 1991). CNS neurons have also been shown to extend fibers in vivo through lesion sites containing implants of fetal CNS tissue (Bjorklund and Stenevi 1979; Kromer et al. 1981; Bjorklund 1991), as well as  108 implants consisting of fibroblasts genetically modified to express growth factors (Fisher and Gage 1993; Tuszynski et al. 1994). In addition, CNS motor neurons are capable of regenerating their peripheral projecting axon if axotomized within the PNS (Ramon y Cajal 1959). These findings indicate that CNS neurons retain intrinsic axonal growth programs which enable long-distance axonal regeneration in the presence of a favorable extraneuronal environment. Several axonal growth inhibitors have been identified in the developing and mature CNS. Myelin-associated proteins that inhibit the anatomical growth of axons in vitro as well as the regrowth of axotomized corticospinal fibres in vivo have been identified in rat spinal cord (Caroni and Schwab 1988). Neutralization of these proteins with functionally-blocking antibodies facilitates anatomical regeneration following axotomy (Schnell and Schwab 1990). Oligodendrocytes also express the inhibitory molecule janusin, which is present in myelin of the CNS. Janusin expression coincides with the developmental onset of myelination (Wintergerst et al., 1993; Bartsch et al., 1993) and has been shown to be a repulsive substrate for neuronal cell bodies as well as growth cones (Pesheva et al., 1989; Taylor et al., 1993). Astrocytes may also contribute to the inhibitory nature of the mature CNS by expressing tenascin which, as a repulsive substrate for neuronal cell bodies and growth cones, may form either pathways or boundaries to neurite outgrowth (Faissner and Kruse, 1990; Taylor et al., 1993). Adult CNS astrocytes also express lower levels of N CAM as compared to embryonic astrocytes (Silver and Rutishauser 1984).  109 Further evidence of the inhibitory nature of the mature CNS comes from studies of regenerating sensory neurons. Sensory neurons within the dorsal root ganglia have a peripheral axon projecting to sites in the skin, as well as a central-projecting axon which enters the dorsal horn of the spinal cord and contacts interneurons and motor neurons. Although lesions of both peripheral or central projections result in regeneration within the PNS, central-projecting neurites stop growing when they reach the dorsal root entry zone of the spinal cord (Ramon y Cajal 1958; Stenaas et al. 1987). Peripheral neurons transplanted into the CNS are also incapable of axonal extention within the environment of the CNS (Ramon y Cajal 1958). Thus, it would appear that the unfavorable extraneuronal environment of the CNS accounts in large part for the poor regenerative capacity of central neurons. Studies which identify the particular inhibitory factors and the relative contribution of these factors to the inhibitory nature of the CNS are important in efforts to evoke regeneration of CNS tissue following injury. The developmental onset of myelination coincides with a loss of regenerative capability of injured spinal cord (see chapter 2); this suggests that myelin may inhibit the regrowth of injured axons. Immunological myelin-suppression (see chapter 3) serves as a tool with which to investigate the effects on myelin on the ability of the developing spinal cord to regenerate following injury. This chapter illustrates that developmental myelin-suppression in vivo facilitates regeneration of transected spinal cord in late embryonic chicks. Chick spinal cord transections  110 during late embryonic development in normally-myelinated animals result in no repair whatsoever. These findings demonstrate that myelin is inhibitory to the regeneration of transected spinal cord in embryonic chick and constitute the first demonstration of functional recovery following complete spinal cord transection.  111 MATERIALS AND METHODS Developmental Myelln-Suppression See page 65. Spinal Cord Transection Transections consisted of a pinch lesion of the mid to high-thoracic spinal cord using sharpened Dumont #5-45 forceps (Fine Science Tools, North Vancouver, British Columbia #11253-25). A #00 pin marked to the appropriate depth of the cord for that stage of development, was then passed across the entire width of cord through the lesion to ensure that the spinal cord was completely severed (for details, see Keirstead et al. 1992). In addition, embryos were randomly selected from batches of transected animals and perfused intracardially with O.1M PBS containing 2500 USP units of heparin per 50 mIs PBS, pH 7.4 (at 37°C) followed by perfusion with 4% paraformaldehyde in O.1M phosphate buffer, pH 7.4 (at 4-10°C). The dissected tissue was then postfixed for 24 hours at 4°C and subsequently embedded in paraffin (see below). Parasagittal 10 um sections were cut and mounted on gelatin-coated slides, stained with Toluidine blue, and examined under a light microscope to ensure complete transection. Lastly, some hatchling chicks received lumbar injections of 1 .0 ul of tetramethylrhodamine-labelled dextran amine (RDA; see ‘neuroanatomy’ below) at the time of thoracic spinal cord transection. After hatching, they were perfused as outlined above and then postfixed for 24 hours at 4°C. The dissected brainstems were transferred to 30% sucrose in O.1M  112  PBS, pH 9.0 (4°C) for 24 hours. Each brainstem was sectioned in the transverse plane using a Leitz liquid CO 2 freezing microtome and examined for the presence of retrogradely-labelled brainstem-spinal neurons. The dissected spinal cords were subsequently embedded in paraffin (see below). Parasagittal 10 urn sections were cut and examined for evidence of the injection site and transection site. Paraffin Embedding See page 29 Immunohistochemistry  See page 30. Neuroanatomy Birds were anesthetized with an intramuscular injectipn of ketamine hydrochloride (30 mg/kg) plus xylazine (Rompun, 3 mg/kg). After removal of the dorsal vertebrae overlying the rostral lumbar cord, 0.2-1 .0 ul of 25% tetramethylrhodamine-labelled dextran amine (RDA; Molecular Probes Inc., 10000 MW, #D-1817) in 2.5% Triton X-100 diluted in 0.1M tris-buffer (pH 9.0) was directly injected into the spinal cord using a glass micropipette (tip diameter =  40-50 urn) attached to a Picospritzer II pump. Previous studies have indicated  that this amount of RDA, injected at this level of the cord, remains confined to the lumbar level (ie. does not diffuse rostrally to or above the site of transection), and within 24-48 hr is retrogradely transported via brainstem spinal axons to the cell bodies of origin, with no trans-synaptic transport to  113 brainstem neurons not having spinal projections (Hasan et al. 1991; Keirstead et al. 1992; Hasan et al. 1993). After 48 hours, the P4 birds were given a lethal intramuscular injection of anesthetic (sodium pentobarbital, 75 mg/kg) and then perfused prior to cardiac arrest and fixed as outlined above. The dissected brains were subsequently postfixed for another 24hr and then transferred to 30% sucrose in 0.1M PBS, pH 9.0 (4°C). Each brainstem was sectioned in the transverse plane using a Leitz liquid CO 2 freezing microtome. The number and position of retrograde labelled brainstem-spinal neurons, for each brainstem section, were then noted and photographed under a microscope equipped for epifluorescence.  114 RESULTS Complete transections of the El 5 thoracic spinal cord were confirmed by randomly selecting embryos from experimental batches after surgery and sectioning the tissue for light microscopic histological examination. Complete transections were confirmed in all cases (22 sacrificed immediately after surgery, 16 sacrificed on E16). Additionally, 3 myelin-suppressed embryos received lumbar injections of 1 .0 ul tetramethylrhodamine-labelled dextran amine (RDA) solution at the time of the thoracic spinal cord transection. Only a completely transected spinal cord would prevent the retrograde transport of this neuroanatomical tracer to the brainstem. Subsequent examination of the brainstem and cervical spinal cord on posthatching day (P) 5 showed a complete lack of RDA rostral to the transection site (Fig. 4-lA), although neuronal and axonal labelling was evident near the area of injection in the lumbar spinal cord (Fig. 4-1B). This confirms that the transection procedure reliably severs the entire thoracic spinal cord. Myelin-suppression was confirmed in those animals randomly selected from batches of myelin-suppressed, El 5 transected embryos (see above). lmmunohistochemical analysis revealed a complete lack of MBP immunoreactivity in all areas of the spinal cord excluding the most rostral segments (Fig. 4-2). These findings confirm that immunological myelin suppression reliably delays the developmental onset of myelination until late stages of embryonic development (i.e. E17; see chapter 3).  115 Figure 4-1. The transection procedure reliably severs the entire thoracic spinal cord. Control embryos received lumbar injections of tetrarnethylrhodamine labelled dextran amine (RDA) solution at the time of the thoracic spinal cord transection. Only a completely transected spinal cord would prevent the retrograde transport of this neuroanatomical tracer to the brainstem. A: Ventromedial reticular formation of the caudal pons in a posthatching day (P) 5 chick that received a thoracic transection and a lumbar injection of RDA on embryonic day (E) 15; note the absence of retrogradely-labelled gigantocellular reticulospinal neurons. B: Lumbar spinal cord of the same hatchling chick in parasaggital section; note the neuronal and axonal labelling evident near the previous injection site. This confirms that the transection procedure reliably severs the entire thoracic spinal cord. (Bars  =  5Oum for A, 100 urn for B).  117 Figure 4-2. Developmental myelin-suppression in the thoracic spinal cord of the embryonic chick in parasaggital section. Developmental myelin-suppression (and complete transections) were confirmed in animals randomly selected from experimental batches immediately after surgery. A: Unoperated (normally myelinated) control embryonic day (E) 15 spinal cord in parasaggital section showing extensive myelin basic protein (MBP) immunoreactivity within white matter. B: MBP immunofluorescence staining of an E15 spinal cord, randomly selected from an experimental batch of animals, each of which recieved a single injection of polyclonal galactocerebroside (GalC) antibodies plus complement proteins on El 1 and a complete thoracic spinal cord transection on El 5; note the absence of myelin. In all photographs the outer edge of the spinal cord lies on the right hand side, with the white matter adjacent. (Bars  =  lOOum).  119 Neuroanatomical or functional (see Discussion) assessments were conducted on: a) 21 myelin-suppressed, El 5 transected embryos; b) 8 normally myelinated (ie. uninjected) E15 transected control animals; c) 6 immunological control El 5 transected animals (see Materials and Methods) and d) 11 normally myelinated and untransected control animals. Neuroanatomical and functional assessments were often carried out on the same animal. Neuroanatomical regeneration following transection was assessed by counting the brainstem-spinal neurons labelled by the retrograde transport of RDA injected into the lumbar spinal cord after hatching (Fig. 4-3). There was a similar number and distribution of retrogradely labelled reticulospinal neurons in the 11 normally-myelinated, untransected control animals (Fig. 4-3A) and the 21 myelin-suppressed, E15-transected experimental animals (Fig. 4-3B) following a post-hatching injection into the lumbar spinal cord. In the ventromedial reticular formation of the pons, the myelin-suppressed El 5-transected animals averaged 1003 retrogradely labelled reticulospinal neurons per animal (range: 920-1292 cells). Normally myelinated, untransected control animals averaged 1043 retrogradely labelled reticulospinal neurons per animal (range: 692-1311 cells). Comparable numbers and distributions of retrogradely labelled neurons were also noted for other brainstem-spinal nuclei with projections to the lumbar spinal cord including the vestibular nucleus (Fig. 4-3C and D), locus ceruleus, subceruleus nucleus and raphe nucleus. In contrast, 8 normally myelinated and 6 immunological control embryos (see above) transected on E15 showed no  120 Figure 4-3. Neuroanatomical regeneration of brainstem-spinal projections. Photomicrograghs of retrogradely labelled neurons within the brainstem in posthatching day (P) 4 chicks. Brainstem-spinal neurons were labelled by the retrograde axonal transport of tetramethyirhodamine-labelled dextran amine (RDA) injected into the lumbar spinal cord on P2 and allowed two days for transport. A: RDA labelled gigantocellular reticulospinal neurons within the ventromedial reticular formation of the caudal pons in an unoperated (normally myelinated) control P4 chick. B: RDA labelled gigantocellular reticulospinal neurons within the ventromedial reticular formation of the caudal pons in a P4 experimental hatchling that was subjected to developmental myelin-suppression on El 1, followed by a complete thoracic spinal cord transection on El 5; note that the number and distribution of retrogradely labelled neurons is comparable to A. C: RDA labelled vestibulospinal neurons within the lateral vestibular nucleus of the dorsolateral pons in an unoperated (normally-myelinated) control P4 chick. D: RDA labelled vestibulospinal neurons within the lateral vestibular nucleus of the dorsolateral pons in a P4 experimental hatchling that was subjected to developmental myelin-suppression on El 1, followed by a complete thoracic spinal cord transection on E15; note that the number and distribution of retrogradely labelled neurons is comparable to C. Comparable neuroanatomical repair was evident for other brainstem-spinal projections. E: Ventromedial reticular formation of the caudal pons in a P4 hatchling that was (normally myelinated and) transected on E15; note the absence of retrogradely labelled  121 neurons. F: Lateral vestibular nucleus of the dorsolateral pons in a P4 hatchling that was (normally-myelinated and) transected on El 5; note the absence of retrogradely labelled neurons. A similar lack of neuroanatomical repair was observed in animals that received immunological control solutions on El 1 followed by a complete thoracic spinal cord transection on E15. (Bars  =  5Oum).  125 retrogradely labelled brainstem-spinal neurons within the ventromedial reticular formation of the pons (Fig. 4-3E), the vestibular nucleus (Fig. 4-3F) or any other brainstem nucleus with projections to the lumbar spinal cord. Axonal repair/regeneration of descending brainstem-spinal projections was also observed in 3 myelin-suppressed animals injected with cascade bluelabeled dextran amine (CBDA) into the lumbar spinal cord at the time of the El 5 thoracic transection, and a second retrograde fluorescent tracer (RDA) into the lumbar spinal cord on P4 (Fig. 4-4). The animals were sacrificed 48 hours later and their brains and spinal cords processed for light microscopic analysis. Although CBDA was present in the lumbar spinal cord (Fig. 4-4A), there was no evidence of CBDA-labelled neurons within the brainstem, confirming that the E15 transection was complete. RDA was present in the lumbar spinal cord (Fig. 4-4B) as well as the ventromedial reticular formation of the pons (Fig. 4-4C), vestibular nucleus, locus ceruleus, subceruleus nucleus and raphe nucleus. Behavioral observations of hatchling chicks previously transected on El 5 during myelin-suppression indicated complete recovery of locomotor capabilies (results not shown). Myelin-suppressed, El 5-transected hatchling chicks were capable of initiating locomotion in response to a positive stimulus (eg. food, water or the presence of other chicks). In addition, the speed and frequency of movement as well as the righting capability of all hatchling chicks previously myelin-suppressed and El 5 transected were indistinguishable from unoperated control hatchlings of a similar age. Hatchling chicks that had received an E15  126 Figure 4-4. Neuroanatomical regeneration of brainstem-spinal projections in myelin-suppressed animals injected with cascade blue labelled dextran amines (CBDA) at the time of the El 5 thoracic transection. To further ensure complete transections in animals displaying neuroanatomical regeneration, several embryos received lumbar injections of CBDA at the time of the thoracic spinal cord transection. Only a completely transected spinal cord would prevent the retrograde transport of this neuroanatomical tracer to the brainstem. Neuroanatomical regeneration was evidenced by the retrograde axonal transport of tetramethylrhodamine-labelled dextran amine (RDA) injected into the lumbar spinal cord on P4 and allowed two days for transport. A: Lumbar spinal cord of a hatchling chick that was subjected to developmental myelin-suppression on El 1, followed by a complete thoracic spinal cord transection and lumbar injection of CBDA on E15; note the CBDA neuronal and axonal labelling evident at the previous (El 5) injection site. No CBDA was present within the brainstem. This confirms that the transection reliably severed the entire thoracic spinal cord. B: Lumbar spinal cord from the same animal; note the RDA neuronal and axonal labelling evident at the previous (P4) injection site. C: RDA labelled gigantocellular reticulospinal neurons within the ventromedial reticular formation of the caudal pons in the same animal; note that the number and distribution of retrogradely labelled neurons is comparable to unoperated control labelling (see Fig. 4-3; Bars  =  100 urn for A and 8; 5Oum for C)  128 transection plus no injection or an immunological control injection on E9-E12 exhibited no locomotor capabilities whatsoever.  129  DISCUSSION The experiments reported here examined the influence of myelin on the ability of chick spinal cord axons to regenerate following complete embryonic spinal cord transection. Embryonic chicks lose the ability to repair their injured spinal cord on or about embryonic day (E) 13 of the 21 day developmental period in ovo. Spinal cord transections prior to E13 result in partial or complete repair (Shimizu et al. 1990; Hasan et al. 1991). Transections after E13 result in little or no repair, rendering the animal incapable of voluntary locomotion after hatching (Sholomenko and Steeves 1987; Eidelberg 1981; Shimizu et al. 1990; Hasan et al. 1991; Keirstead et al. 1992). This loss of regenerative capability is coincident with the developmental onset of myelination within the spinal cord (Bensted et al. 1957; Hartman et al. 1979; MackIm  and Weill 1985; Keirstead et  al. 1992). Chapter 4 of this thesis outlined an immunological method of delaying the developmental onset of myelination until E17. The studies reported here demonstrate that immunological myelin-suppression extends the ‘permissive’ period for spinal cord repair until later stages of development. Transections as late as E15 in myelin-suppressed animals resulted in complete anatomical regeneration. Furthermore, El 5 transections in myelin-suppressed animals resulted in complete locomotor recovery after hatching; behavioral observations indicated that voluntary locomotion was completely indistinguishable from that of unoperated control hatchlings of a similar age. These findings illustrate that myelin is inhibitory to the functional regeneration  130 of transected spinal cord in embryonic chick (Keirstead et al. 1992). Physiological assessments of post-hatching chicks previously myelin suppressed and transected on E15 also indicate complete recovery of locomotor capabilities (Fig. 4-5; Keirstead et al. 1992). These studies were conducted in collaboration with Dr. Gillian D. Muir (a Postdoctoral Fellow working under the supervision of Dr. John D. Steeves, University of British Columbia, Departments of Zoology and Anatomy, Vancouver, B.C., Canada). EMG recordings from leg muscles during post-hatching walking were obtained from normally myelinated, untransected control chicks and myelin-suppressed, El 5 transected chicks. The pattern of leg muscle activity obtained from myelin-suppressed, El 5-transected chicks did not differ from those obtained from normally-myelinated, untransected control chicks (Fig. 4-5A and B). As expected during walking, the same muscle (eg. lateral gastrocnemius muscle, an ankle extensor muscle) in the right and left leg showed alternating periods of activity. In addition, an antagonist muscle of the right lateral gastrocnemius, the sartorius (a knee extensor/hip flexor muscle) also exhibited activity that alternated with the right lateral gastrocnemius. The right iliofibularis (knee flexor/hip extensor) burst concurrently with the right lateral gastrocnemius and alternated with the right sartorius. None of the normally-myelinated, El 5-transected chicks were capable of locomotion or even unsupported standing. The relationships between muscle activity (burst duration) and step cycle duration for normally-myelinated, untransected control and myelin-suppressed,  131 Figure 4-5. Physiological recovery of brainstem-spinal projections. Simultaneous electromyographic (EMG) recordings from four leg muscles during overground walking by an unoperated (normally-myelinated) control chick (A) and a myelin suppressed E15-transected chick (B). The myelin-suppressed E15-transected chick shows the same muscle activity patterns as the control chick. C: Regression of muscle activity (burst) duration versus step cycle duration for lateral gastrocnemius muscle (open squares) and sartorius muscle (filled squares) during overground walking by a normally-myelinated, untransected hatchling chick. The burst duration of the lateral gastrocnemius muscle increases with increasing cycle duration, while the burst duration of the sartorius muscle remains constant as cycle duration increases. D: Regression of burst duration versus step cycle duration for lateral gastrocnemius muscle (open squares) and sartorius muscle (filled squares) during overground walking by a myelin-suppressed El 5-transected hatchling chick. This animal displays the same relationships as the control animal in C. The slopes of corresponding regression lines in C and D are not significantly different. All regressions are 2 for lateral gastrocnemius and sartorius are 0.58 and significant to p<O.05. R 0.04 in (C) and 0.59 and 0.08 in (D).  A  R. Lat.  4jøb 4*4$4 R. Sari. i  fr4  R. Iliofib.  L. Lat Gastroc.  R. Lat.Gastroc. -  R. Sart. *1Øl  WA.-  -*JL4.  R. Iliofib. t14  L. Lat. Gastroc.  (D 0  0 Ii  Ut  ‘1 p)  (D  0•1  o  w  0  I.  U  i  Iii  0  I  I  0  0  0  ii III  1111111  II  I  0  0  Ut I-,.  Ii ‘1  (D  0 0  0  0  0  0 0  0  0  0  burst duration (sec.)  0  Lii  w  0  0  I  II  U I  0  II  I  M  0  ii  00  0  ii  U  0  i  I  0  I  0  i  0  11111  burst duration (sec.)  II  0  C,  X’  134 E15-transected chicks were also similar (Fig. 4-5C and D). The lateral gastrocnemius muscle is active during the weightbearing phase (stance phase) of the step cycle. As cycle duration increases (ie. the animal’s velocity decreases), the duration of the stance phase increases, as does the burst duration of the lateral gastrocnemius muscle (Hollyday and Hamburger 1977). Conversely, the sartorius muscle is active during the non-weightbearing phase (swing phase) of the step cycle. As cycle duration increases, the duration of the swing phase remains relatively constant, as does the burst duration of the sartorius muscle (Hollyday and Hamburger 1977). This supports the observation that the suppression of the onset of myelination extends the permissive period for functional spinal cord repair in the embryonic chick (Keirstead et al. 1992). Normally-myelinated control animals or immunological control animals, transected on E15, showed no functional recovery. It is arguable that the locomotor recovery observed in the myelin suppressed, E15 transected chicks is not dependent on the functional repair/regeneration of brainstem-spinal projections, but due to intrinsic activity of neural networks confined to the lumbar spinal cord (Sholomenko and Steeves 1987; Eidelberg 1981; Steeves et al. 1987). If this were the case, however, it is unlikely that the locomotor abilities would have been so equivalent (Sholomenko and Steeves 1987). In addition, myelin-suppressed, E15-transected hatchling chicks were capable of initiating locomotion in response to a positive stimulus (eg. food, water or the presence of other chicks). Further evidence comes from  135 previous demonstrations in late embryos or hatchling chicks that direct focal stimulation of brainstem-spinal neurons within the gigantocellular reticular formation (an identified brainstem locomotor region), only elicits locomotor activity in an animal transected prior to E13 (Hasan et al. 1991; Hasan et al. 1992; Valenzuela et al. 1990). All the available evidence suggests the high quality of functional locomotor recovery in myelin-suppressed, El 5-transected animals is due to the functional repair/regeneration of brainstem-spinal connections. Normally-myelinated or immunological control injected embryos that received thoracic transections on El 5 exhibited no anatomical or functional spinal cord repair after hatching. Retrograde tract tracing studies revealed no anatomical regrowth of severed axons across the transection site (Fig. 4-3E and F), and behavioral and physiological observations indicated no voluntary locomotor capabilities after hatching (Fig. 4-5A and C). The lack of spinal cord repair in immunological control injected, E15transected animals demonstrates that the spinal cord injection itself on E9-E1 2 did not ‘prime’ the spinal cord to regenerate. Pre-conditioning injuries have been shown to facilitate axonal regeneration in several systems (Richardson and Issa 1984; Richardson and Verge 1986; Richardson and Verge 1987). E15transected animals that received E9-E1 2 spinal cord injections of either non specific antibody plus complement, GaIC antibody only, complement proteins only, vehicle only (0.1M PBS, pH 7.4) or the mouse GaIC antibody plus heat-  136 inactivated serum showed no anatomical or functional recovery (Fig. 4-3; Fig. 45). This demonstrates that both the myelin-specific antibody plus complement are required to facilitate functional repair of injured spinal cord. It is most probable that the functional repair of myelin-suppressed, E15transected spinal cord was due to true axonal regeneration. Neurogenesis in the developing chick embryo is complete by E5 (McConnell and Sechrist 1980; Sechrist and Bronner-Fraser 1991). Therefore it is unlikely that recovery was due to projections from newly differentiated neurons. Chick brainstem-spinal projections complete their axonal projections to lumbar levels of the spinal cord by E10-E12 (Okado and Oppenheim 1985). By E12, brainstem-spinal projections throughout the spinal cord are equivalent in number and distribution to those in a hatchling chick (Okado and Oppenheim, 1985). Therefore, it is unlikely that subsequent development could contribute to recovery from spinal cord transections during the final week of embryonic development. Direct proof that the functional repair of myelin-suppressed, El 5-transected spinal cord was due to true axonal regeneration could be obtained from double-labelling studies. In a double-labelling paradigm, the neuronal population with projections to the lumbar spinal cord would be maximally labelled on E12 with a retrograde tract tracer conjugated to a specific fluorochrome. After myelin-suppression, El 5 thoracic transection, and a period for repair, a second retrograde tract tracer conjugated to a different fluorochrome would then be injected into the lumbar spinal cord. Only truely regenerated axons would be labelled with both  137 fluorochromes. Although theoretically sound, this approach could not be applied in practice by this investigator. The additional operations required by a doublelabelling study decreased the viability of experimental animals so as to render the study impractical and unethical. During early embryonic development, CNS neural projections have a high capacity for plastic changes in response to injury (So et al. 1981; Shimizu et al. 1990; Treherne et al. 1992). It is possible that developmental myelin suppression may have enhanced and prolonged the capacity of developing axonal projections for plastic changes. Plasticity of neural projections is often associated with an increased expression of growth-associated protein GAP-43; GAP-43 expression in growing neurites typically decreases after contact with the target cell (Meiri et al. 1986; Skene 1989; Baizer and Fishman 1987). If myelination in the rat lumbar spinal cord is prevented by neonatal X-irradiation, the expression of GAP-43 within the spinal cord as assessed by immunohistochemistry and immunoblotting is extended to later stages of development (Kapfhammer and Schwab 1994). These studies suggest that myelin may directly or indirectly suppress the capacity of the developing CNS for plastic changes such as sprouting and fibre growth. Developmental myelin suppression of the chick embryo may have similarly prolonged the capacity of developing axonal projections for plastic changes. This possibility could be investigated by an immunohistochemical analysis of GAP-43 expression within the normal and myelin-suppressed chick spinal cord.  138 Myelin has been shown to inhibit the cellular attachment of, and neurite outgrowth from neuroblastoma cells in vitro (Savio and Schwab 1989; Savio and Schwab 1990). Myelin-associated proteins that inhibit the anatomical growth of axons in vitro (Caroni and Schwab 1988) as well as the regrowth of axotomixed corticospinal fibers in vivo (Schnell and Schwab 1990) have been identified in rat spinal cord. Myelin also expresses the growth inhibiting molecule janusin (Wintergerst et al. 1993; Bartsch et al. 1993). Janusin has been shown to be a repulsive substrate for neuronal cell bodies as well as growth cones (Pesheva et al. 1989; Taylor et al. 1993). Thus, it would appear that myelin may inhibit axonal regeneration by suppressing the capacity of the developing CNS for plastic changes, as well as providing an inhibitory substrate for growing neurites. In conclusion, these findings are the first demonstration that suppression of myelination results in both neuroanatomical repair and functional CNS recovery after an embryonic spinal cord injury. The present data clearly confirm and extend the proposition that the presence of CNS myelin contributes to the inhibition of neuronal repair after an adult CNS injury (Ramon y Cajal 1959; Caroni and Schwab 1988; Schnell and Schwab 1990). This suggestion is also indirectly supported by the demonstration that lamprey, which do not have myelinated CNS axonal fiber tracts, are capable of functional regeneration after either a larval or adult spinal cord injury (McClellan 1990).  139  CHAPTER 5  NEUROANATOMICAL REPAIR AND PHYSIOLOGICAL RECOVERY FOLLOWING TRANSECTION AND IMMUNOLOGICAL DEMYELINATION OF THE HATCHLING CHICK SPINAL CORD  140 INTRODUCTION  Myelin within the embryonic chick spinal cord provides an inhibitory environment for the functional regeneration of axotomized brainstem-spinal projections concerned with locomotion (Keirstead et al. 1992). Chapter 3 of this thesis outlines an immunological method of delaying the developmental onset of myelination in vivo (developmental myelin-suppression) until later stages of embryonic development. Spinal cord transections on embryonic day (E) 15 in a myelin-suppressed animal result in complete anatomical regeneration and functional repair (see chapter 4). Spinal cord transections on E15 in a normally myelinated animal result in no repair whatsoever, rendering the animal completely incapable of voluntary locomotion after hatching. These studies indicate that myelin is inhibitory to the functional regeneration of transected spinal cord in embryonic chick (Keirstead et al. 1992) and suggest that demyelination of hatchling spinal cord may facilitate repair following hatchling spinal cord transections. This chapter discusses the development and characterization of a protocol effective in demyelinating the hatchling chick spinal cord in vivo (immunological demyelination). Additional experiments reported here demonstrate that complete transection and concurrent immunological demyelination of the hatchting chick spinal cord result in partial neuroanatomical regeneration of brainstem-spinal projections. Collaborative experiments reported in the chapter discussion indicate that this partial  141 neuroanatomical regeneration of demyelinated and transected hatchling spinal cord is followed by functional synapse formation such that stimulation of brainstem locomotor regions elicits electrical activity in the muscles of the leg. Methods of in vivo demyelination of mature central nervous system (CNS) tissue include X-ray irradiation (Blakemore 1977), drug administration (Blakemore 1978: Ludwin 1978: Eames et al. 1977), viral infection (DalCanto and Lipton 1980; Herndon et al. 1977), nerve compression (Bunge et al. 1961; Clifford-Jones et al. 1980) and cell-mediated immunological reactions (Lampert 1968; Raine and Bornstein 1970). A brief review of these alternative demyelination methods may serve to illustrate the uniqueness of, and advantages offered by, the immunological demyelination technique used in the experiments reported here. X-ray irradiation of living tissue results in mitotic interference and ultimately the death of actively dividing cells. Because neogenesis of spinal cord neurons is complete by birth in the rat and mouse, X-ray irradiation during neonatal or adult life targets the glial population of the spinal cord. Irradiation of the rat spinal cord has been reported to result in an inhibition of neonatal myelin formation, as well as demyelination of adult myelinated spinal cord via destruction of oligodendrocytes (Blakemore 1977). X-ray irradiation also results in failure of the astrocyte proliferation necessary to maintain the integrity of the glial limitans (Sims et al. 1985). The absence of oligodendrocytes, combined with defects in the glial limitans, leads to Schwann cell myelination of CNS  142 axons (Sims et al. 1985). Primary demyelination of adult CNS can also be obtained via administration of drugs including cuprizone (Ludwin 1978), ethidium bromide (Blakemore 1992), lysolecithin (Blakemore 1978), 6-aminonicotinamide (Blakemore 1975) and diptheria toxin (Eames et al. 1977). Demyelination in this manner is relatively long-term (greater than 6 months for lysolecithin in rabbits) and may or may not be followed by oligodendrocyte remyelination (Blakemore 1978). Remyelination is often accomplished by invading Schwann cells following drug-induced demyelination (Blakemore 1982). Although effective in evoking oligodendrocyte cell death and primary demyelination, drug administration is often also accompanied by a loss of astrocytes (Blakemore 1992; Blakemore 1978). Cuprizone-induced demyelination, on the other hand, is accompanied by proliferation of astrocytes characterized by swollen processed and a ‘watery’ cytoplasm (Ludwin 1978). In the case of diptheria toxin, demyelination is accompanied by extensive scarring and Wallerian-type degeneration of nerve fibres surrounding the injection site (Eames et al. 1977). Models of virally-induced demyelination include Theiler’s murine encephalomyelitis virus (TMEV) infection (Dal Canto and Lipton 1980) and JHM mouse hepatitis virus infection (Herndon et al. 1977). Both of these models result in cytolytic infection of oligodendroglia and subsequent myelin breakdown accompanied by prominent gliosis and extensive inflammatory cell infiltrates (Dal Canto and Lipton 1980; Herndon et al. 1977). Demyelination induced by  143 TMEV infection lasts for as long as 8 months and remyelination is predominantly due to Schwann cells (Dal Canto and Lipton 1980). Demyelination induced by JHM virus is relatively short-lived; remyelination begins within two weeks postinfection and is accomplished primarily by generation of new oligodendrocytes (Herndon et al. 1977). Both TMEV and JHM viral infection can kill the host (approximately 50% mortality with JHM) and suffer from a lack of predictability as to the site of the lesion (Dal Canto and Lipton 1980; Herndon et al. 1977). Other methods of inducing primary demyelination in vivo include nerve compression (Bunge et al. 1961; Clifford-Jones et al. 1980) and experimental allergic encephalomyelitis (Raine and Bornstein 1970; Lampert 1968). Although effective in producing myelin loss, these demyelinating methods are complicated by extensive proliferation of astrocytes and inflammatory cells as well as axonal degeneration (Clifford-Jones et al. 1980; Raine and Bornstein 1970). In order to study the specific effects of myelin on the ability of the hatchling chick spinal cord to regenerate following injury, it was necessary to employ a method of demyelination that does not produce non-specific pathological changes such as a chronic breach of the glial limitans, astrogliosis, intense inflammation or axonal degeneration. Unfortunately, each of the demyelination methods outlined above suffer significantly from one or more of these non-specific complications. This prompted the development of the immunological method of myelin suppression outlined in this chapter and chapter 3. The experiments presented here illustrate that the injection of  144 galactocerebroside (GaIC) antibodies or 04 antibodies plus complement proteins into the hatchling spinal cord results in transient demyelination. Furthermore, the time course of immunological demyelination can be precisely controlled. Immunological methods similar to the one described here have been used to demyelinate guinea pig optic nerve (Sergott et al. 1982; Ozawa et al. 1989), cat optic nerve (Carroll et at. 1984; Carroll et at. 1985) and rat spinal cord (Mastaglia et al. 1989) in vivo. Data presented in this chapter and chapter 3 illustrate that immunological demyelination is not accompanied by alterations of the axonal or astroglial population of the spinal cord. Details regarding the interaction of complement plus GaIC or 04 antibody with the oligodendrocyte and myelin membranes are discussed in chapter 3 of this thesis.  -  Immunological demyelination was employed in the present experiments in order to test the hypothesis that the removal of spinal cord myelin would facilitate neuroanatomical and. functional regeneration by providing a more permissive extraneuronal environment for regenerating brainstem-spinal projections within the transected hatchling chick spinal cord. CNS myelin and cultured oligodendrocytes are strong inhibitors of neurite outgrowth in vitro (Schwab and Caroni 1988). Myelin and myelin-associated proteins have also been shown to inhibit the functional regeneration of transected spinal cord in the embryonic chick (Keirstead et al. 1992) as well as the neuroanatomical regeneration of corticospinal fibres in the rat (Schnell and Schwab 1990). The hypothesis outlined above assumes that hatchling chick spinal cord  145 neurons have the intrinsic ability to regenerate, given a permissive extraneuronal environment. Although vertebrate CNS axons will not regrow in the environment of the adult spinal cord (Eidelberg 1981; Sholomenko and Steeves 1987), peripheral nerve grafts into the CNS provide a favorable environment through which CNS axons will regenerate (Ramon y Cajal 1959; David and Aguayo 1981; Aguayo et al. 1991). CNS neurons have also been shown to extend fibres in vivo through lesion sites containing implants of fetal CNS tissue (Bjorklund and Stenevi 1979; Kromer et al. 1981; Bjorklund 1991), as well as implants consisting of fibroblasts genetically modified to express growth factors (Fisher and Gage 1993; Tuszynski et al. 1994). In addition, motor neurons residing within the ventral horn of the spinal cord are capable of regenerating their peripheral projecting axon if axotomized within the PNS (Ramon y Cajal 1959). These findings indicate that adult CNS neurons retain intrinsic axonal growth programs which enable long-distance axonal regeneration in the presence of a favorable extraneuronal environment. The experiments presented here illustrate that complete transection of the hatchling chick spinal cord in vivo followed immediately by 2-3 weeks of immunological demyelination results in partial regeneration of brainstem-spinal projections. Retrograde tract tracing studies indicate that 5-15% of brainstem spinal projecting neurons (as compared to the maximal number of retrogradely labelled brainstem-spinal projecting neurons obtained from untransected control hatchlings) regenerate processes through the previous thoracic transection site  146 to lumbar levels of the spinal cord. Evidence presented in the chapter discussion illustrates that these regenerated fibres make functional synaptic connections with motorneurons of the lumbar spinal cord.  147 MATERIALS AND METHODS Immunological Demyellnation Birds between the ages of post-hatching day (P) 1 and P9 were anesthetized with an intramuscular injection of ketamine hydrochloride (30 mg/kg) plus xylazine (Rompun, 3 mg/kg). After removal of the dorsal vertebrae overlying the low thoracic spinal cord, direct spinal cord injections were performed using a glass micropipette (tip diameter  =  30-40 urn; A-M Systems,  Everett, Washington #6045) connected to a Picospritzer II pressure injection system (General Valve Corp., Fairfield, New Jersey). Each animal received a total volume of 10 ul, over 1-4 penetrations. Alternatively, solution was delivered over a longer time period by inserting into the exposed low thoracic spinal cord a canula connected to a 7 day (model #1007D) or 14 day (model #2ML2) osmotic mini-pump (Alzet Corp., 950 Page Mill Road, P.O. Box 10950, Palo Alto, CA 94303-0802) which was placed under the skin on the dorsal surface of the neck. The pumps used in these experiments deliver solution at a rate of 0.5 ul per hour, or 12 ul per day. Immunological demyelination was 1 rabbit GaIC antibody (Chemicon International Inc., evoked with either an lgG Temecula, California #AB142) at a dilution of 1:5 with 33% guinea pig complement (Gibco BRL, Burlington, Ontario #19195-01 5) in O.1M phosphate buffered saline (PBS), pH 7.4 or an 1gM polyclonal 04 antibody (a gift from Melitta Schachner, Wayne State University School of Medicine, U.S.A.) at a dilution of 1:5 with 33% guinea pig complement (Gibco BRL, Burlington, Ontario  148 #19195-01 5) in 0.1M PBS pH 7.4. Immunological control hatchlings were administered either: 1) GalC antibody only, 2) 04 antibody only, 3) guinea pig complement only or, 4) vehicle only (0.1M PBS, pH 7.4). These immunological control solutions were delivered by direct spinal cord injection as well as by pump. Animals not undergoing further experimentation were perfused intracardially with 0.1M PBS containing 2500 USP units of heparin per 50 mIs PBS, pH 7.4 (at 37°C) followed by perfusion with 4% paraformaldehyde in 0.1M phosphate buffer, pH 7.4 (at 4-10°C). The dissected tissue was then postfixed for 24 hours at 4°C and subsequently embedded in paraffin for immunohistochemical analysis (see below). Spinal Cord Transection Birds between the ages of post-hatching day (P) 4 and P9 were anesthetized with an intramuscular injection of ketamine hydrochloride (30 mg/kg) plus xylazine (Rompun, 3 mg/kg). After removal of the dorsal vertebrae overlying the low thoracic spinal cord, the spinal cord was transected using a sharpened corneal blade (Fine Science Tools, North Vancouver, British Columbia) with a diameter roughly equivalent to the inside diameter of the hatchling chick spinal cord. The corneal blade was inserted through the spinal cord to the inner ventral surface of the spinal column and gently rotated. To ensure complete transection of the spinal cord, hatchling chicks were randomly selected immediately after transection and their spinal cords fresh-  149 dissected and examined under a dissecting microscope. In addition, some hatchling chicks received lumbar injections of 0.5 ul of tetramethyirhodamine labelled dextran amine (RDA; see ‘neuroanatomy’ below) at the time of thoracic spinal cord transection. 21 days after RDA injection, they were perfused intracardially with 0.1M PBS containing 2500 USP units of heparin per 50 mIs PBS, pH 7.4 (at 37°C) followed by perfusion with 4% paraformaldehyde in 0.1M phosphate buffer, pH 7.4 (at 4-10°C). Others received lumbar injections of 0.5 ul of RDA 17 days after the thoracic spinal cord transection. 48 hours after RDA injection, they were perfused as outlined above. The dissected tissue was then postfixed for 24 hours at 4°C. The dissected brainstems were transferred to 30% sucrose in 0.1M PBS, pH 9.0 (4°C) for 24 hours. Each brainstem was 2 freezing microtome sectioned in the transverse plane using a Leitz liquid CO and examined for the presence of retrogradely-labelled brainstem-spinal neurons. The dissected spinal cords were subsequently embedded in paraffin (see below). Parasagittal 10 urn sections were cut and examined for evidence of the injection site and transection site. Paraffin Embedding See page 29. Immunohistochemistry See page 30. Neuroanatomy See page 112.  150 RESULTS Immunological Demyellnation The removal of spinal cord myelin from the hatchling chick was achieved by administering complement proteins plus an oligodendrocyte-specific, complement-binding antibody directly into the low thoracic spinal cord between posthatching day (P) 1 and P9 (immunological demyelination). The state of spinal cord myelination within experimental and control animals was assessed with myelin basic protein (MBP) immunohistochemistry. Polyclonal galactocerebroside (GaIC) antibody and polyclonal 04 antibody are oligodendrocyte-specific, complement-binding antibodies that, when combined with guinea pig complement, caused demyelinating lesions within the spinal cord that extended for approximately six (6) spinal cord segments either side of the injection site in the majority of cases (n = 69, 60 with single injections and 9 with osmotic mini-pumps). Of the 69 demyelinated lesions examined, 14 were restricted to one side of the spinal cord only, 28 were limited to 2-4 spinal cord segments either side of the injection site, and 2 were accompanied by an additional demyelinated lesion in the cervical region of the spinal cord. The extent and degree of spinal cord demyelination was similar when the complement proteins and antibodies were administered on either P1, P2, P3, P4, P5, P6, P7, P8 or P9 (Fig. 5-1). Approximately one half of the demyelinated spinal cords contained ‘clumps’ of MBP immunoreactivity homogeneously distributed throughout the myelin lesion (Fig. 5-iC).  151 Figure 5-1. Immunological demyelination in the thoracic spinal cord of the hatch ling chick in parasaggital section. A: Unoperated (normaUy-myelinated) control posthatching day (P) 2 chick showing extensive myelin basic protein (MBP) immunoreactivity within the spinal cord white matter. B: MBP immunofluorescence staining of a P7 spinal cord from an animal that received a single injection of polyclonal galactocerebroside (GaIC) antibodies plus complement on P6; note the absence of myelin. C: MBP immunofluorescence staining of a P7 spinal cord from an animal that received a single injection of polyclonal galactocerebroside (GaIC) antibodies plus complement on P6; note the presence of myelin ‘clumps’. In all photograghs the outer edge of the spinal cord lies on the right hand side, with the white matter adjacent. (Bars 1 OOum).  =  “1  153 Immunological control hatchlings were administered either: 1) GaIC antibody only (n  =  12), 2) 04 antibody only (n  =  12), 3) guinea pig complement  only (n=12) or, 4) vehicle only (0.1M PBS, pH 7.4; n=12) on P6. From each group of immunological control hatchlings, four animals were sacrificed on each day, 1, 5 and 1 2 days after treatment. MBP immunohistochemical analysis of their spinal cords revealed no evidence of demyelination (Fig. 5-2). Single micropipette injections of complement proteins plus oligodendrocyte-specific antibody into the spinal cord were performed on a total of 102 hatchlings (91 animals with GaIC antibodies and 11 animals with 04 antibodies). See table 1. There was no evidence of immunological demyelination 6 or 9 hours after injection (Fig. 5-3A), however, decreased MBP  -  immunoreactivity was evident in 3 of 5 animals sacrificed 12 hours after injection (Fig. 5-3B). Immunological demyelination was evident 24 and 48 hours after injection (Fig. 5-3C) and in the majority of animals sacrificed 3 days after injection. No signs of immunological demyelination was detected in animals sacrificed 4 and 5 days after injection (Fig. 5-3D). Long-term infusion (with 7- and 14-day Aizet osmotic mini-pumps) of complement plus GaIC antibody into the spinal cord was performed on a total of 23 hatchlings. Immunological demyelination was clearly evident in 5 animals sacrificed 5 days after installation of a 7-day pump. Immunological demyelination was also evident in 4 animals sacrificed 12 days after installation of a 14-day pump (Fig. 5-4A). No evidence of immunological demyelination was  154 Figure 5-2. Pattern of myelin basic protein (MBP) immunoreactivity in immunological control thoracic spinal cords in parasaggital section. A: MBP immunofluorescence staining of a posthatching day (P) 7 spinal cord from an animal that received a single injection of polyclonal galactocerebroside (GaIC) antibodies only on P6; note that myelin is unperturbed. B: MBP immunofluorescence staining of a posthatching day (P) 7 spinal cord from an animal that received a single injection of complement only on P6; note that myelin is unperturbed. In all photograghs the outer edge of the spinal cord lies on the right hand side, with the white matter adjacent. (Bars  =  lOOum).  Table 5-1: Summary of results from single injection of complement proteins plus GaIC or 04 antibodies directly into the hatchling chick spinal cord  Sacrifice Time Post-  Total  Injection  Number  Number Normally-  Demyelinated  Myelinated  6hours  4  0  4  9hours  4  0  4  l2hours  5  3  2  1 day  36  30 (8 with 04 Ab)  6  2days  11  8  3  3 days  31  19 (3 with 04 Ab)  12  4days  7  0  7  5days  4  0  4  157 Figure 5-3. Time course of immunological demyelination and remyelination following a single injection of polyclonal galactocerebroside (GaIC) antibodies plus complement. A: Myelin basic protein (MBP) immunofluorescence staining of a hatchling chick spinal cord in parasaggital section 9 hours after the injection of GaIC antibodies plus complement; note the presence of myelin. B: MBP immunofluorescence staining of a hatchling chick spinal cord in parasaggital section 12 hours after the injection of GaIC antibodies plus complement; note that myelin is decreased. C: MBP immunofluorescence staining of a hatchling chick spinal cord in parasaggital section 24 hours after the injection of GaIC antibodies plus complement; note the absence of myelin. D: MBP immunofluorescence staining of a hatchling chick spinal cord in parasaggital section 5 days after the injection of GaIC antibodies plus complement; note the presence of myelin. In all photograghs the outer edge of the spinal cord lies on the right hand side, with the white matter adjacent. (Bars  =  lOOum).  160 Figure 5-4. Immunological demyelination and remyelination with a 14 day osmotic mini-pump containing polyclonat galactocerebroside (GaIC) antibodies plus complement. A: Myelin basic protein (MBP) immunofluorescence staining of a hatchling chick thoracic spinal cord in parasaggital section 12 days after the installation of an osmotic mini-pump containing GaIC antibodies plus complement; note the absence of myelin. B: MBP immunofluorescence staining of a hatchling chick thoracic spinal cord in parasaggital section 21 days after the installation of an osmotic mini-pump containing GaIC antibodies plus complement; note the presence of myelin. In all photograghs the outer edge of the spinal cord lies on the right hand side, with the white matter adjacent. (Bars =  lOOum).  162 detected in 6 animals sacrificed 21 days (Fig. 5-4B), and 8 animals sacrificed 24 days, after installation of a 14-day pump. The astrocyte population of the spinal cord did not appear to be disturbed by immunological demyelination, as assessed by glial fibrillary acidic protein (GFAP) immunohistochemistry (Fig. 5-5). The astrocyte number and distribution in immunologically demyelinated spinal cords (Fig. 5-5B) was similar to that of unoperated control tissue of the same age (n=8; Fig. 5-5A). Immunohistochemical analysis of spinal cords from animals sacrificed 24 hours after the injection of complement proteins and GaIC antibodies on P2 (n=3) and P5 (n=3), revealed no evidence of astogliosis. Additionally, individual astrocytes in demyelinated spinal cords did not appear to express higher levels of GFAP than individual astrocytes from unoperated control tissue of the same age. Direct pressure injection of control and experimental solutions into the thoracic spinal cord did not result in significant damage to the spinal cord tissue. The injected solution did not detectably displace spinal cord tissue or result in an area of necrosis at the injection site. Likewise, canula insertion into the spinal cord was not followed by necrosis or cavity formation. Three weeks after pump installation, the hole in the spinal cord initially created by the canula did not exceed the diameter of the canula itself. Transection Transection of the hatchling chick spinal cord did not result in the  163 Figure 5-5. Pattern of glial fibrillary acidic protein (GFAP) immunoreactivity during immunological demyelination. A: Unoperated (normally-myelinated) control posthatching day (P) 6 spinal cord showing extensive GFAP immunoreactivity within the white matter. B: GFAP immunofluorescence staining of a P6 spinal cord from a hatchiing chick that received a single injection of polyclonal galactocerebroside (GaIC) antibodies plus complement on P5; note that GFAP immunoreactivity is comparable to A, indicating that immunological demyelination does not induce an astrogliotic response. In all photograghs the outer edge of the spinal cord lies on the right hand side, with the white matter adjacent. (Bars  =  lOOum).  165 formation of a large cavity between the proximal and distal segments of the transected spinal cord (Fig. 5-6). In all of the 43 spinal cords examined 1 5-24 days after complete transection, the proximal and distal segments were intimately juxtaposed at the transection site. Autofluorescent inflammatory infiltrates at the transection site were minimal. To ensure complete transection of the spinal cord, 1 2 hatchling chicks were randomly selected immediately after spinal cord transection, fresh dissected, and examined under a dissecting microscope. In all cases a complete spinal cord transection was confirmed. In addition, 6 animals received a lumbar injection of RDA at the time of thoracic spinal cord transection. Only a complete spinal cord transection would prevent the retrograde axonal transport of the tracer to the cell bodies of origin in the brainstem (Fig. 5-7). Twenty-one (21) days after RDA injection, they were sacrificed and their brains examined for the presence of retrogradely-labelled brainstem-spinal neurons. In no case was there evidence of retrograde transport of RDA to the brainstem (Fig, 5-7A). In all cases analysis of the spinal cords revealed a discrete area of RDA-Iabelled cells within the lumbar spinal cord and no evidence of RDA transport to any region of the spinal cord rostral to the previous transection site (Fig. 5-7B). Other normally-myelinated control animals (n=3) received lumbar injections of 0.5 ul of RDA 17 days after the thoracic spinal cord transection. Again, only a complete spinal cord transection would prevent the retrograde  166 Figure 5-6. Spinal cord transection site in parasaggital section 20 days after complete thoracic transection and 14-day immunological demyelination. Note that the proximal and distal segments of the spinal cord are intimately juxtaposed at the transection site. Cavity formation was never observed in spinal cords examined 15-24 days post-transection and autofluorescent inflammatory infiltrates were minimal. (Bars  =  5Oum).  168 Figure 5-7. The transection procedure reliably severs the entire thoracic spinal cord. Control embryos received lumbar injections of tetramethylrhodamine labelled dextran amine (RDA) solution at the time of the thoracic spinal cord transection. Only a completely transected spinal cord would prevent the retrograde transport of this neuroanatomical tracer to the brainstem. A: Ventromedial reticular formation of the caudal pons in a posthatching day (P) 26 chick that received a thoracic transection and a lumbar injection of RDA on P5; note the absence of retrogradely-labelled gigantocellular reticulospinal neurons. B: Lumbar spinal cord of the same hatchling chick in parasaggital section; note the neuronal and axonal labelling evident near the previous injection site. This confirms that the transection procedure reliably severs the entire thoracicspinal cord. Similar results were observed when RDA was injected 17 days after complete thoracic spinal cord transection of a normally-myelinated (control) hatchling chick. (Bars  =  5Oum for A, 100 urn for B).  170 axonal transport of the tracer to the cell bodies of origin in the brainstem. 48 hours after RDA injection, they were sacrificed and their brains examined for the presence of retrogradely-labelled brainstem-spinal neurons. In no case was there evidence of retrograde transport of RDA to the brainstem. Analysis of the spinal cords revealed in all cases a discrete area of RDA-labelled cells within the lumbar spinal cord and no evidence of RDA transport to any region of the spinal cord rostral to the previous transection site. Demyellnation and Transection Spinal cord transections and osmotic mini-pump installations (containing demyelinating or control solutions) were performed an a total of 48 hatchling chicks aged P4-P9. In all cases, demyelinating or control solutions were  -  administered using a 14-day osmotic mini-pump connected via catheter to a canula inserted 2 spinal cord segments caudal to the transection site. Neuroanatomical assessments of spinal cord regeneration were conducted on all 48 of these animals. Physiological assessments of spinal cord regeneration (in addition to the neuroanatomical assessments) were conducted on 8 of these animals (see chapter discussion). In all cases, the spinal cord transection preceded the osmotic mini-pump installation by approximately 15 minutes (the time required for the surgery itself). Neuroanatomical regeneration was assessed 15-24 days after spinal cord transection and immunological demyelination by counting the brainstem-spinal neurons labelled by the retrograde transport of RDA injected into the lumbar  171 spinal cord 24 hours prior to sacrifice (Fig. 5-8). In transected, demyelinated hatchling chicks the number of retrogradely-labelled neurons within the reticular formation averaged 148 cells (range= 71-202; n=21; Fig. 5-8B) which is approximately 14% of the number of retrogradely-labelled neurons within the reticular formation of RDA-injected, unoperated controi hatchlings of the same age (average= 1043; range= 692-1311; n= 11; Fig. 5-8A). Comparable numbers and distributions of retrogradely-labelled neurons were also noted for other brainstem-spinal nuclei with projections to the lumbar spinal cord including the vestibular nucleus (Fig. 5-8C and D), locus ceruleus, subceruleus nucleus and raphe nucleus. In contrast, transected hatchlings that were administered control solutions over a 2-week period showed no signs of neuroanatomical regeneration 15-24 days post-transection (Fig. 5-8E and F). No retrogradely labelled brainstem-spinal projecting neurons were observed in transected hatchling chicks that were administered vehicle only (O.1M PBS, pH 7.4; n  =  10), polyclonal GaIC only (n  =  3) or guinea pig complement only (n  =  3) over  a 2-week period. Prior to sacrifice, hatchling chicks that had been transected and demyelinated for 2 weeks did not show any behavioral signs of locomotor recovery. From the day of spinal cord transection, all animals remained incapable of standing or supporting their own weight. Recumbancy was sternal  in most cases. Alternating leg movements were often observed. A decrease in  172 Figure 5-8. Neuroanatomical regeneration of brainstem-spinal projections. Photomicrograghs of retrogradely labelled neurons within the brainstem in posthatching day (P) 28 chicks. Brainstem-spinal neurons were labelled by the retrograde axonal transport of tetramethylrhodamine-labelled dextran amine (RDA) injected into the lumbar spinal cord on P26 and allowed two days for transport. A: RDA labelled gigantocellular reticulospinal neurons within the ventromedial reticular formation of the caudal pons in an unoperated (normally myelinated) control P28 chick. B: RDA labelled gigantocellular reticulospinal neurons within the ventromedial reticular formation of the caudal pons in a P28 experimental hatchling that was subjected to immunological demyelination and complete thoracic spinal cord transection on P5; note that the number and distribution of retrogradely labelled neurons is substantially less than A. C: RDA labelled vestibulospinal neurons within the lateral vestibular nucleus of the dorsolateral pons in an unoperated (normally-myelinated) control P28 chick. D: RDA labelled vestibulospinal neurons within the lateral vestibular nucleus of the dorsolateral pons in a P28 experimental hatchling that was subjected to immunological demyelination and complete thoracic spinal cord transection on P5; note that the number and distribution of retrogradely labelled neurons is substantially less than C. Comparable neuroanatomical repair was evident for other brainstem-spinal projections. E: Ventromedial reticular formation of the caudal pons in a P28 hatchling that was transected on P5 and received galactocerebroside (GaIC) antibodies only over a 14 day period; note the  173 absence of retrogradely labelled neurons. F: Lateral vestibular nucleus of the dorsolateral pons in a P28 hatchling that was transected on P5 and received galactocerebroside (GaIC) antibodies only over a 14 day period; note the absence of retrogradely labelled neurons. A similar lack of neuroanatomical repair was observed in animals that received other immunological control solutions over a 14 day period and a complete thoracic spinal cord transection on P5. (Bars  =  5Oum).  177 the size of the leg muscles post-transection was observed in all cases. In order to ensure that the neuroanatomical recovery of transected, demyelinated hatchling chicks was indeed due to true axonal regeneration (and not neogenesis and\or subsequent development), a double labelling paradigm was adopted (Fig. 5-9). On P6 a maximal number of brainstem-spinal projections was labelled with a lumbar spinal cord injection of RDA. After 24 hours to allow for retrograde transport, the thoracic spinal cords were completely transected and an osmotic mini-pump containing GaIC antibodies and complement proteins installed 2 segments caudal to the transection site. A second retrograde tract tracer (cascade blue labelled dextran amine; CBDA) was injected into the lumbar spinal cord 20 days later. Analysis of the brainstem 24 hours later revealed double-labelled neurons within the major locomotor regions of the brainstem with projections to the lumbar spinal cord including the reticular formation, vestibular nucleus, locus ceruleus, subceruleus nucleus and raphe nucleus (n=3; Fig. 5-9A and B). Specific RDA and CBDA labelling was confirmed by viewing the brain sections with a non-specific filter. When viewed with a fluoresceine filter, no cells were visible, indicating that the RDA and CBDA labelling was indeed specific, and not due to autofluorescence (Fig. 5-9C). Less than 10 cells per nucleus were double-labelled. Previous studies have indicated that, even in an untransected chick, the maximal number of double-labelled neurons obtainable from the intraspinal injection of 2 different retrograde tract tracers is 30% of the number labelled by either tracer alone (Hasan et at. 1993).  178 Figure 5-9. Double labelling of brainstem-spinal projections. Photomicrographs of retrogradely double-labelled reticulospinal neurons within the ventromedial reticular formation of the caudal pons in the chicken. Brainstem-spinal neurons were first labelled by retrograde axonal transport of rhodamine-labelled dextran amine (RDA) injected into the lumbar spinal cord on P6. The thoracic spinal cord was completely transected on P8. The infusion of complement plus galactocerebroside (GaIC) antibodies into the thoracic cord (2 segments caudal to the transection site) was begun immediately thereafter via an osmotic pump for the next 14 days. On P28, the second retrograde tracer, cascade bluelabelled dextran amine (CBDA), was injected into the lumbar spinal cord and the animal was sacrificed two days later on P30. A: Gigantocellular reticulospinal neurons labelled prior to spinal transection with the first retrograde tracer, RDA. B: Giganotcellular reticulospinal neurons subsequently labelled with the second tracer, CBDA, after immunological demyelination and spinal transection. NOTE: some of the same brainstern-spinal neurons are double-labelled with both RDA and CBDA. C: Fluorescence photomicrograph of the same photographic field using a (green) fluorosceine filter. This indicates that the double-labelling of reticulospinal neurons (RDA + CBDA) was not an autofluorescent artifact. (Bars =  50 urn).  A  180 DISCUSSION The results presented here demonstrate that the transient removal of myelin from the hatchling chick spinal cord by immunological means facilitates partial neuroanatomical regeneration of axotomized brainstem-spinal projections  in vivo. Collaborative efforts described below further indicate that this partial neuroanatomical regeneration is accompanied by functional synaptogenesis, such that brainstem stimulation of locomotor regions in transected, demyelinated hatchlings elicits electrical activity within the muscles of the legs. These studies demonstrate that myelin is inhibitory to the regeneration of axotomized brainstem-spinal projections in the post-hatching chick. The injection of galactocerebroside (GalC) antibodies or 04 antibodies and complement proteins directly into the spinal cord of the P1-P9 hatchling chick results in immunological demyelination which extends several segments either side of the injection site (Fig. 5-1; Fig. 5-3; Fig. 5-4). The astrocyte population of the spinal cord is undisturbed by the demyelination procedure (Fig. 5-5). Immunological demyelination takes place within 24 hours (Fig. 5-3) and persists for as long as the antibodies and complement proteins are administered (the longest trial period was 14 days; Fig. 5-3; Fig. 5-4). Remyelination of the spinal cord takes place within 4-7 days of the cessation of treatment (Fig. 5-3; Fig. 54). Neither antibody alone, complement proteins alone, nor vehicle (PBS) alone caused demyelination when injected into the spinal cord, indicating that both the oligodendrocyte-specific antibody and complement proteins are necessary in  181 order to evoke immunological demyelination (Fig. 5-2). GaIC antibodies and complement proteins were delivered to the hatchling chick spinal cord over a 14-day period in an attempt to rescue previously axotomized brainstem-spinal projections. Osmotic mini-pumps containing GaIC antibodies and complement proteins were installed shortly after spinal cord transection, and neuroanatomical regeneration of brainstem-spinal projections was assessed 15-24 days later by counting the brainstem neurons retrogradely labelled by an injection of tetramethyirhodamine-labelled dextran amine (RDA) into the lumbar spinal cord (Fig. 5-8). In transected, demyelinated animals the number of retrogradely-labelled brainstem-spinal neurons was approximately 515% of the number of retrogradely-labelled brainstem-spinal neurons in RbA injected, untransected control hatchlings of the same age. Transected animals that received no treatment or control solutions (GaIC antibody only, complement proteins only or vehicle only) over a 2-week period showed no signs of neuroanatomical regeneration. True axonal regeneration contributed to the neuroanatomical recovery of transected, demyelinated hatchling chicks as evidenced by double-labelling of brainstem-spinal projecting neurons (Fig. 5-9). Prior to transection and initiation of immunological demyelination, the existing population of brainstem-spinal projecting neurons were maximally labelled with a lumbar injection of RDA. A second retrograde tract tracer (cascade blue labelled dextran amine; CBDA) was injected into the lumbar spinal cord 20 days later. Analysis of the brainstem 48  182 hours later revealed double-labelled neurons within the major locomotor regions of the brainstem with projections to the lumbar spinal cord. This demonstrates that axons labelled with the first tracer (RDA) regenerated through the transection site to pick up the second tracer (CBDA). This finding is supported by studies which indicate that neurogenesis of brainstem-spinal projecting neurons in the chick is complete by embryonic day (E) 5 (McConnell and Sechrist, 1980; Sechrist and Bronner-Fraser, 1991) and the development of brainstem-spinal projections is complete to all levels of the spinal cord by ElO or Eli (Okado and Oppenheim 1985). In all cases, RDA was injected approximately 8mm caudal to the previous transection site. Prior studies have indicated that RDA remains confined to the area of injection (ie. does not diffuse rostrally to or above the site of transection; Hasan et al. 1991; Keirstead et al. 1992; Hasan et al. 1993). Transected and demyelinated .hatchling chicks lived, on average, 21 days prior to neuroanatomical assessments of regeneration, which indicates a rate of axonal regeneration of approximately 0.4mm per day. The rate of axonal regeneration reported here is much slower than those reported in other studies, which document rates of axonal outgrowth of 1-2mm per day (Ramon y Cajal 1959; Schnell and Schwab 1990). This discrepancy may be accounted for by an initial period of axonal retraction immediately following spinal cord transection, which the present techniques are incapable of discerning. The relatively severe nature of the lesion used in the present studies may be  183 associated with a greater degree of secondary degenerative events which may extend the period of axonal retraction and/or impede the growth of regenerating axons. Alternatively, the rates of axonal outgrowth in the chick CNS may be inherently slower than those of the rat and mouse. The neuroanatomical regeneration of transected, demyelinated hatchlings summarized above did not result in any behavioral signs of locomotor recovery. After spinal cord transection, all animals remained incapable of standing or supporting their own weight. Although alternating leg movements were often observed, this does not indicate supraspinal-mediated voluntary control of locomotion. When supported on a treadmill, transected animals are capable of locomotion, mediated by the intinsic activity of neural networks confined to the lumbar spinal cord (Sholomenko and Steeves 1987; Eidelberg 1981; Steeves et al. 1987). The lack of behavioral recovery in transected, demyelinated hatchlings may have been due to insufficient leg musculature. A decrease in the size of the leg muscles during the recovery period post-transection indicated extensive muscular atrophy in all cases. Alternatively, the lack of behavioral recovery in transected, demyelinated hatchlings may have been due to a lack of synaptogenesis of regenerated brainstem-spinal axons with spinal cord motoneurons or interneurons, ultimately innervating the leg muscles. However, the experiments described below negate this possibility. Physiological assessments of locomotor capabilities of transected and demyelinated or control-treated (PBS only or GalC only) hatchling chicks were  184 undertaken in collaboration with Dr. Gerry Sholomenko (a postdoctoral fellow under the supervision on Dr. Kerry Delany, Simon Fraser University, Vancouver, B.C.; Fig. 5-10, Fig. 5-11). Three weeks after spinal cord transection and the initiation of immunological demyelination, brainstem stimulation (3OHz/0.5msec monophasic pulses) of decerebrate, hatchling chicks evoked motor activity both above (pectoralis; PECT) and below the site of transection (n  =  3).  Electromyograms from the sartorius muscles (SART; major hip flexor) illustrate alternating periods of activity in the right and left legs (Fig. 5-10). Following electromyographic recording, the animals were paralysed and unidirectionally ventilated to prevent movement artifact. Electroneurograms from an episode of brainstem stimulation show the presence of rhythmic SART and PECT activity (Fig. 5-1 1). In transected animals treated with GaIC only (n = 2) or PBS only (n =3) for a 2-week period, brainstem stimulation resulted in electromyographic activity above the transection site but no electromyographic or electroneurographic activity was present be!ow the transection site. All of these animals were subsequently included in the neuroanatomical studies outlined above. These findings indicate that neuroanatomical regeneration of brainstem spinal projections following spinal cord transection in immunologically demyelinated hatchlings is accompanied by functional synaptogenesis with spinal cord neurons which directly or indirectly innervate the muscles of the legs. The proper connectivity of growing neurites with their targets presumably  185 Figure 5-10. Electromyograms (EMG) from the sartorius (SART; major hip flexor) and pectoralis (PECT; major wing depressor) muscles of a decerebrate brainstem stimulated 27 day chick subjected to immunological demyelination and transection on posthatching day (P) 9. Brainstem stimulation at 5OuA (3OHz/O.5msec monophasic pulses) evoked motor activity both above and below the transection site. In this case the wings were held to prevent movement artifact in the leg myograms.  L. SART  R. SART  L. PECT I  -  I  LL  III  LI  “ia II-I[.  ii  II11111 II  I  Lh  1I1’FhIIliiI I  LiftJ LllIkJ  ii irr  11h g 1  II  J  •11  p  T’  J,I 1  111  1  hIALI i 1 I  ,  —  ali  1I  iIII  II1tj  iA  I  I  1SEC  a-I-.  I  [--  I.  1  I  187 Figure 5-11. Electroneurograms (ENG) from an episode of brainstem stimulated (300uA) activity in the same chick as Fig. 5-10. The animal was paralyzed and unidirectionally ventilated to prevent movement artifact. At the start of stimulation one of the left toes was held for ten seconds and released. The top two traces show the start of the episode with a slow time base to demonstrate the onset of rhythmic pectoralis (PECT) activity and mainly tonic sartorius (SART) activity. The expanded bottom two traces show the presence of rhythmic SART activity superimposed upon the increase in tonic activity below the transection site.  A  PECT  LirJIU Ii  JiiI iijr  I’ j 1 ItII 1 iJ  1 ‘r  I.  Il)JL I’ II  ‘I •I  SART  iiJ IIii I’ ‘1  I ‘II  i .1.  jLLid [II  rj  1’  B  j1 iIi  j’  ‘r  PECT  SART  1. liii  ii  ,I  i  liii I  II••l  I! L II ‘ ‘I  •1  II••III  1i  I  I  ii  189 arises as a result of transient spaciotemporal patterns of guidance factors during development, or to the presence of complementary guidance factors which are permanent features of the growing neurites, the pathway(s) and the target tissue(s). The neuroanatomical regeneration and functional synaptogenesis observed following transection and demyelination of the hatchling chick (ie. mature) spinal cord would support the later scenario. These two scenarios are not mutually exclusive. Neuroanatomical regeneration and functional synaptogenesis with appropriate targets has also been observed following axotomy of the fibre connections between the entorhinal area and the hippocampal complex (Li et al. 1994). In horizontal slices of rat postnatal day 9 and 10 tissue, severed fibre projections between the entorhinal cortex and the hippocampal complex regenerated in both directions and re-established correct laminar, pathway and target specificity. This study lends further support to the suggestion that guidance factors are permanent features of growing neurites, pathway(s) and target tissue(s). The onset of immunological demyelination of the hatchling spinal cord is extremely fast (immunological demyelination takes place within 24 hours of treatment; see Table 1; Fig. 5-3). The antibody-mediated, complement dependent nature of immunological demyelination may account for its rapid onset. Complement fixation by cell-surface binding antibodies has been shown to compromise the ionic homeostasis of many different cell types in culture within minutes or hours of treatment (Mayer 1972; Morgan 1989). GalC  190 mediated, complement-dependent injury of oligodendrocyte myelin in the guinea pig optic nerve in vivo (Sergott et al. 1984) is detectable within 1-2 hours of treatment. Remyelination following immunological demyelination is also extremely fast, occurring within one week of the cessation of treatment (Fig. 5-3; Fig. 54). The speedy onset of remyelination following immunological demyelination itself suggests that oligodendrocyte cell bodies survive the immunological demyelination procedure, and are a likely origin of subsequent myelin. Complement membrane attack of neutrophils in culture is accompanied by a inhibition of energy-requiring processes, followed by complete biochemical and functional cell recovery when the cells are transferred to complement-free medium (Morgan 1988). In oligodendrocytes, complement membrane attack in vitro is also followed by a complete restoration of cellular energy stores (Scolding et al. 1989). More direct proof of oligodendrocyte cell body survival following immunological demyelination was obtained from collaborative studies undertaken with Dr. Alan Peterson (Mount Royal Hospital, Monteal, Quebec). Immunological demyelination as described above was performed on MBP-LacZ transgenic mice (described in Foran and Peterson 1993; Fig. 5-12). The oligodendrocyte cell bodies of this transgenic mouse line contain the LacZ gene product, fl-galactosidase, which can be detected with the histological stain Blue X-Gal (Foran and Peterson 1993). The injection of GaIC antibodies and  191 Figure 5-12. Immunological demyelination of the MBP-Lac Z, dlO-8O transgenic  mouse spinal cord. This animal received an injection of galactocerebroside (GaIC) antibodies plus complement directly into the spinal cord on postnatal day (PN) 12 and was sacrificed on PN13. A: /J-galactosidase positive oligodendrocyte distribution on the surface of the spinal cord; note the focal decrease in fl-galactosidase positive oligodendrocytes at the site of injection. This suggests that oligodendrocyte cell bodies are destroyed at the site of injection. B: Photomicrograph of toluidine blue stained, ,8-galactosidase reacted transverse section of the spinal cord two segments rostral to the injection site; note the virtual absence of myelinated axons in the vicinity of oligodendrocytes (stars). Myelin is absent or dramatically decreased for several segments around the area of injection. This section was taken from the same spinal cord shown in whole mount above (A). C: Photomicrograph of toluidine blue stained,  fi  galactosidase reacted transverse section of an unoperated control spinal cord; note the abundance of myelinated axons (arrows) in the vicinity of oligodendrocytes (stars).  C /  /  193 complement proteins into the spinal cord of postnatal day 12 transgenic mice resulted 24 hours later in oligodendrocyte cell death which was restricted to the immediate injection site (most probably due to the injection itself; Fig. 5-1 1A). Toluidine blue staining of lum transverse sections of these spinal cords 2 segments rostral to the injection site indicated a dramatic decrease in the number of myelinated axons, however, Bluo-Gal stained oligodendrocyte cell bodies were clearly present (n=3; Fig. 5-11B). Toluidine blue staining of lum transverse sections of unoperated control spinal cords reveal densely compacted myelin profiles in the vicinity of Bluo-Gal stained oligodendrocyte cell bodies (Fig. 5-1 1C). These preliminary studies suggest that oligodendrocyte cell bodies survive immunological demyelination and are a likely source of  -  subsequent myelin. It is also possible that de-differentiation of astrocytes or astrocyte precursors may contribute to spinal cord myelination following immunological demyelination. In demyelinating lesions of the cat optic nerve, reactive glial cells which have begun to differentiate along the astrocyte lineage (GFAPI may reverse their direction of differentiation, evidenced by the coexpression of both astrocyte- and oligodendrocyte-specific markers (GFAP, GaIC; Kim 1985; Carroll et al. 1987). Switching glial cultures from one type of culture media to another favoring the alternative direction of differentiation results in the transient coexpression of both astrocyte and oligodendrocyte phenotypes (Ingraham and McCarthy 1989). Cells of the 02A lineage may actually exhibit  194 greater plasticity in demyelinating/remyelinating conditions. Cells coexpressing astrocyte and oligodendrocyte phenotypes can be isolated from the spinal cords of young adult mice demyelinated by corona virus infection (Armstrong et al. 1990). In addition, cells coexpressing astrocyte and oligodendrocyte phenotypes have been reported in vivo in a number of demyelinating situations (Bunge et al. 1961; Carroll et al. 1987; Godfraind et al. 1989). Although the results presented in this chapter suggest that remyelination following immunological demyelination is facilitated by surviving oligodendrocyte cell bodies, it is possible that dedifferentiation of astrocytes may additionally contribute to the recovery of spinal cord myelinating oligodendrocytes. Pending double labelling studies of immunologically demyelinated spinal cords using both astrocyte- and ol igodend rocyte-specific antibodies, the possibility of astrocyte dedifferentiation and redifferentiation into myelinating oligodendrocytes can not be excluded. Following complete transection, the proximal and distal segments of the severed hatchling chick spinal cord remained intimately juxtaposed (Fig. 5-6). Fifteen to 21 days after spinal cord transection, light microscopic analysis of sectioned spinal cords indicated a lack of cavity formation at the transection site and an absence of autofluorescent inflammatory infiltrates separating the proximal and distal segments. In contrast, mouse spinal cord transection results in cavity formation at the lesion site (Schnell and Schwab 1990) that is often associated with a robust inflammatory response. The lack of secondary damage  195 in the hatchling chick spinal cord after transection may be due to the unique method of transection. Alternatively, this discrepancy may be due to differences between species in the immunological response to spinal cord transection and the corresponding breach of the blood-brain barrier. In conclusion, the findings presented here demonstrate that the injection of oligodendrocyte-specific antibodies plus complement proteins into the hatchling chick spinal cord results in transient immunological demyelination that persists for as long as these compounds are administered. Furthermore, these findings demonstrate that complete transection of the hatchling chick spinal cord in vivo followed immediately by 2-3 weeks of immunological demyelination results in neuroanatomical regeneration of brainstem-spinal projections that is accompanied by functional synapse formation. These findings constitute the first demonstration of functional regeneration following injury to the mature spinal cord and clearly demonstrate that hatchling chick spinal cord myelin is inhibitory to the regeneration of axotomized brainstem-spinal connections involved in locomotion.  196  CHAPTER 6  GENERAL DISCUSSION  197 The studies outlined in the preceding chapters began with the observation that the developmental onset of myelination in the embryonic chick spinal cord is coincident with a loss of regenerative ability following spinal cord transection (chapter 2; Keirstead et al. 1992; Okado and Oppenheim 1985; Hasan et al. 1991). Other studies have identified several different proteins expressed by central nervous system (CNS) oligodendrocytes and oligodendrocyte-produced myelin that inhibit neurite outgrowth in vitro (Pesheva et al. 1993; McKerracher and David, unpublished observations) and in vivo (Schnell and Schwab 1990). These findings suggested that myelination within the embryonic chick spinal cord may inhibit regeneration following injury, and prompted the development of the developmental myelin-suppression procedure used in the subsequent studies described here. To assess a potential inhibitory role for myelin in the functional regeneration of severed brainstem-spinal projections in the embryonic chick, I developed a method of delaying the developmental onset of spinal cord myelination until embryonic day (E) 17 of the 21 day developmental period in ovo (chapter 3). Myelination within the embryonic chick spinal cord normally begins on E13 (Bensted et al. 1957; Hartman et al. 1979; MackIm  and Weill  1985; Keirstead et al. 1992). In order to evoke developmental myelin suppression, oligodendrocyte cell surface-binding antibodies were injected directly into the thoracic spinal cord, along with a source of complement proteins. Galactocerebroside (GaIC) and 04 antibodies were utilized due to their  198 specificity for oligodendrocytes and myelin, as well as their ability to fix complement (Ranscht et al. 1982; Sommer and Schachner 1982). Immunological control solutions (GaIC antibodies only, complement proteins only, vehicle only, non-specific antibody plus complement or GalC antibodies plus heat-inactivated serum) had no discernable effects on spinal cord myelin development, indicating that both the oligodendrocyte-specific antibodies and complement proteins are necessary to evoke myelin-suppression. Developmental myelin-suppression specifically targeted spinal cord myelin, apparently leaving the oligodendrocyte cell bodies intact, and did not appear to elicit an astroglial response or alter the dendritic architecture of the neuronal population of the spinal cord. Remyelination following myelin-suppression was shown to be fully compensatory. Transections of the myelin-suppressed spinal cord during the previously defined restrictive period for spinal cord repair then serve as a direct test of the inhibitory properties of CNS myelin to regenerating axons. Transections as late as El 5 in myelin-suppressed embryos resulted in complete neuroanatomical and physiological repair, enabling functional locomotor capabilities after hatching comparable to an untransected control chick (chapter 4). Transections on E15 in normally-myelinated, or immunological control embryos resulted in no anatomical or physiological repair, rendering such an animal completely incapable of voluntary locomotion after hatching. These findings indicated that myelin within the spinal cord of an embryonic chick inhibits functional regeneration following spinal cord injury. In addition, these  199 studies suggested that demyelination of the (myelinated) hatchling chick spinal cord may also be an effective means of promoting regeneration following spinal cord injury. A method of demyelinating the hatchling chick spinal cord was developed by adapting the method of developmental myelin-suppression so as to effectively demyelinate the hatchling spinal cord over a two week period (immunological demyelination; chapter 5). Osmotic mini-pumps containing complement proteins plus polyclonal GaIC or 04 antibodies were implanted under the skin and attached to a canula which directly infused the low thoracic spinal cord. Immunological control solutions (GaIC antibodies only, complement proteins only or vehicle only) had no discernable effects on spinal cord myelin, indicating that both the oligodendrocyte-specific antibodies and complement proteins are necessary to evoke demyelination. Immunological demyelination in this manner did not disturb the astrocyte population of the spinal cord, nor did it produce any signs of necrosis at the infusion site. Remyelination of the spinal cord, following immunological demyelination, took place within one (1) week of cessation of treatment (exhaustion of the pump reservoir) and was fully compensatory. GaIC antibodies and complement proteins were delivered to the hatchling chick spinal cord in an attempt to rescue previously axotomized brainstem-spinal projections (chapter5). Immunological demyelination was initiated shortly after complete thoracic transection of the spinal cord, and neuroanatomical  200 regeneration of brainstem-spinal projections was assessed 1 5-24 days later by counting the brainstem neurons retrogradely-labelled by an injection of tetramethylrhodamine-labelled dextran amine (RDA) into the lumbar spinal cord. In transected, immunologically demyelinated animals the number of retrogradely labelled brainstem-spinal neurons was approximately 5-15% of the number of retrogradely-labelled brainstem-spinal neurons in RDA-injected, untransected control hatchlings of the same age. Transected animals that received no treatment or control solutions (GalC antibody only, complement proteins only or vehicle only) over a 2-week period showed no signs of neuroanatomical regeneration. Double-labelling studies indicated that true axonal regeneration contributed to the neuroanatomical recovery of transected and immunologically demyelinated hatchling chicks. Collaborative studies undertaken with Dr. Gerry Sholomenko (a postdoctoral fellow under the supervision on Dr. Kerry Delany, Simon Fraser University, Vancouver, B.C.) indicated that the neuroanatomical regeneration of transected, immunologically demyelinated hatchling chicks was followed by functional synaptogenesis (chapter 5). Three weeks after spinal cord transection and subsequent initiation of immunological demyelination, brainstem stimulation of decerebrate hatchlings evoked motor activity both above (pectoralis muscles) and below (sartorius muscles) the site of transection. Electromyographic recordings from the sartorius muscles illustrated alternating periods of activity in the right and left legs. Subsequent electroneurographic recordings also showed  201 the presence of rhythmic sartorius and pectoralis activity. In transected animals treated with immunological control solutions over a 2-week period, brainstem stimulation resulted in electromyographic activity above the transection site but no electromyographic or electroneurographic activity was present below the transection site. These findings indicate that neuroanatomical regeneration of brainstem-spinal projections following spinal cord transection in immunologically demyelinated hatchlings is accompanied by functional synaptogenesis with spinal cord neurons which directly or indirectly innervate the muscles of the legs. Immunological myelin-suppression was also effective in removing (already present) myelin from the neonatal mouse spinal cord (chapter 5). These results indicate that immunological myelin-suppression is not species restricted. It remains to be determined whether this intervention will improve the repair and recovery of function after injury to the neonatal mouse spinal cord. Axonal regeneration following spinal cord transection in both late embryonic and hatchling chicks was restricted to those animals subjected to the immunological suppression of CNS myelin. However, the degree of functional recovery accompanying neuroanatomical regeneration differed substantially in embryonic and hatchling chicks. It is conceivable that the dramatic functional recovery noted in the myelin-suppressed, El 5 transected embryos, as compared to the transected, demyelinated hatchlings, may be due to; 1) an enhanced intrinsic ability for axonal extention, 2) a relative abundance of growth-  202 promoting factors, and/or, 3) a relative paucity of growth-inhibitory factors. Several studies have indicated that neurons in culture display an inherent decrease in speed of neurite extention, axonal regeneration as well as a decrease in terminal sprouting with age (Argiro et al. 1984; Collins and Lee 1982; Black and Lasek 1979; Pestronck et al. 1980). Age-related differences in axonal growth potential are most obvious in culture systems that simulate a growth-inhibitory environment. One such system is the in vitro correlate of the glial scar, the three dimensional astrocyte culture system (Fawcett et al. 1989). In this culture system, embryonic retinal ganglion cells and sensory neurons are capable of extending axons through the astrocyte matrix, whereas projections from postnatal retinal ganglion cells and sensory neurons fail to penetrate the astrocyte matrix (Fawcett et al. 1989a). Other studies have indicated that the developmental programme of gene expression is not recapitulated during regeneration attempts in the adult. Microtubule-associated protein (MAP) la, one of the many developmentally regulated MAPs which control the assembly and stabilization of microtubules behind the axon growth cone, is not re expressed during regeneration (Woodhams et al. 1989). Oligodendrocytes are also an unfavorable substrate for growing postnatal axons, which cease growth or are repelled upon contact (Schwab 1990; Fawcett et al. 1989b). In contrast, embryonic retinal ganglion cells and dorsal root ganglion cells are apparently insensitive to the inhibitory properties of oligodendrocytes, growing into contact with, and extend neurites over oligodendrocytes (Ard et al. 1991). Thus, as a  203 result of an apparent insensitivity to glial-derived inhibitory molecules, embryonic neurons may be intrinsically more capable of regenerating severed axons. Developmental changes in the abundance of growth-promoting factors have also been reported. Extracellular matrix (ECM) molecules such as laminin, fibronectin and proteoglycans, essential for cell attachment, migration and process extention, are widely expressed in the developing central nervous system (CNS) and are found in a more restricted distribution in the adult (Sheppard et al. 1991; Carbonetto 1984; Steindler et al. 1990). Cell adhesion molecules, although present in both the embryonic and adult CNS, decrease their neurite-outgrowth promoting ability during development and are also distributed in a more restrictive fashion in the adult (Doherty et al. 1990b). As one might imagine, integrin receptors for many ECM molecules are also functionally down-regulated during development (Reichardt and Tomaselli 1991). Growth factors are perhaps the most influential neurite growth-promoting factors available to the developing CNS. Growth factors regulate the size of developing neuronal populations (Harper and Thoenen 1981), promote maturation and cell survival (Hamilton and Levi-Montalcini 1949), prevent cell death (Arenas and Persson 1994) and enhance sprouting (Schnell et al. 1994). Many growth factors and their receptors are developmentally regulated (Knusel et al. 1994), suggesting that injured adult neurons may not receive the benefits  204 that growth factors provide to developing, and perhaps injured, embryonic neurons. Attempts to increase the degree of functional recovery of transected, demyelinated hatchling chicks may be expedited by identifying and providing the specific growth factor(s) that brainstem-spinal projecting neurons are responsive to. The limited functional recovery of transected, demyelinated hatchling chicks (compared to El 5-transected, developmentally myelin-suppressed embryos) may also be due to neurite growth-inhibitory factors present in the mature CNS, but absent in the developing system. Spinal cord transections in the hatchling chick result in a pronounced astroglial response that is confined to one spinal cord segment either side of the injury (Fig. 6-1). The proliferation and hypertrophy of astrocytes in and around a site of CNS injury has been suggested to provide a physical barrier to axonal growth (Reier and Houle 1988; Hertz et al 1990). Furthermore, astrocytes express several anti-adhesive molecules including tenascin (Brodkey et al. 1993) and chondroitin sulfate, dermatan sulfate and keratan sulfate proteoglycans (Herndon et al. 1990). In contrast, spinal cord transections in the embryonic chick are not accompanied by an astrogliotic response (Shimizu et al 1990), despite the presence of astrocytes in the late embryonic spinal cord (chapter 2). A lack of astrogliosis following embryonic CNS injury has also been noted in other systems (Berry et  205 Figure 6-1. Astrogliosis following thoracic transection of the hatchling chick spinal cord. Photomicrographs of glial fibrillary acidic protein (GFAP) immunoreactivity in parasaggital section. A: Gray matter two segments rostral to the transection site shown in B; note the lack of astrogliosis. B: Photomontage through the transection site; note that astrogliosis is restricted to approximately one segment either side of the transection. (Bars  lOOum).  207 al. 1968; Maxwell et al. 1990). This implies a developmentally-regulated change in the response of astrocytes to CNS injury. Methods of reducing or eliminating astrogliosis following adult spinal cord injury would be useful for investigating the relative contribution of astrogliosis to the lack of spinal cord repair in transected hatchling chicks. Despite age-related differences in the inherent capability of axons to regenerate, as well as developmental changes in the abundance and distribution of growth-promoting and growth-inhibiting factors (reviewed above), myelin clearly plays a major role in determining the regenerative success of axotomized embryonic and hatchling chick brainstem-spinal projecting neurons. Only those animals subjected to immunological myelin-suppression showed any signs of neuroanatomical regeneration and physiological recovery following spinal cord injury. Functional recovery has also been observed following CNS injury to invertebrates as well as several lower vertebrate systems such as the lamprey spinal cord, which is unmyelinated and is capable of functional regeneration following injury to either the larval or adult animal (McClellan 1990). Considering the inhibitory nature of CNS myelin for regenerating axons, a philosophical question arises: Why, over the course of evolution, has a system developed that prohibits regeneration following CNS injury? It is likely that the inhibitory nature of CNS myelin for neurite outgrowth evolved for reasons other than to inhibit neural regeneration following injury. One might imagine natural selection pressures contributing to the development  208 of CNS protective structures, such as the shock-absorbing cerebrospinal fluid layer or the protective enclosure provided by the cranium and vertebral column. Individuals with less well developed CNS protective structures would be far more likely to suffer serious CNS injury, which would in turn decrease or eliminate their chances of contributing to the gene pool. However, the CNS itself may be immune to natural selection pressures which would favor CNS regeneration. Injury to the CNS is almost exclusively associated with extensive damage to extraneural protective structures and is commonly associated with a loss of sympathetic and parasympathetic control (including control of reproductive organs) or an immediate cessation of vital functions. If the wheels of evolution, genetic recombination and mutation, did generate mechanisms which could facilitate CNS regeneration, this phenotype may not be selected for as a result of these various extraneural complications. Therefore, it is arguable that the CNS is immune to natural selection pressures concerned with regeneration. The inhibitory nature of CNS myelin for neurite outgrowth may have evolved for developmental reasons that incidently affect regenerating neurons. Myelination may serve to suppress abberent sprouting in nerve fibre tracts. The developmental onset of CNS myelination in higher vertebrates generally takes place after the developmental period of axonal process outgrowth and target recognition. In the embryonic chicken, for example, the developmental onset of spinal cord myelination takes place on embryonic day (E) 13 (Keirstead et al.  209 1992; Bensted et al. 1957; Hartman et al. 1979; MackIln and Weill 1985); brainstem-spinal neurons complete their projections to all levels of the spinal cord by E12 (Okado and Oppenheim 1985). Oligodendrocytes appear in the hamster optic tract and rostral superior colliculus by postnatal day (PN) 5 and spread throughout most of the stratum opticum by PN9, after retinocollicular axons have innnervated the superior colliculus (Schneider et al. 1990; Jhaveri et al. 1992). In the rat spinal cord, the corticospinal tract descends during the first postnatal week and begins to be myelinated at PN1O-1 1 (Schreyer and Jones 1982). Furthermore, neutralization of myelin-associated neurite growth inhibitors in the rat optic nerve (Colello et al. 1991) and hamster optic tectum (Kapfhammer et al. 1992) leads to increased sprouting. Given the permissive extraneuronal environment for developing neurites, the nonpermissive properties of CNS myelin may serve to inhibit aberrant collateral branching and terminal arborization once proper axonal connections have been made. CNS myelin may also serve a boundary function in development. Myelination occurs at different times in different tracts of the developing CNS (Bensted et al. 1957; Rozeik and Von Keyserlingk 1987). Late-developing tracts may project through a region of the CNS that is bordered by myelinated tracts, which may provide ‘boundaries’ to the late-projecting axons. Such a scenario is seen in the developing corticospinal tract (CST) of the rat. The CST descends through the dorsal funniculus of the spinal cord during the first postnatal week, and is intimately surrounded by the myelinating fasciculus cuneatus and gracilis  210 (Schreyer and Jones 1982). Removal of spinal cord oligodendrocytes and myelin with X-ray irradiation, or application of activity-blocking antibodies to identified myelin-associated neurite growth inhibitors results in abberent growth of CST axons outside of the anatomical region that the CST normally occupies (Schwab and Schnell 1991). These findings imply that late developing fibres may be indirectly guided to their targets by the non-permissive nature of adjacent myelinated tracts. The postulated developmental roles for neurite growth inhibition by CNS myelin (outlined above) clearly affect regenerative efforts by injured axons within myelinated regions of the CNS. Injured central fibres undergo a period of extensive sprouting that recapitulates neurite outgrowth in development (Ramon y Cajal 1959). Unlike developing axons, however, regenerating axons exist in an extremely hostile extraneuronal environment, created in part by the presence of myelin. The studies outlined in this thesis demonstrate that the regenerative success of transected hatchling and late-embryonic chick spinal cord is determined in part by the presence or absence of myelin. This suggestion is supported by the demonstration that oligodendrocytes are responsible for restricting innervation of the superior colliculus following postnatal injury (Kapfhammer et al. 1992). In a normal hamster, retinocollicular axons arrive at the superior colluculus prior to myelination, and innervate the superficial gray as well as the deeper stratum opticum. If the outer layers of one superior colliculus are destroyed, the lesioned retinal fibres will cross the tectal midline and  211 innervate the contralateral superior colliculus. The recrossing retinal fibres will terminate throughout the entire mediolateral extent of the superior colliculus if the eye that normally innervates this side is removed. However, the terminals formed by the recrossing fibres are restricted to the superficial gray, which is unmyelinated at the time when the recrossing terminals arrive. The deeper stratum opticum of the superior colliculus is actively myelinating at this time and remains uninnervated by recrossing fibres. However, in the presence of antibodies that neutralize the myelin-associated neurite inhibitory activity, recrossing retinal fibres terminate within the stratum opticum as well as the superficial gray. These studies indicate that myelin restricts plasticity within the injured CNS. The inhibitory nature of CNS myelin for regenerating axons may be a consequence of its developmental roles. Despite the hostile extraneuronal environment for regenerating CNS axons, immunological myelin-suppression in the late-embryonic or hatchling chick spinal cord facilitates partial neuroanatomical regeneration of brainstem spinal projections that is accompanied by functional synaptogenesis (chapters 4 and 5). These studies indicate that CNS myelin is inhibitory to the regeneration of transected spinal cord in late-embryonic and hatchling chick. Furthermore, the different degrees of regeneration observed after injury in the myelin suppressed embryonic and hatchling chick spinal cords suggest that other therapies may be required in more mature animals, therapies that provide regenerating brainstem-spinal projections with a more ‘embryonic-like’  212 environment. Identification of those extraneuronal substrates and growth factors that brainstem-spinal axons are responsive to is a first step in constructing alternative therapies for injured axons. 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