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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 ONCNS REPAIR AFTER INJURYByHANS STEGMANN KEIRSTEADB.Sc., University of British Columbia, 1990A THESIS SUBMITTED IN PARTIAL FULFILLMENT OFTHE REQUIREMENTS FOR THE DEGREE OFDOCTOR OF PHILOSOPHYinTHE FACULTY OF GRADUATE STUDIES(ZOOLOGY)We accept this thesis as conformingto he required standard.:.THE UNIVERSITY OF BRITISH COLUMBIASeptember 1994©Hans Stegmann Keirstead, 1994In presenting this thesis in partial fulfillment of therequirements for an advanced degree at the University of BritishColumbia, I agree that the Library shall make it freely availablefor reference and study. I further agree that permission forextensive copying of this thesis for scholarly purposes may begranted by the head of my department or by his or herrepresentatives. It is understood that copying or publication ofthis thesis for financial gain shall not be allowed without mywritten permission.Department ofThe University of British ColumbiaVancouver, CanadaDate SIIABSTRACTIn higher vertebrates, axons within the differentiated central nervoussystem (CNS) possess a very limited capacity for repair after injury. Thefollowing experiments were designed to determine the contributions of CNSmyelin to the lack of regeneration observed following transection of the lateembryonic and hatchling chick spinal cord. The developmental onset ofmyelination in the chick begins at embryonic day (E) 13 of the 21 daydevelopmental period. Spinal cord transections after the developmental onset ofmyelination 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 anunidentified antigen on oligodendrocyte progenitors) plus complement betweenE9-E12 results in a delay in the onset of myelination until E17 (developmentalmyelin-suppression). A subsequent transection of the spinal cord as late as El 5(i.e. during the normal restrictive period for repair) results in completeneuroanatomical regeneration and functional recovery. Spinal cord transectionson El5 in a normally-myelinated embryo result in no neuroanatomicalregeneration or functional recovery. These findings indicate that CNS myelin isinhibitory to the functional regeneration of transected spinal cord in embryonicchick (Keirstead et al. 1992).These studies also suggest that myelin suppression might also facilitateregeneration after adult spinal cord injury. Hatchling chickens are precocial andIIItheir brainstem and spinal cord can be considered in all respects adult-like.Administration of complement-binding GaIC antibodies or 04 antibodies pluscomplement to the hatchling spinal cord results in the transient removal ofspinal cord myelin (immunological demyelination). The thoracic cord ofposthatching day (P)2-P10 chickens were completely transected andimmunological demyelination was simultaneously initiated. Fourteen to 28 dayslater, retrograde tract tracing, including double-labeling studies, indicated thatapproximately 5-15% of the brainstem-spinal projections had regenerated acrossthe transection site to lumbar levels. Even though voluntary locomotion was notobserved after recovery, focal electrical stimulation of identified brainstemlocomotor regions evoked either stepping movements or ‘fictive’ stepping inparalysed animals (collaborative studies, see chapter 5). This indicates that thetransient demyelination of injured hatchling (i.e. mature) chick spinal cordfacilitated axonal regeneration resulting in some functional synaptogenesis withspinal neurons.ivTABLE OF CONTENTSABSTRACTTABLE OF CONTENTS ivLIST OF TABLES viLIST OF FIGURES viiLIST OF ABBREVIATIONS ixACKNOWLEDGEMENTS xiCHAPTER 1 General introduction 1CHAPTER 2 Myelin development in the embryonic chick spinal cord 18INTRODUCTION 19MATERIALS AND METHODS 29RESULTS 34DISCUSSION 50CHAPTER 3 Developmental myelin-suppression in the embryonic 55chick spinal cordINTRODUCTION 56MATERIALS AND METHODS 65RESULTS 67DISCUSSION 90VCHAPTER 4 Neuroanatomical repair and functional recovery of 104transected spinal cord in embryonic chickINTRODUCTION 105MATERIALS AND METHODS 111RESULTS 114DISCUSSION 129CHAPTER 5 Neuroanatomical repair and physiological recovery 139following transection and immunological demyelinationof the hatchling chick spinal cordINTRODUCTION 140MATERIALS AND METHODS- 147RESULTS 150DISCUSSION 180CHAPTER 6 General discussion 196REFERENCES 213viLIST OF TABLESTable 5-1 Summary of immunological demyelination with 156single injectionviiLIST OF FIGURESChapter 2Figure 2-1 GaIC during development 35Figure 2-2 CNP during development 37Figure 2-3 MBP during development 40Figure 2-4 MAG during development 42Figure 2-5 Toluidine blue E15 spinal cord 45Figure 2-6 GFAP during development 47Chapter 3Figure 3-1 Developmental myelin-suppression (MBP, MAG, CNP) 68Figure 3-2 Developmental myelin-suppression (toluidine blue) 73Figure 3-3 Onset of myelination following developmental 76myelin-suppressionFigure 3-4 Hatchling levels of myelination following developmental 79myelin-suppressionFigure 3-5 Immunological control injections 82Figure 3-6 GFAP during and after developmental myelin-suppression 85Figure 3-7 MAP-2 during developmental myelin-suppression 88Figure 3-8 Schematic representation of mechanism of developmental 91myelin-suppressionFigure 3-9 MBP mRNA during developmental myelin-suppression 96VIIIChapter 4Figure 4-1 Spinal cord transection control 115Figure 4-2 Confirmation of developmental myelin-suppression 117Figure 4-3 Neuroanatomical regeneration in the embryo 120Figure 4-4 Neuroanatomical regeneration with transection control 1 26Figure 4-5 Physiological recovery 131Chapter 5Figure 5-1 Immunological demyelination 151Figure 5-2 Immunological control injections 154Figure 5-3 Time course of immunological demyelination 157Figure 5-4 14-day immunological demyelination and remyelination 160Figure 5-5 GFAP during immunological demyelination 163Figure 5-6 Transection site 20 days post-transection 166Figure 5-7 Spinal cord transection control 168Figure 5-8 Neuroanatomical regeneration in the hatchling 172Figure 5-9 Neuroanatomical regeneration in hatchling- double labelling 178Figure 5-10 Physiological recovery- EMG 185Figure 5-1 1 Physiological recovery- ENG 187Figure 5-12 Immunological demyelination in mice 191Chapter 6Figure 6-1 Astrogliosis following hatchling spinal cord transection 205ixLIST OF ABBREVIATIONSbFGF Basic fibroblast growth factorCAM Cell adhesion moleculeCBDA Cascade blue labelled dextran amineCNP 2’,3’-cyclic nucleotide 3’-phosphodiesteraseCNS Central nervous systemE Embryonic dayEAE Experimental allergic encephalomyelitisECM Extracellular matrixEGF Epidermal growth factorEMG ElectromyogramENG ElectroneurogramFGF Fibroblast growth factorGalC GalactocerebrosideGFAP Glial fibrillary acidic proteinhr HourHSPG Herarin sulfate proteoglycanMAP-2 Microtubule-associated protein-2MBP Myelin basic proteinMAG Myelin-associated glycoproteinmRNA Messenger ribonucleic acidNCAM Neural cell adhesion moleculexNGF Nerve growth factorNTF Neurotrophic factor02A Oligodendrocyte, Type-2 AstrocyteP Posthatching dayPDGF Platelet derived growth factorPECT PectoralisPN Postnatal dayPNS Peripheral nervous systemRDA Rhodamine labelled dextran amineSART SartoriusTGF Transforming growth factorpm MicronxiACKNOWLEDGEMENTSI will begin by acknowledging three people whose influence led me toembark on the academic road that I find myself on. My father Kenneth EugeneKeirstead has shown me by example that a diligent work ethic unfailingly yeildsrewards. I have learnt to identify the price to pay for every action, and so doingam able to make new beginnings with an informed and realistic perspective. Mymother Sandra Marlene Keirstead has enforced in me a ‘geshtalt’ method ofviewing existing and foreseen situations. She has also provided unfailingemotional support for which I am eternally grateful. David John Roberts hasbeen a friend and inspiration to me from a very young age. I recognized manyyears ago that it would take more than a Ph.D. to equal his creative and activemind.I thank my wife Melanie Gaye ter Borg for all of the love, balance andsupport that she has provided. Melanie, you have shown me that it is possibleto enjoy a rich and diverse lifestyle, even during times of chaos and confusionat the workplace. Thank-you for making my life so much fun. John DouglasSteeves has taught me far more than research skills during my graduate years.You have been both mentor and friend. Thank-you for your wisdom andperspective.I would also like to thank Gillian Dawn Muir for her help with aspects ofmy thesis, but more importantly for making me smile so much at work. Gillian, itxl’has been nothing but fun working with you. Thanks also to Diane Henshel,David M. Pataky and Gerald Sholomenko for their advice and collaborativeefforts. This thesis would not have been possible without the technical supportof Karen Goh, John McGraw, Karin Mathias and Ania B. Wisniewska. I cannotthank these individuals enough for their commitment, interest and hard work.Finally, I would like to thank the Natural Sciences and EngineeringResearch Council of Canada and the Network of Centres of Excellence forNeural Regeneration and Functional Recovery for their scholarship support.1CHAPTER 1GENERAL INTRODUCTION2Traumatic injury to the central nervous system (CNS) sets into motion amyriad of intracellular and intercellular events involved in the immune response,cellular proliferation, axonal degeneration and, in few cases, neuronalregeneration. Successful neuronal regeneration requires the re-extension ofsevered axons, axonal pathfinding, synapse formation, and ultimately therestoration of physiological functions. Different types of neurons within andbetween species differ in their ability to regenerate following injury. Forexample, CNS neurons of invertebrates and lower vertebrates are able toregenerate axonal processes. Severe injury to the tectum of goldfish (Grant andKeating 1986), the spinal cord of goldfish (Bernstein 1964) and axolotl (Grimm1971; Holder et al. 1982), the retinotectal projections of fish and amphibians(Easter 1983) or the VIlIth cranial nerve of Rana pip/ens (Zakon and Capranica1 981) results in successful neuronal regeneration and restoration of function.The ability of CNS neurons to regenerate has been gradually lost over thecourse of evolution, such that regeneration is generally restricted to anamniotesin a state of continuous growth. Perhaps the best studied example in thiscontext is the regenerative ability of the optic nerve and tectal efferents of Ranapip/ens (Easter 1983). The retinotectal system of post-metamorphic Ranapip/ens undergoes continuous neurogenesis of retinal ganglion cells, followed byaxonal projection and functional synapse formation (Easter 1983). In contrast tothe dramatic regenerative ability of the Rana pip/ens optic nerve, tectal efferentsfail to regenerate following injury (Lyon and Stelznêr 1987). Neurogenesis in the3frog tectum stops at metamorphosis (Grant and Keating 1986). Thus, Ranapipiens may exist at an intermediate stage in the phylogenetic trend towards aloss of regenerative capabilities.The adult CNS of higher vertebrates has a very limited capacity forregeneration following injury. CNS regeneration in higher vertebrates isrestricted 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 theadult CNS results in traumatic degeneration of both the proximal and distalstumps of the severed axons (Ramon y Cajal 1959). Proximal stumps undergoan initial stage of retrograde degeneration (usually back to the nearest collateralbranch) followed by an aborted attempt at regeneration (growth cone formationand elongation of approximately 1-2mm; Cajal 1928). Due to a lack of trophicsupport, distal axonal stumps undergo Wallerian degeneration and are ultimatelyresorbed by glial cells (WaIler 1852; Ranvier 1871, 1873; Ramon y Cajal 1959).The lack of regeneration exhibited by adult CNS neurons of highervertebrates does not imply that these neurons lack the intrinsic capability toregenerate following injury, If peripheral nerve segments containing Schwanncells are grafted to a site of CNS injury, severed CNS axons are able to growout and make functional synaptic connections with their targets (Ramon y Cajal1959; David and Aguayo 1981; Aguayo et al. 1991). Similarly, axotomized CNSneurons will extend axons in vivo through implants of fetal CNS tissue4(Bjorklund and Stenevi 1979; Kromer et al. 1981; Bjorklund 1991), or implantsconsisting of fibroblasts genetically modified to express growth factors (Fisherand Gage 1993; Tuszynski et al. 1994). In addition, motor neurons residingwithin the ventral horn of the spinal cord are capable of regenerating theirperipheral projecting axon if the axotomy took place within the PNS (Ramon yCajal 1959). These findings indicate that CNS neurons retain intrinsic growthprograms which enable long-distance axonal regeneration in the presence of afavorable extraneuronal environment.The factors contributing to the poor regenerative capacity of the matureCNS of higher vertebrates can be divided into two general categories: 1) growthpromoting factors present in the developing system which are either absent oraberrently expressed following adult injury, and 2) growth inhibitory factorswhich are chronically expressed in the adult CNS or are upregulated followingadult injury.Growth Promoting FactorsGrowing, regenerating or mature axons in the adult state are constantlyexposed to an extremely complex molecular environment. Extracellular matrix(ECM) molecules facilitate cell attachment, cell migration and process extensionduring development and are in a position to intimately influence regeneratingaxons. Fibronectin is a widely distributed ECM component which ispredominantly expressed by radial glia during a limited period of neuritic growth5in development (Sheppard et al. 1991). Astrocytes, however, fail to upregulatefibronectin following injury to the adult CNS (Egan et al. 1991). Laminin is alsopresent in the developing ECM of virtually all higher vertebrates, providing asubstrate for cell migration and anchorage (Carbonetto 1984). However, likefibronectin, laminin does not appear to be detectably upregulated following CNSinjury (Sosale et al. 1988). Laminin upregulation has been observed followingoptic nerve transection, however, injury-induced sprouting was confined tolaminin(-) areas (Giftochristos and David 1988). Finally, heparin sulfateproteoglycan (HSPG; one of several proteoglycans which form the majorconstituents of CNS ECM) has been shown to promote cell attachment andspeading. Again, HSPG is only seen transiently in development during the periodof pattern formation and connectivity (Steindler et al. 1990).Cell adhesion molecules (CAMs) constitute another set of adhesiveproteins that are fundamental to the motility of the growth cone. CAMs aredivided into two groups, the Ca2- independent immunoglobulin superfamily andthe Ca2- dependent, homophilically-interacting cadherin family. Moleculesbelonging to the immunoglobulin superfamily share structural motifs referred toas immunoglobulin domains, each consisting of approximately 100 amino acidslooped by a disulfide bridge (Cunningham et al. 1987). Members of theimmunoglobulin superfamily include Li, Ng-CAM, contactin, Fl 1 /F3, Tag1/axonin-1, MAG,P0(Grumet 1991), Nr-CAM (Grumet et al. 1991), SC1/DM-GRASP (Tanaka et al. 1991) as well as the phylogenetically oldest and best-6characterized member, neural cell adhesion molecule (N-CAM; reviewed inCarbonetto and David 1993). N-CAM isoforms are expressed byoligodendrocytes (Bhat and Silberbeg 1986), astrocytes (Noble et al. 1985) andneurons (Nybroe et al. 1989) and bind in a homophilic or heterophilic manner tocomponents of the ECM including heparin sulfate proteoglycan (Cole andAkeson 1989). A threshold level of N-CAM expression is required before neuriteoutgrowth can occur; small increases in N-CAM levels beyond this thresholdresult in great increases in neurite outgrowth-promoting ability (Doherty et al.1990a). During development, however, the neurite outgrowth-promoting abilityof 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 beattributed to the reduction in polysialic acid content, which increases the avidityof N-CAM-N-CAM interactions (Doherty et al. 1990b). An advancing filopodiummust make contacts that are adhesive enough to support the contractile forcesthat 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 interactintracellularly with actin microfilaments and cytoplasmic proteins called catenins(McGee and Buxton 1991) and share several common structural featuresincluding Ca2- binding sites (Takeichi 1991). All of the various types ofcadherins interact homophilically with like cadherins on another cell to generate7adhesion (Takeichi 1991). Cadherins are expressed early in the developingnervous system (Takeichi 1991). CNS regeneration- or degeneration-inducedchanges in cadherin expression have yet to be documented.Integrins are a superfamily of cell-surface receptors for ECM moleculesthat are found in abundance on growth cones (Carbonetto 1984). lntegrins areheterodimers that share a common beta subunit and are classified into familiesaccording to their alpha subunit. The presence of appropriate ECM receptors isclearly a prerequisite for the advancement of developing or regeneratingneurites. Although integrins are found in many developing systems (Reichardtand Tomaselli 1991), they are functionally down-regulated during development,which may partially account for the reduced axonal regeneration seen in vivo asthe CNS matures.In order to facilitate axonal advancement, ECM degradation may beinitiated by proteases such as plasminogen activators (Pittman 1 985), proteasenexins (Rovelli et al. 1992) and metalloproteases (Machida 1991). Althoughproteases have been associated with axonal growth in many CNS areas (Sumiet al. 1992), an in vivo role can not be firmly established since studies ofproteases have generally been limited to culture assays. Nonetheless, it hasbeen suggested that proteases may facilitate axonal regeneration by degradingor altering the ECM in advance of the growth cone (Brodkey et al. 1993).Perhaps the most important group of molecules influencing CNSdevelopment and regeneration are neurotrophic factors (NTFs), or growth8factors. Over 40 years ago Hamburger and Levi-Montalcini proposed thatneurons are overproduced during development and, during maturation of thenervous system, compete for a target-derived factor or factors. Due to a limitedsupply of these NTFs, only a portion of the original population of neuronssurvive. Thus, it was postulated that specific NTFs promote the survival,differentiation and maturation of different populations of neurons indevelopment (Hamilton and Levi-Montalcini 1949). These early experimentsfacilitated the discovery and characterization of nerve growth factor (NGF),which has been shown to regulate the size of specific neuronal populationsduring 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 toNGF, while the other members of the neurotrophin family, brain-derivedneurotrophic factor (BDNF), neurotrophin (NT)-3 and NT-4/5 have morewidespread effects on central and peripheral neurons (Thoenen 1991). Forexample, NT-3 has been shown to prevent the death of noradrenergic neuronsof the locus coeruleus (Arenas and Persson 1994), enhance sprouting incorticospinal tract axons during development and after spinal cord injury(Schnell et al. 1 994), and play a role in the survival of spinal proprioceptiveafferents and their peripheral sense organs (muscle spindles and Golgi tendonorgans) in development (Ernfors et al. 1994).9Other NTFs not belonging to the neurotrophin family also influenceneuronal survival during development and after CNS injury. Transforminggrowth factor (TGF)-beta, platelet-derived growth factor (PDGF), fibroblastgrowth factor (FGF) and epidermal growth factor (EGF) have all been shown tohave 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 ofthe CNS as well, such as gliogenesis, the induction of macrophage motility,stimulation of angiogenic growth, or facilitation of a new glial limitans formationaround a site of CNS injury (Brodkey et al. 1993).NTF receptor distribution is also an important consideration in determiningthe 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 trkproto-oncogenes for signal transduction (Meakin and Shooter 1992). Clearly,attempts to manipulate the NTF environment of regenerating neurons must alsoconsider the expression of appropriate NTF receptors by the neuronal populationof interest.Growth Inhibitory FactorsThe growth of neurites to their targets during nervous systemdevelopment clearly requires the appropriate spacial and temporal expression ofadhesion molecules. While the adhesion molecules described above direct10neurite growth by enhancing cellular adhesion, several adhesion molecules havebeen described that direct neurite growth by providing a non-permissivesubstrate. Anti-adhesive molecules may increase the efficiency of surfacerecognition processes for directed neurite outgrowth.Injury to the CNS results in the local release of cytokines and NTFs frominjured neurons and glial cells (Hugh Perry et al. 1993; Nakajima and Kohsaka1993). These factors stimulate astrocytes directly or indirectly (via thestimulation of microglia) to assume a reactive state, characterized by theupregulation of glial fibrillary acidic protein and expression of several cell-surfacemolecules that are normally not expressed, or are expressed at low levels (Hertzet al. 1990; Reier and Houle 1988). Tenascin is one such molecule that isupregulated by astrocytes after injury (Brodkey et al. 1993). Tenascin isexpressed in the CNS during development (Chiquet 1989) and, as a result of itsanti-adhesive properties, may guide growing neurites by growth cone diversion(Martini and Schachner 1991). In stripe culture assays, neurons tend to avoidareas containing tenascin, preferring to grow on laminin, fibronectin or tenascinfree polyornithine (Faissner and Kruse 1990). Developing astrocytes alsoexpress several anti-adhesive proteoglycans including chondroitin sulfate,dermatan sulfate and keratan sulfate, which promote cell detachment andinhibition of attachment (Herndon et al. 1990). The neurite-inhibitory propertiesof chondroitin sulfate proteoglycan define functional boundaries duringdevelopment in the rodent somatosensory barrel field (Sheppard et al. 1991)11and the roof plate of the spinal cord (Snow et al. 1990). Lesions to the adultCNS result in an upregulation of proteoglycans (McKeon et al. 1991). Thus,injury to the adult CNS appears to induce astrocytes to re-express severaldevelopmentally-regulated anti-adhesive molecules including tenascin andvarious proteoglycans.Reactive astrocytes may also inhibit neurite growth through a CNS lesionsite by providing a physical barrier to growing neurites. Astrocytes undergoextensive proliferation and hypertrophy in and around a site of CNS injury (Hertzet al. 1990; Reier and Houle 1988). It has been proposed that the densenetwork of astocytes which forms at the lesion site provides a barrier throughwhich growing neurites are unable to navigate (reviewed in Reier and Houle1988). The complexly interdigitating astrocytic processes often exhibit anincreased number of junctional complexes, which may contribute to theformation of an impenetrable physical barrier to growing neurites. In thehibernating ground squirrel, however, axonal regeneration fails to occur despitethe lack of glial scar formation following spinal cord transection (Guth et al.1981).Oligodendrocytes and oligodendrocyte-produced myelin also expressneurite-inhibitory molecules. Myelination within the developing CNS generallytakes place after neuronal projections have reached their targets (Schreyer andJones 1982; Okado and Oppenheim 1985; Schneider et al. 1990; Keirstead etal. 1992; Jhaveri et al. 1992). It has thus been hypothesized that one of the12functions of myelin within the CNS is to stabilize newly-formed neuralconnections (Kapfhammer and Schwab 1994). One such molecule expressed bydeveloping and mature CNS oligodendrocytes is janusin, originally referred to asJ1-160/180 (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 NI-250 that account for much of the neurite-inhibitory activity in CNS myelin(Caroni and Schwab 1988). The identification of these inhibitors began with theobservation that neurons, astrocytes and fibroblasts in culture preferred to growon substrates other than oligodendrocytes and their radial, highly-branchedprocess networks (Schwab and Caroni 1988). In contrast, frozen sections fromneonatal rat spinal cords treated with mitotic inhibitors or X-irradiated (toprevent myelin formation) proved to be permissive substrates for growingneuroblastoma cells (Savio and Schwab 1989; Savio and Schwab 1990). TheCNS myelin inhibitory activity was largely recovered after protein separation inSDS-PAGE and reconstitution into liposomes, and was subsequently identifiedas 35 kDa (Nl-35) and 250 kDa (Nl-250) proteins (Bandtlow et al. 1990). Whenadded to favorable substates, these proteins inhibited neurite growth.Functionally-blocking monoclonal antibodies to these proteins (IN-35 and IN250) were then generated (Caroni and Schwab 1988). Application of theseantibodies (by implantation of antibody-secreting hybridoma cells near the lateralventrical) in rats with transections of the corticospinal tract resulted in limited13anatomical regrowth of axons approximately 5mm caudal to the lesion site 2-3weeks after injury (Schnell and Schwab 1990). Despite the limited regenerationobtained with this approach, these studies identified specific proteins in CNSmyelin 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. McKerracheret al. 1994). Experiments involving protein fractionation of bovine CNS myelinand in vitro testing of growth inhibitory activity have identified MAG as aninhibitor of process extention for differentiated PC12 cells. Furtherexperimentation, including the generation and use of functionally-blockingantibodies, is necessary to elucidate the relative contribution of MAG to theneurite-inhibitory activity of CNS myelin.Strategies to evoke substantial regeneration following injury to the CNSmust take into consideration the many growth-promoting and growth-inhibitingfactors that may influence a particular population of injured neurons. Precludingthe formulation of such strategies, however, is information regarding the relativecontribution of each of these factors to the ability of injured neurons toregenerate. Although neurite-inhibitory proteins have been identified in CNSmyelin, any in vivo attempts to neutralize the inhibitory activity have resulted invery limited regeneration (Schnell and Schwab 1990), possibly due to limitationsin the neutralization technique itself. The experiments outlined in this thesiswere designed to address the question: to what extent does CNS myelin14contribute to the lack of regeneration observed following spinal cord injury tothe late embryonic and hatchling chick?The chick offers several advantages as an experimental model. Thedevelopmental stages of the chicken Gallus domesticus have been thoroughlydocumented (Hamburger and Hamilton 1951). Embryonic surgery in vivo isdifficult to perform on mammalian embryos due to maternal complications,however, the avian egg provides for easy and uncomplicated access to theembryonic chick at any stage of development. In addition, the structure andfunction of the vertebrate brainstem and spinal cord has remained virtuallyunchanged throughout evolution (Sarnat and Netsky 1981). In particular, thebrainstem-spinal control of locomotion in birds very similar to that of othervertebrates, including mammals (Bekoff 1976; Okado and Oppenheim 1985;Sholomenko and Steeves 1987; Steeves et al. 1987; Webster and Steeves1988, 1991; Sholomenko et al. 1991a,b,c; Steeves and Jordan 1980;Armstrong 1986). Following injury to the adult spinal cord, birds suffer thesame 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 theability to recover from spinal cord injury is lost in development (Okado andOppenheim 1985; Hasan et al. 1991). Transections of the developing spinalcord early in embryonic development result in complete functional recoveryassessed after hatching. Transections of the spinal cord after embryonic day (E)1513 of the 21 day developmental period result in no anatomical or physiologicalrepair, rendering such an animal completely incapable of voluntary locomotionafter hatching. Thus, there appears to be a transition during development of theembryonic chick, from a permissive to a restrictive period for spinal cord repair.Investigations of extraneuronal changes during development that are coincidentwith the transition from permissive to restrictive repair periods may provideinsights into the lack of spinal cord repair following injury to the adult spinalcord.I began the studies outlined in this thesis with an investigation of myelindevelopment within the spinal cord of the chick. Histological andimmunohistochemical analysis of the developing chick spinal cord revealed thatthe developmental onset of myelination is coincident with the transition frompermissive to restrictive repair periods (chapter 2). This finding, taken togetherwith other studies outlined above indicating the neurite-inhibitory properties ofCNS myelin, suggested that myelin may inhibit the regeneration of transectedspinal cord in embryonic chick.In order to test this hypothesis, a method of delaying the developmentalonset of myelination in vivo was developed (chapter 3). Due to the presence ofseveral different putative neurite-inhibitors in CNS myelin (see above) a methodof complete myelin ablation was desirable. The studies outlined in chapter 3indicated that direct spinal cord injections of complement proteins pluscomplement-fixing antibodies to oligodendrocyte cell surface-specific antigens16resulted in a delay of the developmental onset of myelination until El 7. Thistechnique was also shown to be effective in suppressing the developmentalonset of myelination in the neonatal mouse spinal cord. Transections of thespinal cord during developmental myelin-suppression then serve as a direct testof whether myelin inhibits the regeneration of transected spinal cord inembryonic chick.Complete spinal cord transections as late as E15 in myelin-suppressedembryonic chicks resulted in complete neuroanatomical and physiological repair(Chapter 4). Normally-myelinated embryos transected on E15 exhibited noanatomical or physiological repair after hatching. These studies indicated thatmyelin inhibits the functional regeneration of transected spinal cord in -embryonic chicks and suggested that spinal cord myelin may likewise inhibitregeneration following injury to the adult spinal cord.In order to test the inhibitory properties of adult CNS myelin, a method oftransiently demyelinating the adult spinal cord was developed. The proceduredeveloped for myelin-suppression in the embryo was modified so as totransiently demyelinate the adult chick spinal cord over a 2-3 week period.Immunological demyelination was also shown to be effective in removing myelinfrom the mouse spinal cord, indicating that this procedure is not speciesrestricted. Complete transection of the hatchling chick spinal cord, followed bydemyelination over a 2-3 week period resulted in partial neuroanatomicalregeneration of axotomized fibres (chapter 5). Collaborative studies outlined in17Chapter 5 demonstrate that this neuroanatomical regeneration is accompaniedby functional synaptogenesis within the spinal cord. Brainstem stimulation oftransected and demyelinated hatchling chicks 2-4 weeks after surgery elicitedelectrical activity within the muscles of the legs. These studies indicate thatadult spinal cord myelin inhibits the regeneration of transected spinal cord in thehatchling chick. Furthermore, these findings constitute the first demonstration inhigher vertebrates of functional regeneration following spinal cord injury.In contrast to the complete regeneration observed in the myelinsuppressed embryo, transection and demyelination of the hatchling chick spinalcord elicited only partial regeneration. This observation suggests that otherfactors affecting regeneration, that may be present in the embryo, may beabsent or below threshold levels of effectiveness in the hatchling chick.Nevertheless, myelin appears to be a major factor affecting the ability of theembryonic and hatchling chick spinal cord to regenerate following injury.18CHAPTER 2MYELIN DEVELOPMENT IN THE EMBRYONIC CHICK SPINAL CORDÑ19INTRODUCTIONMyelin is a highly differentiated membrane structure produced byoligodendrocytes in the central nervous system (CNS). Although the chief roleof 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 ionchannel distribution in axons (Joe and Angellides 1992), restriction of targetarea innervation by developing neuronal projections (Schwab and Schnell 1991)and several CNS diseases including multiple sclerosis, optic neuritis andexperimental allergic encephalomyelitis (Quarles et al. 1989). Recently, CNSmyelin has been shown to inhibit the regeneration of transected spinal cordaxons in rat (Schnell and Schwab 1990) and embryonic chick (Keirstead et al.1992). Developmental studies of myelination have proven to be a useful meansof elucidating the various functions of myelin in the adult animal.Oligodendrocyte precursors originate from the ventricular zone of theneural tube, and express mRNA encoding the larger of the two isoforms of2’,3’-cyclic nucleotide 3’-phosphodiesterase (CNP; Yu et al. 1994; Schrerer etal. 1994). Oligodendrocyte precursors isolated from the optic nerve expressantigens labelled by GD3 and A2B5 antibodies (Raff 1989: Reynolds and Wilkin1988). Culture studies of oligodendrocyte progenitors isolated from the rat opticnerve have defined these cells as oligodendrocyte-type-2 astrocyte (02A)progenitor cells which differentiate in vitro into oligodendrocytes or type-2astrocytes, depending on the culture conditions (Raff 1989). When no serum is20provided in the culture medium, all 02A progenitor cells promptly differentiateinto oligodendrocytes. This constitutes the ‘default’ pathway of 02A progenitordifferentiation. When serum is included in the culture medium, the majority of02A progenitor cells differentiate into astrocytes. Ciliary neurotrophic factor hasbeen identified as the factor in astrocyte-conditioned medium that is responsiblefor inducing 02A progenitor cells to differentiate along the astrocyte lineage.Stable differentiation of astrocytes also requires an unidentified component ofthe extracellular matrix (reviewed in Williams and Price 1992). It should benoted, however, that several investigators have found no evidence of astrocytedifferentiation from freshly-dissociated 02A progenitor cells, or from animmortalized 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 intodemyelinated lesions of the rat spinal cord, blue X-gal staining of the spinal cordat various time points after injection revealed only ,8-galactosidase-positiveoligodendrocytes. No fl-galactosidase-positive astrocytes were identified. Thesefindings suggest that the differentiation of 02A progenitor cells into astrocytesis a phenomenon unique to in vitro experiments.Oligodendrocyte progenitors undergo a set number of cell divisions (about8) before differentiating into oligodendrocytes (Raff et al. 1985; Raff 1989).Platelet derived growth factor (PDGF) and basic fibroblast growth factor (bFGF)21have been identified as factors involved in the division of oligodendrocyteprogenitors prior to differentiation (reviewed in Goldman 1992). PDGFupregulates the expression of bFGF receptors on oligodendrocyte progenitors,inducing their responsiveness to the mitogenic effects of bFGF. It is theorizedthat an internal clock controls the timely differentiation of oligodendrocytes fromtheir progenitors in the presence of bFGF and PDGF (Raff et al. 1985). Thetiming mechanism of the internal clock may have two separate components; acounting component driven by mitogens (like bFGF), and an effector componentdriven by hydrophobic signalling molecules (like thyroid hormone, retinoic acidand glucocorticoids) that induce oligodendrocytes to withdraw from the cellcycle (Barres et al. 1994). Receptors for thyroid hormone, retinoic acid andglucocorticoids are able to inhibit the activity of AP-1 transcription factors,which are formed by the heterodimerization of Jun and Fos proteins andmediate the proliferative response to growth factors (Barres et al. 1994;Ransone and Verma 1990). As one might imagine, hyperthyroidism acceleratesthe deposition of myelin, whereas hypothyroidism delays it (Walters and Morell1981; Legrand 1986; Dussault and Ruel 1987). It is thus hypothesized thatoligodendrocyte mitogens induce cellular division, which decreases the AP-1activity with each division. When AP-1 activity drops below a threshold level,hydrophobic signals are then able to further reduce the activity below a levelrequired to keep the cell dividing, and division ceases.Subsequent to cessation of cellular proliferation, cellular differentiation22takes place (Barres et al. 1994). PDGF and bFGF are detectable in the rat,mouse and chick around the time of oligodendrocyte differentiation (Richardsonet 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 developmentalonset of oligodendrocyte differentiation (Kalcheim and Neufeld 1990).Furthermore, the rat thyroid gland becomes active around birth, and beginssecreting thyroid hormone at a time when oligodendrocytes first appear in theCNS (Samel 1968; Puymirat 1992). CNS myelination in the human also takesplace shortly after the thyroid gland becomes active, at about the 1 2th week ofgestation (Friede 1989).The ganglioside GM3 may also play a role in oligodendrocytedifferentiation. Differentiation of control cells in vitro is associated withincreased metabolic labelling of endogenous GM3, suggesting a role for GM3 asprecursors differentiate towards the more mature stages of myelin production(Vim et al. 1994). Furthermore, addition of exogenous GM3 to cultures enrichedfor 02A cells promotes differentiation through a pre-myelinating stage (labelledby 04 antibodies which react with sulfatide, seminolipid and an unidentifiedantigen on oligodendrocyte progenitors; Sommer and Schachner 1982) to amyelinating stage, characterized by process formation and expression of myelinrelated constituents such as GaIC, sulfatide and myelin-associated glycoprotein(Yim et al. 1994). Gangliosides have been shown to promote or inhibit thephosphorylation of many neural proteins including myelin basic protein23(Goldenring et al. 1985; Kim et al. 1986). Because these myelin-relatedconstituents are expressed in the later stages of 02A differentiation, the precisestage(s) at which GM3 may exert its effects are unknown.Concurrent with the differentiation of oligodendrocytes fromoligodendrocyte progenitors is the expression of galactocerebroside (GaIC) bynewly differentiated oligodendrocytes (Benjamins et al. 1987). Newlydifferentiated pre-myelinating oligodendrocytes also begin to express mRNAencoding 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 andCNP have not been elucidated. CNP immunoreactivity is found in thecytoplasmic compartments of the oligodendrocyte, mainly in the soma andparanodal loops of the myelin sheaths (Braun et al. 1988). Although extractedCNP 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 thelipid membranes of the oligodendrocyte (Benjamins et al. 1987). GalC is linkedto the cytoskeleton and may play a role in transmembrane signalling (Benjaminsand Dyer 1990).Pre-myelinating oligodendrocytes and their progenitors are highlymigratory (Small et al. 1987). Early in development these cells migrate fromtheir place of origin in the subventricular zone and infiltrate the fasciculatedaxonal tracts of the CNS where they differentiate into oligodendrocytes (Warf et24al. 1991; NolI and Miller 1993; Yu et al. 1994). The signal for these newly-differentiated oligodendrocytes to begin myelination of axons has not beendetermined, although axonal contact has been implicated (Doyle and Colman1993). Cyclic AMP has been implicated in the activation of myelin genes in botholigodendrocytes and Schwann cells (Goda et al. 1989; Monuki et al. 1989).The O4, GalC oligodendrocyte begins myelination by sending out a processthat contacts the axon then begins wrapping around the axon. Myelinassociated glycoprotein (MAG) may be involved in the adhesive interactionsbetween the oligodendrocyte process and the axonal membrane (reviewed inQuarles 1988). MAG levels are high during the initial stages of myelination anddecrease considerably thereafter. In the mature CNS, MAG immunoreactivity isrestricted 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, twomajor myelin proteins are expressed that play a significant role in thecompaction process. Myelin basic protein (MBP) mRNA is translated within thecell body and oligodendrocyte processes and incorporated into the myelinmembranes on the cytoplasmic surface (reviewed in Mikoshiba et al. 1991).During the compaction process, homophilic interactions of MBP bring thecytoplasmic surfaces of the myelin membranes together, resulting in theextrusion of the cytoplasm and the formation of the major dense line. In crosssection, the major dense line appears under electron microscopy as a dark spiral25originating at the axonal surface. Proteolipid protein (PLP) appears just prior tocompaction as a transmembrane protein with a large extracellular domain on thesurface of the myelin membrane (reviewed in Mikoshiba et al. 1991). Duringcompaction, the extracellular surfaces are brought together as facing PLPextracellular domains homophilically interact. This results in the formation of theintraperiod line which is seen under electron microscopy as a thin line spirallingbetween the major dense lines.The mature myelin sheath is interrupted at approximately 1-2 mmintervals along the length of the axon at nodes of Ranvier. A singleoligodendrocyte may send out myelinating processes to 30 or more internodalsegments on neighboring axons. The compacted myelin sheath increases themembrane resistance and decreases the membrane capacitance of the axon(reviewed in Morell et al. 1989). These two features of myelinated axons resultin less decremental decay of the electrical charge as it travels between theNodes of Ranvier. The myelin sheath may also play a role in determining thedistribution of voltage-gated sodium channels along the length of the axon.Although direct causation has not been convincingly demonstrated, myelinatedaxons consistantly display a characteristic sodium channel distribution quitedifferent from that of an unmyelinated axon. Voltage-gated sodium channels areclustered around the nodes of Ranvier and the adjacent regions under theparanodal loops of the myelin sheath. Voltage-gated sodium channels are absentor very sparse in the internodal segments of the myelinated axon. These26properties of myelinated axons result in super-threshold stimulation of the actionpotential only at the nodes of Ranvier, and highly efficient conduction of theresulting electrical signal within the myelinated internodal segment. Thisphenomenon is known as ‘saltatory conduction’.Saltatory conduction is disturbed in nervous system diseases whichinvolve 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 displayingabnormally large numbers of inflammatory cells (T-cells, B-cells, monocytes andmacrophages/microglia). Physiological deficits during periods of exacerbations ofthe disease are related to the CNS area displaying the demyelinated lesion.Analysis of the cerebrospinal fluid (CSF) of MS patients also reveal ‘oligoclonalbanding’, which indicates an abnormally high concentration of antibodies to oneor very few unidentified antigens. Although an immunological regulatorymalfunction is clearly present in MS, it has not been determined whether theautoimmunity is causitive or correlative. Both genetic and environmental factorshave been implicated in triggering the disease (reviewed in livanainen 1981). Apopular animal model of MS is the experimental allergic encephalomyelitis (EAE)mouse (reviewed in Quarles et al. 1989). This pathogenic condition is induciblein several species by the injection of CNS tissue or MBP into the circulatorysystem. The presence of myelin antigens in the bloodstream induces anautoimmune reaction which results in the formation of demyelinated lesions27within the CNS. Interestingly, neonatal exposure to MBP protects mice fromdeveloping EAE when challenged as an adult (Adorini et at. 1990). In such ananimal, the developing thymus of the neonatal mouse generates T-cells whichrecognize the MBP antigen as ‘self’.More recently, myelin has been shown to display an activity whichinhibits the growth and regeneration of neurites. Growing neurites in culture willavoid white matter when an alternative substrate is available (Schwab andCaroni 1988). When plated directly onto CNS white matter, cells fail to extendprocesses beyond a few millimeters. This inhibitory activity has been isolatedand found to reside in 35kD and 250kD proteins isolated from rat CNS whitematter (Bandtlow et al. 1990). Antibodies to these inhibitory proteins have beengenerated and, when administered to the corticospinal lesioned rat, facilitatepartial anatomical regeneration of corticospinal axons (Schnell and Schwab1990). Although no functional repair follows such an intervention, this studyclearly demonstrates that CNS myelin contains a component that inhibits theregeneration of axotomized corticospinal fibres. Furthermore, myelin-associatedinhibitors have been suggested to play a developmental role in axonalpathfinding (Schwab and Schnell 1991). The following chapters of this thesisalso describe the inhibitory properties of myelin for the regeneration ofaxotomized brainstem-spinal projections (also see Keirstead et al. 1992).Recognizing that myelin may be inhibitory to the growth and regenerationof spinal cord axons, I asked the question; when does the process of28oligodendrocyte differentiation and myelin development occur relative to axonaldevelopment in the chick spinal cord? Neurogenesis of brainstem-spinalprojecting neurons is complete by embryonic day (E)3- E5 (McConnell andSechrist 1980), and brainstem-spinal projections controlling locomotion arecomplete to all levels of the spinal cord by E12 (Okado and Oppenheim 1985). Ihypothesized that myelination of the spinal cord would begin after neuronaldevelopment and axonal projections were complete. In order to determine thedevelopmental onset of spinal cord myelination, I conducted a series ofimmunohistochemical investigations using antibodies to various myelin-specificantigens including GalC, CNP, MBP and MAG (described above).29MATERIALS AND METHODSFertilized White Leghorn eggs were incubated at 37°C in an automaticrotating incubator. Prior to surgery or sacrifice, all eggs were developmentallystaged using accepted protocols (Hamburger and Hamilton 1951).Paraffin EmbeddingAnimals were perfused intracardially at the appropriate developmentalstage (see results) with O.1M phosphate buffered saline (PBS) containing 2500USP units of heparin per 50 mIs PBS, pH 7.4 (at 37°C) followed by perfusionwith 4% paraformaldehyde in 0.1M phosphate buffer, pH 7.4 (at 4-10°C). Thedissected spinal cords were then immersed in the same fixative for 24hrs at 4°Cand subsequently returned to PBS pH 7.4 (at 4-10°C) until further processing. Inpreparation for paraffin embedding, the spinal cords were immersed in 50%ethanol for two hours then transferred to 70% ethanol where they remainedovernight. The following morning the spinal cords were transferred toembedding cartridges and placed in an automated histomatic tissue processor.The histomatic tissue processor was adjusted so as to sequentially expose thetissue 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 undervacuum, and a final 60 minutes paraffin at 50-60°C under vacuum. The tissuewas then transferred from embedding cartridges to embedding blocks andsurrounded by hot paraffin which was then allowed to cool and harden. Theparaffin blocked tissue was cut on a Leitz microtome into 10pm parasagittal30sections and mounted on gelatin-coated slides for immunohistochemicalprocessing.ImmunohistochemistryAntigens were localized using indirect immunofluorescence.Galactocerebroside immunohistochemistry was performed Ofl Gyrostat sectionedtissue that was perfused as outlined above. All other antigens were localized onparaffin-embedded tissue sections. The paraffin-embedded tissue sections weresequentially immersed in two, 60 second xylene baths to remove the paraffinthen brought up to 100% hydration with two minute immersions in 100%ethanol, 95% ethanol, 70% ethanol, 50% ethanol and a five minute immersionin PBS pH 7.4. In order to block non-specific background staining, excess PBSwas 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 fluidwas 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-X100) was applied (200-400 p1 per slide) and incubated for 3 days at 4 degreesCelcius. After this 3 day incubation period, excess primary antibody wasremoved with 3 washes of 5 minutes each in 1 % goat serum in PBS containing3% Triton-X 100. The secondary antibody (diluted in 1% goat serum in PBScontaining 3% Triton-X 100) was applied (200-400 p1 per slide) and incubatedfor one hour at room temperature. Slides were rinsed in PBS and fitted withmicroscope cover glass in 1:2 PBS, glycerol prior to microscopic analysis. For31long-term storage, the cover glass was sealed with a small amount of nail polisharound the perimeter.The rabbit anti-human myelin basic protein antibody (MBP; AccurateChemical Scientific Corp., #AXL746), the rabbit anti-cow glial fibrillary acidicprotein (GFAP; Dakopatts Corp., #Z334) and the mouse anti-bovine microtubuleassociated protein- 2 (MAP-2; Amersham #RPN1194) were all used at a dilutionof 1:100 in 1% goat serum in PBS containing 3% Triton-X 100. The mouseanti-bovine Myelin Associated Glycoprotein (MAG; Boehringer Mannheim #1 450972) and the rabbit anti-bovine 2’,3’- Cyclic Nucleotide 3’- Phosphodiesterase(CNP; a gift from Peter Braun, McGill University, Canada) were used at adilution of 1:500 in 1% goat serum in PBS containing 3% Triton-X 100. Therabbit anti-bovine galactocerebroside antibody (Chemicon AB 142) was used ata dilution of 1:10. The secondary antibodies were either goat anti-rabbit FITCconjugated immunoglobulin (Caltag Laboratories, #L42001) diluted 1:100 in 1%goat serum in PBS containing 3% Triton-X 100 or a goat anti- mouse FITCconjugated 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/orsecondary antibodies) were processed alongside tissue sections fromexperimental and control animals. Pre-absorption controls were conducted bythe respective supplier of each antibody. Photomicrographs were taken on aZeiss Axiophot using epifluorescent illumination with the appropriate filters.32Histological StainingAnimals designated for toluidine blue staining were perfused intracardiallyat the appropriate developmental stage (see results) with 0.1M phosphatebuffered saline (PBS) containing 2500 USP units of heparin per 50 mIs PBS, pH7.4 (at 37°C) followed by perfusion with 2.5% glutaraldehyde in O.1Mphosphate buffer, pH 7.4 (at 4-10°C). The dissected tissue was rinsed andstored in 0.1M Na-cacodylate buffer (pH 7.4) overnight. The next morning thetissue 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 withdistilled water and sequentially exposed to 70% ethanol, 85% ethanol, 95%ethanol, 100% ethanol and a final 100% ethanol for 10 minute intervals on arotator. After a 30 minute exposure to propylene oxide on a rotator, the tissueplaced on the rotator for one hour while exposed to 1:1 propylene oxide andSpurr resin. The tissue was exposed to 100% Spurr resin on a rotator fortwenty-four hours, then placed in labelled block moulds with fresh Spurr resinand incubated at 60 degrees Celcius for 16 hours.The hardened blocks were cut into 1pm transverse sections on an ultramicrotome and placed on glass slides for toluidine blue staining. 0.1% toluidineblue stain was filtered and dropped onto sections on a hot plate. The toluidineblue was left on the sections for one or two minutes, long enough for metallicrings to form around the drying toluidine blue drops. The slides were then rinsedwith hot tap water followed by distilled water, then placed on a hot plate to dry.Photomicrographs were taken on a Zeiss Axiophot microscope.3334RESULTSGalactocerebroside (GaIC) immunoreactivity was not detected at any levelof 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 6animals sacrificed on ElO (Fig. 2-1B). GaIC immunoreactivity in these animalswas punctate, and was most often located in the lateral-most regions of thewhite matter tracts. In 3 of 4 animals analysed on El 1, GalC immunoreactivitywas similarly organized. GaIC immunoreactivity was consistently more abundantand 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 myelinationis complete by the time of hatching (n=6 for each day; Fig. 2-iC)2’,3’- cyclic nucleotide 3’- phosphodiesterase (CNP) immunoreactivitywas 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 thespinal cord on Ei2.5 in 6 animals (Fig. 2-28). CNP immunoreactivity wasalways 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 spinalcords were quantitatively similar, suggesting that myelination is complete by thetime 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 (635Figure 2-1. Developmental pattern of galactocerebroside (GaIC)immunoreactivity in the chick spinal cord. A: GaIC immunofluorescence stainingof unoperated control embryonic day (E) 9 thoracic spinal cord in parasaggitalsection showing no GaIC immunoreactivity. B: GalC immunofluorescencestaining of unoperated control ElO thoracic spinal cord in parasaggital sectionshowing the developmental onset of GaIC immunoreactivity. C: GalCimmunofluorescence staining of unoperated control posthatching day (P) 7thoracic spinal cord in parasaggital section showing the hatchling pattern ofGaIC immunoreactivity. In all photographs the outer edge of the spinal cord lieson the right hand side, with the white matter adjacent. (Bars = lOOum).(J%J37Figure 2-2. Developmental pattern of 2’,3’-cyclic nucleotide 3’-.phosphodiesterase (CNP) immunoreactivity in the chick spinal cord. A: CNPimmunofluorescence staining of unoperated control embryonic day (E) 11thoracic spinal cord in parasaggital section showing no CNP immunoreactivity.B: CNP immunofluorescence staining of unoperated control E12.5 thoracicspinal cord in parasaggital section showing the developmental onset of CNPimmunoreactivity. C: CNP immunofluorescence staining of unoperated controlposthatching day (P) 5 thoracic spinal cord in parasaggital section showing thehatchling pattern of CNP immunoreactivity. In all photographs the outer edge ofthe spinal cord lies on the right hand side, with the white matter adjacent. (Bars= lOOum).CL)GD39animals tested with MBP antibody), ElO (6 animals tested with MBP antibody,4animals tested with MAG antibody), Eli (12 animals tested with MBPantibody, 4 animals tested with MAG antibody) or Ei2 (12 animals tested withMBP antibody, 4 animals tested with MAG antibody; Fig. 2-3A; Fig. 2-4A). MBPand MAG immunoreactivity first appeared in the ventrolateral funiculi of thecervical spinal cord on E13 and appeared to proceed in a ventro-dorsal androstral-caudal direction with development (14 animals tested with MBP antibodyand 6 animals tested with MAG antibody; Fig.2-3B; Fig. 2-4B). Comparison ofage-mached El 3 embryos revealed that CNP immunoreactivity was consistentlymore abundant than MBP or MAG immunoreactivity. On E14 all levels of thespinal cord displayed MBP, MAG and CNP immunoreactivity with the ventralfuniculi markedly more dense than other areas (18 animals tested with MBPantibody, 12 animals tested with MAG antibody and 8 animals tested with CNPantibody). By E15 a dense network of immunoreactivity was observed within allspinal cord white matter (28 animals tested with MBP antibody, 8 animalstested with MAG antibody and 8 animals tested with CNP antibody). A dramaticincrease in MBP and MAG immunoreactivity was observed in animals sacrificedon E17 and El8, suggesting a second wave of myelination at this stage ofdevelopment (18 animals tested with MBP antibody for each day, 6 animalstested with MAG antibody for each day). MBP and MAG immunostaining of P1,P3, P5, P7 and PlO spinal cords were qualitatively similar, suggesting thatmyelination is complete by the time of hatching (n=6 for each day, for each of40Figure 2-3. Developmental pattern of myelin basic protein (MBP)immunoreactivity in the chick spinal cord. A: MBP immunofluorescence stainingof unoperated control embryonic day (E) 1 2 thoracic spinal cord in parasaggitalsection showing no MBP immunoreactivity. B: MBP immunofluorescencestaining of unoperated control E13 thoracic spinal cord in parasaggital sectionshowing the developmental onset of MBP immunoreactivity. C: MBPimmunofluorescence staining of unoperated control posthatching day (P) 7thoracic spinal cord in parasaggital section showing the hatchling pattern ofMBP immunoreactivity. In all photographs the outer edge of the spinal cord lieson the right hand side, with the white matter adjacent. (Bars = lOOum).42Figure 2-4. Developmental pattern of myelin-associated glycoprotein (MAG)immunoreactivity in the chick spinal cord. A: MAG immunofluorescence stainingof unoperated control embryonic day (E) 12 thoracic spinal cord in parasaggitalsection showing no MAG immunoreactivity. B: MAG immunofluorescencestaining of unoperated control E13 thoracic spinal cord in parasaggital sectionshowing the developmental onset of MAG immunoreactivity. C: MAGimmunofluorescence staining of unoperated control posthatching day (P) 7thoracic spinal cord in parasaggital section showing the hatchling pattern ofMAG immunoreactivity. In all photographs the outer edge of the spinal cord lieson the right hand side, with the white matter adjacent. (Bars = lOOum).44MBP and MAG; Fig. 2-3C; Fig. 2-4C).Toluidine Blue staining of plastic embedded tissue on E15 revealed denseareas of myelination in all regions of the spinal cord white matter (n=4). At thisstage of development the toluidine blue stained myelin profiles appeared to betightly compacted (Fig. 2-5).In all of the immunohistochemical analyses, myelin-specificimmunoreactivity (for CNP, MBP and MAG) within the spinal cord gray matterwas dramatically reduced compared to myelin-specific immunoreactivity withinthe spinal cord white matter. Only a few sparse fibres were detected in embryosanalysed prior to E15. After E15, all animals consistently displayed myelinspecific immunoreactivity within their spinal cord gray matter. Myelin-specificimmunoreactivity in the adult spinal cord gray matter was consistently denserthan in the embryonic spinal cord gray matter.Glial fibrillary acidic protein (GFAP) immunoreactivity was not detected atany 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 firstdetected in 4 of 6 animals sacrificed on E12 and was almost exclusivelylocalized in the white matter (Fig. 2-6B). On E13 and E14 GFAPimmunoreactivity was evident in both the white and gray matter (n =6 foreach), however, the white matter was far more immunoreactive. GFAPimmunoreactivity increased noticably in both the white and gray matter on El 5,relative to the levels of immunoreactivity in younger animals (n=6). After45Figure 2-5. Toluidine Blue staining of unoperated control embryonic day (E) 15chick spinal cord white matter in transverse section. Notice the denselycompacted profiles of myelinated axons (dark rings; Bars = 25um).47Figure 2-6. Developmental pattern of glial fibrillary acidic protein (GFAP)immunoreactivity in the chick spinal cord. A: GFAP immunofluorescencestaining of unoperated control embryonic day (E) 11 thoracic spinal cord inparasaggital section showing no GFAP immunoreactivity. B: GFAPimmunofluorescence staining of unoperated control E12 thoracic spinal cord inparasaggital section showing the developmental onset of GFAPimmunoreactivity. C: GFAP immunofluorescence staining of unoperated controlposthatching day (P) 5 thoracic spinal cord in parasaggital section showing thehatchling pattern of GFAP immunoreactivity. In all photographs the outer edgeof the spinal cord lies on the right hand side, with the white matter adjacent.(Bars = lOOum). -49hatching, GFAP immunoreactivity increased dramatically. Spinal cords fromhatchlings 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 intenseGFAP immunoreactivity in both the white and gray matter (Fig. 2-6C). Again,the level of GFAP immunoreactivity was greater in the white matter than thegray matter.50DISCUSSIONOligodendrocytes differentiate 2-3 days prior to the developmental onsetof myelination in the embryonic chick spinal cord (Benstead et al. 1957). In vitroexperiments have indicated that the oligodendrocyte-specific expression ofgalactocerebroside (GaIC) is concurrent with the differentiation ofoligodendrocytes from oligodendrocyte progenitors (Benjamins et al. 1987). Thedevelopmental expression of GalC observed in the present in vivo studysupports these findings. GaIC immunoreactivity was first detected in theembryonic chick spinal cord on embryonic day (E) 10, 3 days prior to thedevelopmental onset of spinal cord myelination (described below; Fig. 2-1). Thissuggests that pre-myelinating oligodendrocytes are present within theembryonic chick spinal cord white matter approximately 3 days prior to thedevelopmental onset of myelination.Oligodendrocyte-specific 2’,3’- cyclic nucleotide 3’- phosphodiesterase(CNP) has also been reported to be expressed by pre-myelinatingoligodendrocytes and/or oligodendrocyte progenitors (Braun et al. 1988). It isinteresting in this regard that I observed the developmental onset of CNPimmunoreactivity in the embryonic chick spinal cord on E12.5 (Fig. 2-2), justprior to the onset of myelin basic protein (MBP) and myelin-associatedglycoprotein (MAG) immunoreactivity on E13 (described below). These findingsdo not concur with previous histological studies which indicate that premyelinating oligodendrocytes or oligodendrocyte progenitors appear 2-3 days51prior to myelination (Benstead et al. 1957), nor do they concur with the GaICdevelopmental study outlined above, which indicates that pre-myelinatingoligodendrocytes are present as early as ElO. This discrepency may reflect alow sensitivity of the CNP antibody used in this study. Paraffin processing mayhave additionally contributed to a decreased immunoreactivity of the CNPantigen. Alternatively, oligodendroglial development may be characterized by aGaIC, CNP stage, followed by a GaIC, CNP stage which briefly precedesmyelination.Myelination in the developing chick spinal cord begins in the ventrolateralfuniculi of the spinal cord on E13 and proceeds in a rostral-caudal direction withdevelopment. During the early stages of myelination, MBP, MAG or CNPimmunoreactivity was consistently more abundant in the ventrolateral funiculithan 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 tightlycompacted myelinated axonal profiles (Fig. 2-5). These results are in agreementwith observations of myelin development in the rat and mouse spinal cord,although myelination in these animals begins much later in embryonicdevelopment (Foran and Peterson 1992). By E17, all levels of the myelinatingchick spinal cord showed similar levels of MBP, MAG or CNP immunoreactivity.On E17 a dramatic increase in MBP and MAG immunoreactivity occured in alllevels of the myelinating spinal cord. This second wave of myelination may bedue to an increase in MBP and MAG incorporation into the myelin processes,52extention of myelin processes on or around this stage of development or to anongoing increase in oligodendrocyte number in the early myelinating spinal cordand a corresponding increase in the number of myelin processes at this stage ofmyelination.The onset of myelination in the developing chick spinal cord beginsfollowing the completion of axonal projections from the brainstem to the lumbarlevels of the spinal cord. Brainstem-spinal projections are complete to thecervical 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 theventrolateral funiculi shortly afterwards on E13. The ventrolateral funiculisurround the ventral horns, which are the site of spinal cord motorneurons.Interestingly, E13 also marks the onset of kicking movements of developingembryos. Thus, the developmental onset of spinal cord myelination occursshortly after brainstem-spinal neurons have completed their projections andhave made functional synaptic connections with their targets. The developmentof the corticospinal tract of the rat also follows this temporal sequence. The ratcorticospinal tract is a late-developing tract that begins descending from thebrainstem only after the other tracts of the spinal cord have completed theirdevelopment and have been myelinated (Schreyer and Jones 1982). Myelinationof the corticospinal tract begins only after axonal projections are complete andfunctional connections have been formed.The onset of myelination in the developing chick spinal cord may be53under the control of astrocytes within the spinal cord. In vitro experiments havedemonstrated that oligodendrocyte differentiation is under the control of at leasttwo growth factors, basic fibrillary growth factor (bFGF) and platelet-derivedgrowth 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 beforedifferentiating into oligodendrocytes (Raff et al. 1985; Raff 1989). bFGF alone isa potent mitogen for dividing oligodendrocyte progenitors. PDGF upregulates theexpression of bFGF receptors on oligodendrocyte progenitors, increasing theirsensitivity to this potent mitogen. It is theorized that an internal clock within theoligodend rocyte progenitor controls their timely differentiation intooligodendrocytes (Raff et al. 1985). Oligodendrocyte progenitors may receive anadditional astrocyte-derived signal which over-rides the mitogenic effects ofbFGF and PDGF and prompts oligodendrocyte progenitors to differentiate (SamDavid, McGill University, Canada; unpublished observations). Astrocyteconditioned medium has recently been shown to overcome the antidifferentiative effects of bFGF and PDGF when applied to dividingoligodendrocyte progenitors in culture. When astrocyte-conditioned medium isapplied to bFGF- and PDGF-supplemented cultures of dividing oligodendrocyteprogenitors, the cells promptly exit the cell cycle and differentiate intooligodendrocytes. It is interesting in this regard that I observed that the onset ofastrocyte-specific glial fibrillary acidic protein (GFAP) immunoreactivity occurs54on El 2, just prior to the onset of myelination. Given the results of these twostudies, it is conceivable that astrocytes may directly induce oligodendrocytedifferentiation in the developing chick spinal cord. In the rodent spinal cord,astrocytes appear several days prior to the appearance of oligodendrocytes (Raffet al. 1984). Perhaps the putative astrocyte-derived factor influencingoligodendrocyte differentiation in the rat is present, or reaches threshold levelsof effectiveness, around the time of oligodendrocyte differentiation.The onset of myelination in the developing chick spinal cord is coincidentwith the loss of regenerative capacity of spinal cord axons (Keirstead et al.1992). Complete transection of the embryonic chick spinal cord prior to thedevelopmental onset of myelination at E13, results in complete anatomical andphysiological repair resulting in 100% functional recovery of locomotor activity(Shimizu et al. 1990; Hasan et al. 1991). Double-labelling studies haveillustrated 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 repairextends 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 cordrepair is coincident with the developmental onset of myelination within theembryonic chick spinal cord (Benstead et al. 1957; Hartman et al. 1979;MackIm and Weill 1985; Keirstead et al. 1992). These findings suggest thatmyelin may be inhibitory to the regeneration of transected spinal cord axons,and prompted the studies outlined in the remainder of this thesis.5555oCHAPTER 3DEVELOPMENTAL MYELIN-SUPPRESSION IN THE EMBRYONIC CHICK SPINALCORD56INTRODUCTIONStudies of the regenerative capacity of the developing chick spinal cordhave determined that the spinal cord looses the ability to functionally regeneratefollowing complete transection on or about embryonic day (E) 13 of the 21 daydevelopmental period prior to hatching (Hasan et al. 1991). Transections of thespinal cord after E13 result in little or no neuroanatomical regeneration orfunctional recovery, rendering the animal completely incapable of voluntarylocomotion after hatching (Shimizu et al. 1990; Hasan et al. 1991). Chapter 2of this thesis outlines a series of experiments which led to the observation thatthe developmental onset of myelination in the embryonic chick spinal cordbegins on E13, coincident with the transition from a permissive to a restrictiveperiod for spinal cord repair. This observation suggested that myelin may beinhibitory to the functional repair of transected spinal cord in embryonic chick.In order to test this hypothesis, it was necessary to devise a method ofdelaying the onset of myelination (myelin-suppression) until later stages ofdevelopment (well into the restrictive period for repair). A subsequenttransection 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 adirect test of whether myelin is inhibitory to the functional repair of transectedspinal cord in embryonic chick.This chapter discusses the development and characterization of a57protocol effective in delaying the developmental onset of myelination in theembryonic chick spinal cord.Several mouse models have been developed that show characteristics ofabnormal myelin development. The jimpy mouse is a neurological mutantcharacterized by a deficiency of oligodendrocytes and myelin in the CNS(Sidman et al. 1964). All major components of myelin are reduced in jimpy miceincluding myelin basic protein (MBP; Matthieu et al. 1973), galactolipids(Nussbaum et al. 1969), myelin-associated glycoprotein (MAG; Yanagisawa andQuarles 1986), 2’,3’- cyclic nucleotide 3’- phosphodiesterase (CNP; Sarlieve1976) and proteolipid protein (PLP; Yanagisawa and Quarles 1986). Mutation ofthe PLP gene has subsequently been shown to be responsible for the jimpymutation (Spreyer et al. 1993). The number of oligodendrocytes generated injimpy mice increases over the first two weeks postnatally, and is comparable tothe number of oligodendrocytes generated by normal mice (Ghandour and Skoff1988). 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 reducedcompared to normal mice and, additionally, cannot be maintained in culture aslong as oligodendrocytes from normal mouse brains (Privat et al. 1972). Besidesshowing abnormally high levels of cell death, young jimpy oligodendrocytes aregenerally more highly branched than normal oligodendrocytes (Ghandour andSkoff 1988). This stellate characteristic of young jimpy oligodendrocytes may58adversely affect early oligodendrocyte-neuron interactions leading to decreasedmyelination. In the neonatal jimpy brain, oligodendrocytes often display apolarized morphology, with thicker and stubbier processes as compared tonormal oligodendrocytes (Ghandour and Skoff 1988). Astrocytes are alsoabnormal in jimpy mice, displaying characteristics of a chronic reactive state(Dupouey et al. 1080). Jimpy mice usually survive for a maximum of 2 weekspostnatally (Sidman et al. 1964). Due to these various complications, the jimpyphenotype is an unsuitable model for studies of CNS injury and repair.A dysmyelinated transgenic mouse line termed ‘wonky’ has also beendeveloped, resulting from expression of class 1 histocompatibility molecules inoligodendrocytes (Turnley et al. 1991). Oligodendrocytes do not normallyexpress class 1 histocompatibility molecules (Traugott 1 987; Bartlett et al.1989). Although the mechanism of cellular destruction associated with aberrantMHC molecule expression is unclear, overexpression of class 1histocompatibility molecules in other cell types (eg. pancreatic B cells) leads tononimmune destruction on the cells (Allison et al. 1988). DNA consisting of themurine class 1 H2Kb protein-coding sequences of the genomic clone linked tothe Hind 1 11 fragment of the murine MBP promoter, when microinjected intofertilized eggs, resulted in the ‘wonky’ phenotype (Turnley et al. 1991). Wonkymice are characterized by hypomyelination in the CNS, and exhibit neurologicalsymptoms at 11-14 days of age, just after the onset of myelination in normalmice. Homozygous transgenic mice have a shivering phenotype during59locomoter activity and develop tonic seizures which lead to death at 15-22days. The number of astrocytes is also increased in wonky mice, which mayindicate reactive gliosis. Due to the short life span of the wonky phenotype, thismodel is not suitable for studies of CNS injury and repair.Experimental methods of myelin-suppression in vivo include X-rayirradiation (Blakemore 1977), drugs (Blakemore 1978: Ludwin 1978: Eames etal. 1977), viruses (DalCanto and Lipton 1980; Herndon et al. 1977), nervecompression (Bunge et al. 1961; Clifford-Jones et al. 1980) and cell-mediatedimmunological reactions (Lampert 1968; Raine and Bornstein 1970). Althougheffective in suppressing myelin, none of these methods have demonstrated thecell selectivity and reparative benefits observed in the use of developmentalmyelin suppression.Several in vitro studies have described inhibition of myelin formationmediated by anti-galactocerebroside antibodies or sera from rabbits innoculatedwith whole CNS tissue (Bornstein and Raine 1970; Dorfman et aI.1979; Ranschtet al. 1987). Bornstein and Raine report myelin inhibition via complementmediated cytotoxicity. In this study, anti-CNS anti-serum inhibitedoligodendrocyte differentiation and myelin formation in rat spinal cord cultures(Bornstein and Raine 1970). Exposure of neonatal rat cerebellum cultures toanti-galactocerebroside anti-serum has also been reported to inhibit myelinformation, although this study suggests that complement proteins are notrequired for the inhibition of primary myelination (Dorfman et aI. 1979).60Inhibition of in vitro peripheral myelin formation has also been demonstrated byexposing cultured rat sensory neurons and Schwann cells to monoclonal antigalactocerebroside immunoglobulin (Ranscht 1987). Anti-galactocerebrosideimmunoglobulin prevented elongation of the mesaxon, inhibiting myelinformation by more than 99% as compared to control cultures. This studysuggests that the mechanism of myelin inhibition is the removal ofgalactocerebroside (GaIC) from Schwann cell surfaces by internalization of theGaIC-anti-GaIC antigen-antibody complex. Antisera against GaIC have also beenshown 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 spinalcord in vivo (Mastaglia et al. 1989).Although GaIC is the major sphingolipid produced by oligodendrocytes inthe CNS, its function has not yet been elucidated (Ransht et al. 1982; Dyer andBenjamins 1990). However, several experiments suggest that GalC may beinvolved in transmembrane signalling (Dyer and Benjamins 1990; Benjamins andDyer 1990). GaIC immunostaining of oligodendrocytes in culture revealsconfluent GalC labelling of the oligodendrocyte cell body and membrane sheets(Ghandour and Skoff 1988). In vitro studies have demonstrated that GaICantibodies, when applied to oligodendrocyte cultures, cause patching of GaICover internal domains of MBP (Dyer and Benjamins 1988). GaIC patching is thenfollowed by microtubule disruption in oligodendroglial membrane sheets and thefusion of MBP domains. The depolymerization of microtubules is mediated by an61influx of calcium through plasma membrane channels (Dyer and Benjamins1990). Extracellular EGTA (a calcium chelator) blocks anti-GaIC inducedmicrotubule dissassembly, suggesting that an extracellular source of calciummediates this effect. Microtubule depolymerization is believed to occur byphosphorylation of tubulin and its associated proteins by calcium, calmodulindependent kinases. Actin monomers, normally colocalized with microtubules,reorganize and form filaments in the absence of microtubular structures. As aresult of these cellular alterations, blebbing of the membrane surface andprocess contraction eventually lead to elimination of the oligodendrocyte myelinmembrane sheets. These studies indicate that anti-GaIC antibodies are anefficient means of suppressing myelin in oligodendrocyte cultures and promptedan investigation of their effectiveness in suppressing myelin development in theembryonic chick spinal cord in vivo.The efficiency of GalC antibodies in removing myelin (outlined above) andinhibiting myelin formation suggests that other oligodendrocyte surface-specific,complement binding antibodies may be equally effective for this purpose. Inorder to bind and activate the first component of complement (Cl q), twojuxtaposed surface-bound lgG molecules are required (Law and Reid 1989). Thisnecessitates a high concentration of antigen on the surface of theoligodendrocyte. The efficiency of GaIC antibodies in evoking myelinsuppression may be attributed to the abundance of GaIC on the surface ofoligodendrocyte myelin (GaIC is the major sphingolipid in the myelin membrane).62Pentameric 1gM molecules, on the other hand, are able to bind the firstcomponent of complement and activate the complement cascade when singlybound to a cell surface (Law and Reid 1989). For this reason 1gM-mediated,complement-dependent cellular attack can be initiated with an antibody to arelatively less abundant cell surface antigen and is generally more robust thanan lgG-mediated cellular attack. These advantages prompted a study of theeffectiveness of myelin-suppression initiated by the 04 antibody (developed byMelitta Schachner). The 04 antibody is an 1gM antibody specific for the myelinmembrane (Sommer and Schachner 1981) recognizing an unidentified antigenon the oligodendrocyte progenitor cell surface as well as an antigen present onthe mature myelin sheath (Bansal et al. 1989). The 04 antibody has beendemonstrated to effectively bind complement in vitro (Sommer and Schachner1982).The classical pathway of the complement system is activated by theinteraction of the first component of complement (Cl complex) with the Fcregion of an lgG or 1gM antibody (reviewed in Law and Reid 1989). DifferentlgG isotypes vary in their ability to bind and activate the Cl complex. IgGi andlgG3 isotypes are very active whereas lgG2 is less active and lgG4 is inactive.Although monomeric lgG is capable of activating the Cl complex, the strengthof binding is less than that of multiple Fc regions presented by aggregated lgGin immune complexes. Tight 1gM-Cl complex binding takes place followingbinding of the 1gM antibody to antigen and the concomitant exposure of binding63sites in the many Fc regions of the pentameric antibody. Upon activation, theCl complex (Clq + Cir + Cis) undergoes a conformational change, allowingsequential autoactivation of the Cir and Cis components. The activated Cismolecule then splits the C4 pro-enzyme, liberating the C4a anaphylatoxin andthe large C4b fragment. The activated C4b enzyme then binds and splits the C2pro-enzyme in a Mg2-dependent fashion, producing the C4b2a complex andthe non-catalytic C2b molecule. The C4b2a complex activates C3 by aproteolytic cleavage of C3 into the C3a anaphylatoxin and the highly reactiveC3b molecule. It is at this point in the complement enzymatic cascade that thecomplement system may be irreversibly deposited on the target surface.The C3 molecule is a unique design in molecular architecture. Uponactivation, the removal of C3a results in a conformational change in the C3bmolecule which exposes an internal thiolester. The exposed thiolester isextremely reactive and has the ability to form covalently linked complexes withany nucleophile (a hydroxl or amino group). This implies that C3b can bedeposited on any biological surface. The ability of C3b to bind to any foreign lifeform is clearly an immunological advantage, but protective measures must bepresent to ensure that host cell binding does not take place. The extremelyreactive nature of the thiolester provides its own control. C3b will use water asa nucleophile, limiting the effective range of the activated C3b. Thus C3b isrestricted to binding to the surface of the same cell which triggered itsactivation, or is inactivated by water.64The activated C3b molecule complexes with C4b2a and induces aconformational change in C5, liberating the C5a anaphylatoxin and the C5bmolecule, responsible for initiating the assembly of the membrane attackcomplex (MAC). The formation of the MAC constitutes the terminal steps ofcomplement activity. The activated C5b molecule binds C6 and exposes a C6binding site for C7. In this way C7, C8 and C9 are sequentially bound to theC5b molecule. Binding of C9 initiates a high affinity C9-C9 interaction resultingin the formation of the MAC with a composition of C5b-8(C9), where n mayrange from 1-18. The MAC presumably disrupts the ionic homeostasis of theoligodendrocyte membrane leading to an increase in intracellular Ca2. Asdiscussed above, the increase in intracellular Ca2 results in a disruption ofmicrotubules resulting in retraction of oligodendrocyte processes.This chapter outlines a protocol for, and demonstrates the effectivenessof, complement proteins plus monoclonal GaIC, polyclonal GaIC or 04 antibodiesto delay the developmental onset of myelination in the embryonic chick spinalcord (termed developmental myelin-suppression; Keirstead et al. 1992).Developmental myelin-suppression is documented with MBP, MAG or CNPimmunohistochemistry, toluidine blue staining or a combination of thesetechniques. Furthermore, developmental myelin-suppression is shown to bespecific for oligodendrocyte myelin, leaving the neuronal and astrocyticpopulations of the spinal cord unperturbed.65MATERIALS AND METHODSDevelopmental Myelln-SuppressionThoracic spinal cord injections were performed at embryonic day (E) 9-E12 in the chick embryo using a glass micropipette (tip diameter = 30-40 urn;A-M Systems, Everett, Washington #6045) connected to a Picospritzer IIpressure injection system (General Valve Corp., Fairfield, New Jersey). Myelinsuppression in the chick embryo was evoked by injecting either an lgG3 mousegalactocerebroside (GaIC) antibody (a gift from B. Ranscht, La Jolla CancerResearch 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 oran IgG1 rabbit GaIC antibody (Chemicon International Inc., Ternecula, California#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 04antibody (a gift from Melitta Schachner, Neurobiology ETH-Honggerberg HPME38 CH-8093 Zurich, Switzerland) at a dilution of 1:25 with 20% guinea pigcomplement 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 (2.67mg/mi) which was then diluted 1:25, providing an effective concentration of63.0 ng of GalC hybridoma supernatant injected per gram body weight.To control for non-specific binding of the GaIC antibody effecting ourresults, control embryos were similarly injected with 20% homologous66complement plus a human antibody that does not cross-react with chicken. Wechose a monoclonal antibody to glial fibrillary acidic protein (GFAP), a majorconstituent of astrocytes within the central nervous system (CNS). Otherimmunological control embryos received injections of either: 1) GalC antibodyonly, 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 plushomologous complement, following heat-inactivation of the complement byexposure at 50°C for 30 mm.Those embryos not undergoing a subsequent thoracic spinal cordtransection were perfused intracardially at the appropriate developmental stage(see results) with O.1M PBS containing 2500 USP units of heparin per 50 mIsPBS, pH 7.4 (at 37°C) followed by perfusion with 4% paraformaldehyde inO.1M phosphate buffer, pH 7.4 (at 4-10°C). The dissected brains and/or spinalcords were then immersed in the same fixative for 24hr at 4°C andsubsequently embedded in paraffin using standard protocols.Paraffin EmbeddingSee page 29.ImmunohistochemistrySee page 30.Histological StainingSee page 32.67RESU LTSThe developmental onset of myelination in the embryonic chick spinalcord was delayed by pressure injection of complement proteins plus eithermonoclonal galactocerebroside (GalC) antibodies, polyclonal GaIC antibodies orpolyclonal 04 antibodies directly into the thoracic spinal cord, 1-4 days prior tothe normal developmental onset of myelination (embryonic day 13; see chapter2). Monoclonal galactocerebroside (GalC) antibodies, polyclonal GaIC antibodiesand polyclonal 04 antibodies were utilized due to their abilities to recognizeoligodendrocyte cell-surface antigens and fix complement.Thoracic spinal cord injections of monoclonal GalC antibodies, plus eitherchick serum or guinea pig complement, delayed the developmental onset ofspinal cord myelination until E17 (Fig. 3-1). Immunohistochemical assessmentsof 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 acomplete lack of myelin basic protein (MBP) immunoreactivity throughout thespinal cord excluding the most rostral 1-4 cervical segments.Immunohistochemical assessments of unoperated control El 5 spinal cordsrevealed a dense network of MBP immunoreactivity (Fig. 3-lA; see chapter 2).The extent and degree of developmental myelin-suppression in animalssacrificed on El 5 was similar when the complement proteins and monoclonalGaIC antibodies were administered on either E9, ElO, Ell or El2 (n=3 for eachday).68Figure 3-1. Developmental myelin-suppression in the thoracic spinal cord of theembryonic 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 immunofluorescencestaining of an El 5 spinal cord from a myelin-suppressed animal that received asingle injection of polyclonal galactocerebroside (GaIC) antibodies pluscomplement on El 1; note the absence of myelin. C: Unoperated (normallymyelinated) control El 5 spinal cord showing extensive myelin-associatedglycoprotein (MAG) immunoreactivity within white matter. D: MAGimmunofluorescence staining of an El 5 spinal cord from a myelin-suppressedanimal that received a single injection of GaIC antibodies plus complement onEl 1; note the absence of myelin. E: Unoperated (normally-myelinated) controlEl 5 spinal cord showing extensive 2’,3’-cyclic nucleotide 3’-phosphodiesterase(CNP) immunoreactivity within white matter. F: CNP immunofluorescencestaining of an El 5 spinal cord from a myelin-suppressed animal that received asingle injection of GaIC antibodies plus complement on El 1; note the absenceof myelin. Developmental myelin-suppression is also observed with monoclonalGaIC or 04 antibodies plus complement. The previous injection site isundetectable in B, D and F, indicating that the injection does not causesubstantial damage. In all photographs the outer edge of the spinal cord lies onthe right hand side, with the white matter adjacent. (Bars = lOOum).01:72Thoracic spinal cord injections of polyclonal GaIC antibodies plus guineapig complement on El 1, also delayed the developmental onset of spinal cordmyelination until E17 (Fig. 3-1). lmmunohistochemical assessments of myetinsuppressed 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 1-4cervical segments. Immunohistochemical assessments of myelin-suppressedspinal cords on E13 (n=3), E14 (n=3) and E15 (n=3; Fig. 3-iD) also showed acomplete lack of myelin-associated glycoprotein (MAG) immunoreactivitythroughout 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 of2’,3’-cyclic nucleotide 3’-phosphodiesterase (CNP) immunoreactivity throughoutthe spinal cord excluding the most rostral 1-4 cervical segments.Immunohistochemical assessments of unoperated control El 5 spinal cords (n =3for 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 plusguinea pig complement were sacrificed on E15, and their thoracic spinal cordswere embedded in Spurr resin and transversely microsectioned for totuidine bluestaining (Fig. 3-2). Light microscopic analysis of toluidine blue stained spinalcord tissue revealed a lack of myelin in all regions (Fig. 3-28). In all instances73Figure 3-2. Toluidine Blue staining of chick spinal cord white matter intransverse section. A: Unoperated (normally-myelinated) embryonic day (E) 15spinal cord. Notice the densely compacted profiles of myelinated axons (darkrings). B: E15 spinal cord from a myelin-suppressed animal that received asingle injection of polyclonal galactocerebroside (GaIC) antibodies pluscomplement on El 1; note the virtual absence of myelinated axons. (Bars =25um).75only a very few myelinated axons scattered throughout the spinal cord whitematter were visible. Unoperated El 5 control tissue (n =5) processed in a similarmanner contained an abundance of tightly compacted myelin characteristicallyviewed as rings surrounding the axon profiles (Fig. 3-2A; also see chapter 2).Thoracic spinal cord injections of polyclonal 04 antibodies plus guinea pigcomplement on El 1, also delayed the developmental onset of spinal cordmyelination until El7 (Fig. 3-1). Immunohistochemical assessments of myelinsuppressed 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) immunoreactivitythroughout the spinal cord excluding the most rostral 1-4 cervical segments.lmmunohistochemical assessments of unoperated control El 5 spinal cordsrevealed a dense network of MBP immunoreactivity (Fig. 3-lA; see chapter 2).The 04 1gM antibody consistently produced larger and more complete areas ofmyelin inhibition compared to the monoclonal and polyclonal GaIC IgGantibodies, which were equally effective compared to each other.On or about E17 a period of robust myelination took place in bothunoperated control spinal cords (see chapter 2) and experimental spinal cordssubjected to developmental myelin-suppression (Fig. 3-3). MBPimmunohistochemical analysis of E17 and E18 spinal cords, previously injectedwith monoclonal GaIC antibodies plus guinea pig complement on El 1, revealedimmunoreactivity throughout all levels of the spinal cord (Fig. 3-3B; n 6 foreach day). Similarly, MBP immunohistochemical analysis of E17 and El8 spinal76Figure 3-3. Developmental onset of myelination following developmental myelinsuppression. A: Unoperated (normally-myelinated) control embryonic day (E) 17chick spinal cord showing extensive myelin basic protein (MBP)immunoreactivity within white matter in parasaggital section. B: MBPimmunofluorescence staining of an E17 spinal cord in parasaggital section froma myelin-suppressed chick that received a single injection of polyclonalgalactocerebroside (GaIC) antibodies plus complement on El 1; note that MBPimmunoreactivity is comparable to, or less than, normally myelinated levelsindicated in A. In all photographs the outer edge of the spinal cord lies on theright hand side, with the white matter adjacent. (Bars = lOOum).78cords, previously injected with polyclonal GaIC antibodies plus guinea pigcomplement on El 1, revealed immunoreactivity throughout all levels of thespinal cord (n =4 for each day). Although all myelin-suppressed spinal cordsanalysed on E17 showed obvious MBP immunoreactivity, they appeared to beless intensely stained than unoperated control E17 spinal cord tissue (Fig. 3-3A).MBP immunohistochemical analysis of P2, P4 and P6 spinal cords (n=4for each day), previously injected with monoclonal GaIC antibodies plus guineapig complement on El 1, showed levels of MBP immunofluoresence comparableto unoperated control tissue (Fig. 3-4A and B; n=25). MBPimmunohistochemical analysis of P2, P4 and P6 spinal cords (n=4 for eachday), previously injected with polyclonal GaIC antibodies plus guinea pigcomplement on El 1, also showed levels of MBP immunoreactivity similar to theunoperated control tissue. These results indicate that myelination followingdevelopmental myelin-suppression is fully compensatory.After hatching, animals that had previously been subjected todevelopmental myelin-suppression demonstrated locomotor behavior that wascomparable to unoperated control hatchlings of the same age. Locomotorbehavior of previously myelin-suppressed hatchlings was assessed on P2, P4and P6 (n =8 for each day), prior to sacrifice for immunohistochemical analysis(see above). Visual assessments of postural adjustments, walking, running andrighting responses suggested that neuronal control of locomotion was not79Figure 3-4. Hatchling pattern of myelination following developmental myelinsuppression. 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: MBPimmunofluorescence staining of an P2 spinal cord in parasaggital section from amyelin-suppressed chick that received a single injection of polyclonalgalactocerebroside (GalC) antibodies plus complement on El 1; note that MBPimmunoreactivity is comparable to normally myelinated levels indicated in A. Inall photographs the outer edge of the spinal cord lies on the right hand side,with the white matter adjacent. (Bars = lOOum).oç\081altered in hatchlings that had been subjected to myelin-suppression duringdevelopment.As a control for the possible influence of nonspecific binding of the GaICantibody affecting our results, 5 control embyos were injected in the thoracicspinal cord at El 1 with an antibody to glial fibrillary acidic protein (GFAP; anastrocyte marker) plus guinea pig complement. MBP immunohistochemistry onEl 5 revealed no suppression of myelin development. Other immunologicalcontrol embryos received injections of monoclonal GalC antibody only (Fig. 3-5A; n=6), guinea pig complement only (Fig. 3-5B; n=8), PBS vehicle only(n=4) or monoclonal GaIC antibodies plus heat-inactivated serum (heatingserum prior to use denatures and inactivates the complement proteins; n=8). Inno case was myelin development suppressed or detectably altered, nor wasthere any evidence of anatomical repair or functional recovery after an E15spinal cord transection (see chapter 4).Direct pressure injection of control and experimental solutions into thethoracic spinal cord did not result in significant damage to the spinal cordtissue. The injected solution did not detectably displace spinal cord tissue orresult 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 inE12-E14 spinal cords previously injected on E9-E12. After E14, the injection sitewas undetectable.The developing and mature state of the spinal cord astrocyte population82Figure 3-5. Pattern of myelin basic protein (MBP) immunoreactivity inimmunological control thoracic spinal cords in parasaggital section. A: MBPimmunofluorescence staining of an embryonic day (E) 15 spinal cord from ananimal that received a single injection of GaIC antibody only on El 1; note thatmyelin is unperturbed. B: MBP immunofluorescence staining of an El 5 spinalcord 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 spinalcord lies on the right hand side, with the white matter adjacent. (Bars =1 OOum).84did not appear to be disturbed by developmental myelin-suppression, asassessed by GFAP immunohistochemistry (Fig. 3-6). Immunohistochemicalassessments of myelin-suppressed and unoperated control E14 and E15 (Fig. 3-6A and B) spinal cords (n = 4 for each) showed that the astrocyte number anddistribution were similar for each developmental stage. P6 spinal cord tissuefrom myelin-suppressed animals (n=6) also showed similar astrocyte numbersand distribution as compared to unoperated control P6 spinal cord tissue (Fig. 3-6C and D; n=8). Additionally, individual astrocytes in myelin-suppressed spinalcords did not appear to express higher levels of GFAP than individual astrocytesfrom unoperated controls at any of the developmental stages examined.The neuronal population of the spinal cord did not appear to be disturbedas a result of developmental myelin-suppression (Fig. 3-7). Neuronaldevelopment in the embryonic spinal cord was assessed with microtubuleassociated protein 2 (MAP-2) immunohistochemistry to identify the dendriticmorphology, and thionin staining to identify the neuronal and axonalmorphology. Myelin-suppressed E15 spinal cords analysed with MAP-2antibodies (Fig. 3-78; n = 4) or thioinin staining (n =3) was indistinguishablydifferent from unoperated control El 5 spinal cord analysed with MAP-2antibodies (Fig. 3-7A; n=4) or thionin staining (n=3).85Figure 3-6. Pattern of glial fibrillary acidic protein (GFAP) immunoreactivity inthe myelin-suppressed chick spinal cord in parasaggital section. A: Unoperated(norrnally-myelinated) control embryonic day (E) 15 spinal cord showingextensive GFAP immunoreactivity within the white matter. B: GFAPimmunofluorescence staining of an El 5 spinal cord from a myelin-suppressedanimal that received a single injection of polyclonal galactocerebroside (GaIC)antibodies plus complement on El 1; note that GFAP immunoreactivity iscomparable to A. C: Unoperated (normally-myelinated) control posthatching day(P) 6 spinal cord showing extensive GFAP immunoreactivity within the whitematter. D: GFAP immunofluorescence staining of an P6 spinal cord from amyelin-suppressed animal that received a single injection of polyclonal GaICantibodies plus complement on El 1; note that GFAP immunoreactivity iscomparable to C. In all photographs the outer edge of the spinal cord lies on theright hand side, with the white matter adjacent. (Bars = lOOum for A and B; 50urn for C and D.).c488Figure 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 cordshowing extensive MAP-2 immunoreactivity within the white matter. B: MAP-2immunofluorescence staining of an E15 spinal cord from a myelin-suppressedanimal that received a single injection of polyclonal galactocerebroside (GaIC)antibodies plus complement on El 1; note that MAP-2 immunoreactivity iscomparable to A. In all photographs the outer edge of the spinal cord lies on theright hand side, with the white matter adjacent. (Bars = lOOum).‘4 M:.-‘r’:1‘44-&.,4,:90DISCUSSIONAlthough the neural factors controlling the differentiation anddevelopment of oligodendrocytes within the CNS have not been fullycharacterized, it is nonetheless possible to alter the developmental onset ofmyelination in vivo (Keirstead et al. 1992). By specifically targeting the highlycatalytic and toxic activity of a series of blood enzymes (complement), it ispossible to destroy a cell population of interest (Law and Reid 1989). Targetingoligodendrocytes in this manner necessitates an oligodendrocyte cell surface-specific antibody that is capable of readily binding complement via the Fcreceptor portion of the antibody (Fig. 3-8). The monoclonal galactocerebroside(GaIC), polyclonal GaIC and polyclonal 04 antibodies described above meet bothof these requirements (Ranscht et al. 1982; Sommer and Schachner 1982;Sergott et al. 1984; Ghandour and Skoff 1988; Ozawa et al. 1989; Mastaglia etal. 1989).Direct injection of complement proteins plus monoclonal GaIC, polyclonalGaIC or 04 antibodies into the thoracic spinal cord of an embryonic day (E) 9-El 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 ofthe embryonic chick spinal cord normally begins on E13 (Benstead et al. 1957;Hartman et al. 1979; MackIm and Weill 1985; Keirstead et al. 1992; seechapter 2). Developmental myelin-suppression was confirmedimmunohistochemically using three myelin-specific antibodies (to myelin basic91Figure 3-8. Schematic representation of the postulated mechanism ofdevelopmental myelin-suppression. Oligodendrocyte cell surface-specificantibodies bind to the oligodendrocyte surface. Once bound, they fix the firstcomponent of complement, which in turn activates the complement cascade.The concommitant formation of the membrane attack complex results in adisruption of the ionic homeostasis of the cell.X)I(III1()))1II’.1(It[I)I\IILS)1OI(IJLL)LILJI(IiL1O’)1)‘1)1)S1)!11l1IA/1I’I93protein, MBP, Fig. 3-lA and B; myelin-associated glycoprotein, MAG, Fig. 2-iCand D; and 2’,3’-cyclic nucteotide 3’-phosphodiesterase, CNP; Fig. 3-1E and F)as well as histologically by toluidine blue staining of microthin transversesections of the developing spinal cord (Fig. 3-2). Developmental myelinsuppression also inhibited the expression of several proteins that normallyappear at the developmental onset of myelination in the chicken embryo (resultsnot 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 etal. 1989), this is the first demonstration of an anti-GaIC induced developmentalsuppression of CNS myelin formation. Additionally, this is the firstdemonstration of myelin-suppression in vivo mediated by 04 antibodies.The antigens recognized by the GaIC and 04 antibodies were selecteddue to their oligodendrocyte specificity, the availability of complement-bindingantibodies as well as the temporal appearance of these molecules indevelopment (Ranscht et al. 1982; Sommer and Schachner 1982; chapter 2 ofthis thesis). Although the antigen recognized by the 04 antibody is present onmature oligodendrocytes, a like or related antigen recognized by the 04 antibodyis also expressed by otigodendrocyte precursors just prior to their differentiationinto oligodendrocytes (Sommer and Schachner 1982). GaIC is one of the firstmarkers expressed by oligodendrocytes upon differentiation from their94precursors (Ranscht et al. 1982). Histological studies have demonstrated thatoligodendrocytes differentiate 2-3 days prior to myelination (Benstead et al.1957). In support of these findings, a GaIC immunohistochemical investigationof the developing chick spinal cord indicated that GaIC immunoreactivity wasfirst detected on ElO (see chapter 2 of this thesis). For these reasons, thecomplement proteins plus monoclonal GaIC, polyclonal GaIC or 04 antibodieswere injected on E9-E12.The immunological protocol outlined above may elicit developmentalmyelin inhibition via destruction of all newly-differentiating oligodendrocytes. Ifthis is the case, it follows that the E17 onset on myelination followingdevelopmental myelin-suppression is due to the novel differentiation of -oligodendrocyte progenitors on or about E17. On the other hand, it isconceivable that the immunological protocol outlined above operates viaselective destruction of the myelin sheaths, effectively sparing theoligodendrocyte cell bodies. The E17 onset of myelination followingdevelopmental myelin inhibition would then be a result of the re-extention ofmyelinating processes from the surviving parental cell bodies. These twopossibilities are, of course, not mutually exclusive. Several findings support thelatter scenario. Firstly, in vitro studies of oligodendrocyte attack mediated byGaIC-antisera (Dorfman et al. 1979) or 04 antibodies and complement (Sommerand Schachner 1981), indicate that the addition of these composite solutions tocultured oligodendrocytes results in withdrawl of the myelinating processes and95survival of the oligodendrocyte cell bodies. The subsequent removal of thecomposite solutions from the culture media resulted in the re-extention ofmyelinating processes. Secondly, collaborative efforts undertaken with DavidPataky (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 expressMBP mRNA despite the lack of myelin (Fig. 3-9; Keirstead et al. 1994). BecausemRNA is chiefly located in cell bodies, these findings suggest thatoligodendrocyte cell bodies survive developmental myelin-suppression. Lastly,myelination following developmental myelin-suppression takes place in a speedyand fully compensatory manner (see Results section) suggesting that survivingoligodendrocyte cell bodies contribute to myelination following developmentalmyelin-suppression.MBP mRNA expression in myelin-suppressed spinal cords suggests thatsurviving oligodendrocyte cell bodies continue to express MBP mRNA despitethe loss of myelin processes and sheaths. However, MBP mRNA is found inoligodendrocyte processes as well as oligodendrocyte cell bodies. Thetranslocation of MBP mRNA to oligodendrocyte processes is a reflection of thehigh turnover of MBP within the myelin sheaths (DesJardins and Morell 1983).Therefore, it is arguable that developmental myelin-suppression results in the96Figure 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” meanssacrificed on El 5. B: MBP mRNA expression in El 5 spinal cords four days afterinjection 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 inkilobases.q7AE15BIi 1E15I I28S’2.7k18S’98loss of both the oligodendrocyte cell body and the myelin sheaths; the MBPmRNA detected in myelin-suppressed spinal cords may reside within theoligodendrocyte processes spared by the immunological intervention. Althoughno direct evidence exists to dispute this possibility, it is unlikely that myelinprocesses separated from their parental cell bodies would persist in the spinalcord for four to eight days before being degraded. Therefore it is highly probablethat the MBP mRNA detected within myelin-suppressed spinal cords is locatedwithin oligodendrocyte cell bodies which have survived developmental myelinsuppression.It is also possible that de-differentiation of astrocytes or astrocyteprecursors may contribute to spinal cord myelination following developmentalmyelin—suppression. In demyelinating lesions of the cat optic nerve, reactive glialcells which have begun to differentiate along the astrocyte lineage (GFAP) mayreverse their direction of differentiation, evidenced by the coexpression of bothastrocyte- and oligodendrocyte-specfic markers (GFAP, GaIC; Kim 1985;Carroll et al. 1987). Switching glial cultures from one type of culture media toanother favoring an alternative direction of differentiation results in the transientcoexpression of both astrocyte and oligodendrocyte phenotypes (Ingraham andMcCarthy 1989). Cells of the 02A lineage may actually exhibit greater plasticityin demyelinating/remyelinating conditions (Kim 1985). Cells coexpressingastrocyte and oligodendrocyte phenotypes can be isolated from the spinal cordsof young adult mice demyelinated by corona virus infection (Armstrong et al.991990). In addition, cells coexpressing astrocyte and oligodendrocytephenotypes have been reported in vivo in a number of demyelinating situations(Bunge et al. 1961; Carroll et al. 1987; Godfraind et a!. 1989). Pending doublelabelling studies of developmentally myelin-suppressed spinal cords using bothastrocyte- and oligodendrocyte-specific antibodies, the possibility of astrocytededifferentiation and redifferentiation into myelinating oligodendrocytes can notbe excluded.Both normal and myelin-suppressed spinal cords display a ‘wave’ ofmyelination on or about E17 in development (Fig. 3-3; also see chapter 2 of thisthesis). This period of robust myelination is evidenced by a dramatic increase inthe MBP, CNP and MAG immunoreactivity detected within the spinal cord. It isunclear whether this period of intense myelination is due to an increase inmyelin production by existing oligodendrocytes, or to novel differentiation ofoligodendrocyte precursors and a concomitant extention of myelinatingprocesses. Again, these two possibilities are not mutually exclusive. Afterhatching (E21), levels of MBP, CNP and MAG immunoreactivity in myelinsuppressed spinal cords appear to be equivalent to that of unoperated controlspinal cords (Fig. 3-4). This indicates that myelination following myelinsuppression is fully compensatory, with the E17 ‘wave’ of myelinationcontributing significantly to myelin recovery within the myelin-suppressed spinalcord.Developmental myelin inhibition in vivo requires both complement100proteins and GaIC or 04 antibodies (Fig. 3-5). Immunological control embryosreceiving injections of GalC antibody only, complement proteins only, PBSvehicle only or GaIC antibody plus heat-inactivated serum showed no indicationsof myelin-suppression. In no case was myelin development suppressed ordetectably altered, nor was there any evidence of anatomical repair or functionalrecovery after an E15 spinal cord transection (see chapter 4). This finding issupported by other in vivo studies of anti-GaIC induced demyelination whichwere also shown to be complement-dependent (Sergott et al. 1984; Ozawa etal. 1989). It is interesting that several in vitro studies have demonstrated antiGaIC induced demyelination in the absence of complement (Dorfman et al.1979; Ranscht et al. 1987). The susceptibility of cultured myelin to antibody-mediated attack in the absence of complement may be somewhat analagous tothe susceptibility of myelin proteins to proteolysis. Lysophilized or frozen myelinproteins are rapidly degraded by proteases, whereas freshly isolated myelinproteins are relatively resistant to proteases (Cammer et al. 1986). Thus, themethod of myelin isolation and cultivation in the in vitro studies may alter thenative state of myelin, increasing suscepibility to antibody-mediated attack.Alternatively, the in vivo mechanism of myelin-suppression may differsignificantly from the in vitro mechanism of myelin-suppression.Delaying the developmental onset of myelination does not appear to alterthe development and mature state of the astrocyte population of the spinal cord(Fig. 3-6). Immunohistochemical analysis of E15 myelin-suppressed spinal cords101indicate that glial fibrillary acidic protein (GFAP) immunoreactivity is similar tounoperated control E15 tissue. Developmental myelin-suppression did notinduce detectable changes in astrocyte distribution or individual size. Noevidence of astrogliosis was observed in any of the myelin-suppressed spinalcords examined. Additionally, myelin-suppressed spinal cords examined afterhatching revealed no signs of abnormal GFAP immunoreactivity as compared tounoperated control spinal cords processed in a similar manner.The neuronal population of the spinal cord did not appear to be disturbedas a result of developmental myelin-suppression (Fig. 3-7). Myelin-suppressedEl 5 spinal cords analysed with MAP-2 antibodies to identify dendriticmorphology, and thionin staining to identify neuronal and axonal morphology(results not shown), were indistinguishably different from unoperated controlE15 spinal cords analysed in a similar manner. After hatching, animals that hadpreviously been subjected to developmental myelin-suppression demonstratedlocomotor behavior that was comparable to unoperated control hatchlings of thesame age. Visual assessments of postural adjustments, walking, running andrighting responses suggested that neuronal control of locomotion was notaltered in hatchlings that had been subjected to myelin-suppression duringdevelopment.Myelination has been shown to affect axonal cytoarchitecture in otherdeveloping systems. In the peripheral nervous system (PNS), Schwann cellcontact influences axonal morphogenesis, including the segregation of Na102channels to the nodes of Ranvier (Joe and Angelides 1992) and regulation ofaxonal caliber (de Waegh et al. 1992). The trembler mouse is a peripheralmyelin-deficient mutant that is characterized by marked hypomyelination as aresult of Schwann cell dysfunction. Myelin enwrapment and compaction do notoccur, and axonal caliber, phosphorylation of neurofilaments and slow axonaltransport are significantly decreased (de Waegh et al. 1992). de Waegh et al.(1992) propose a model in which Schwann cells locally regulate neurofilamentphosphorylation via cell-cell contact, mediated perhaps by myelin-associatedglycoprotein (MAG). MAG is a member of the immunoglobulin superfamily ofrecognition/adhesion molecules and is concentrated in the periaxonal space andnodes of Ranvier. MAG may stimulate a neuronal kinase or inhibit aphosphatase, resulting in changes in neurofilament phosphorylation, andtherefore axonal caliber. This relationship is reflected in the adult nerve, whereregions of the axon not in contact with compact myelin, such as the nodes ofRanvier and the Schmidt-Lanterman incisures in the PNS, have smaller axonalcaliber and greater neurofilament packing density than myelinated regions of thesame axon (Price et al. 1990). The apparent insensitivity of chick spinal cordaxons to myelin-suppression may reflect the immature state of the axons at thisstage of development; in a normally-myelinating embryo, myelin-inducedalterations of axonal cytoarchitecture may not take place until later stages ofembryonic development. Thus, analyses of unoperated control and myelinsuppressed El 5 embryos would reveal similar axonal cytoarchitecture.103Alternatively, thionin staining may be insensitive to any alterations produced bydevelopmental myelin-suppression. Perhaps a longer period of chick spinal cordmyelin-suppression or a more detailed analysis of axonal cytoarchitecture mayreveal axonal alterations as a result of the immunological myelin-suppressionprocedure described here.The ability to delay the developmental onset of myelination in the chickembyo may prove to be a useful tool for investigations of myelin developmentas well as interactions of myelin with other cell populations within thedeveloping spinal cord. Perhaps more importantly, developmental myelinsuppression provides a means of assessing the effects of myelin on the abilityof the spinal cord to functionally regenerate following injury. Transections of theembryonic 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 voluntarylocomotion after hatching, suggesting that myelin may be inhibitory to theregeneration of transected axons in the developing embryo. Developmentalmyelin-suppression provides a means of delaying the onset of myelination untillater stages of development; a subsequent transection of the spinal cord duringthe period of myelin-suppression then serves as a direct test of whether myelinis inhibitory to the regeneration of transected spinal cord.104CHAPTER 4NEUROANATOMICAL REPAIR AND FUNCTIONAL RECOVERY OF TRANSECTEDSPINAL CORD IN EMBRYONIC CHICK105INTRODUCTIONTransection of the embryonic chick spinal cord prior to the developmentalonset of myelination at embryonic day (E) 13 results in complete anatomical andphysiological recovery (Shimizu et al. 1990; Hasan et al. 1991). Embryonicspinal cord transections after the developmental onset of myelination result inno recovery, rendering such an animal incapable of voluntary locomotion afterhatching (Shimizu et al. 1990; Hasan et al. 1991; Keirstead et al. 1992). Thesestudies suggest that myelin may be inhibitory to the functional regeneration oftransected spinal cord in embryonic chick. Chapter 3 of this thesis outlines animmunological method of delaying the developmental onset of myelinationwithin the embryonic chick spinal cord until later stages of development.Developmental myelin-suppression then serves as a tool with which toinvestigate the effects of myelin on the ability of the spinal cord to regeneratefollowing injury in later stages of embryonic development. This chapterconcerns itself with the regenerative capacity of the myelin-suppressed chickspinal cord following late embryonic transection in vivo.There are three mechanisms by which an embryonic spinal cord couldeffect repair following complete transection in vivo. Repair of brainstem-spinalprojections following injury to the developing spinal cord may be due toneogenesis of brainstem-spinal projecting neurons within locomotor nuclei of thebrainstem. Although possible following very early embryonic spinal cord106transections, neurogenesis is not likely to play a role in spinal cord repairfollowing late-embryonic transection. Neogenesis within the developing chickbrainstem-spinal projecting nuclei is complete by E5 (McConnell and Sechrist,1980; Sechrist and Bronner-Fraser, 1991). Neurogenesis within the centralnervous system (CNS) during post-development is limited to the retina, spinalcord and tectum of fish and amphibia, the olfactory epithelium and hippocampusof the rodent and the telencephalic vocal control centers of songbirds (Andersonand Waxman, 1985; Holder and Clarke, 1988). Although it is conceivable thatspinal cord injury itself may induce neogenesis of neurons within brainstemlocomotor nuclei, it is unlikely that these cells could contribute to any functionalrecovery of locomotion in hatchlings transected during late embryonicdevelopment. Axons grow at a rate of 1-2 mm/day in vivo (Schnell and Schwab1990). It is unreasonable to assume that late embryonic transection could resultin neogenesis, differentiation and axonal outgrowth exceeding a distance of 400mm within the final 7 days of late embryonic development.Neuroanatomical repair and physiological recovery following injury to thedeveloping spinal cord may alternatively be due to subsequent development ofbrainstem-spinal projections. Brainstem-spinal projections first descend to thespinal cord by embryonic day (E) 3.5 (2) and complete their axonal projectionsto lumbar levels by E10-E12 (Okado and Oppenheim 1985). By E12, brainstemspinal projections throughout the spinal cord are equivalent in number anddistribution to those in a hatchling chick (Okado and Oppenheim 1985).107Therefore, it is reasonable to assume that repair of embryonic spinal cordtransected prior to E13 is in part due to late developing brainstem-spinalprojections. Because brainstem-spinal projections are complete to all levels ofthe spinal cord by E12, however, it is unlikely that subsequent developmentcould contribute to recovery from spinal cord transections during the final weekof embryonic development.Thirdly, repair of brainstem-spinal projections following injury to thedeveloping spinal cord may be due to true axonal regeneration; that is, theextention of a viable neurite from the proximal end of a severed axon. Althoughthe peripheral nervous system (PNS) is capable of true regeneration followinginjury, the damaged CNS typically shows little or no ability to effect anatomicalor functional regeneration (Sholomenko and Steeves 1987: Eidelberg 1981).Several lines of evidence indicate that the poor regenerative capacity of theCNS is due, not to a lack of intrinsic growth programs, but to environmentalinfluences including an absence of trophic factors and the presence of axonalgrowth inhibitors (Ramon y Cajal, 1914; David and Aguayo 1981; Schwab andCaroni 1988). Axons of CNS neurons are able to grow out and make functionalconnections with their targets if peripheral nerve segments containing Schwanncells are grafted to the site of injury (Ramon y Cajal 1914; David and Aguayo1981; Aguayo et al. 1991). CNS neurons have also been shown to extendfibers in vivo through lesion sites containing implants of fetal CNS tissue(Bjorklund and Stenevi 1979; Kromer et al. 1981; Bjorklund 1991), as well as108implants consisting of fibroblasts genetically modified to express growth factors(Fisher and Gage 1993; Tuszynski et al. 1994). In addition, CNS motor neuronsare capable of regenerating their peripheral projecting axon if axotomized withinthe PNS (Ramon y Cajal 1959). These findings indicate that CNS neurons retainintrinsic axonal growth programs which enable long-distance axonalregeneration in the presence of a favorable extraneuronal environment.Several axonal growth inhibitors have been identified in the developingand mature CNS. Myelin-associated proteins that inhibit the anatomical growthof axons in vitro as well as the regrowth of axotomized corticospinal fibres invivo have been identified in rat spinal cord (Caroni and Schwab 1988).Neutralization of these proteins with functionally-blocking antibodies facilitatesanatomical regeneration following axotomy (Schnell and Schwab 1990).Oligodendrocytes also express the inhibitory molecule janusin, which is presentin myelin of the CNS. Janusin expression coincides with the developmentalonset of myelination (Wintergerst et al., 1993; Bartsch et al., 1993) and hasbeen shown to be a repulsive substrate for neuronal cell bodies as well asgrowth cones (Pesheva et al., 1989; Taylor et al., 1993). Astrocytes may alsocontribute to the inhibitory nature of the mature CNS by expressing tenascinwhich, as a repulsive substrate for neuronal cell bodies and growth cones, mayform either pathways or boundaries to neurite outgrowth (Faissner and Kruse,1990; Taylor et al., 1993). Adult CNS astrocytes also express lower levels of NCAM as compared to embryonic astrocytes (Silver and Rutishauser 1984).109Further evidence of the inhibitory nature of the mature CNS comes fromstudies of regenerating sensory neurons. Sensory neurons within the dorsal rootganglia have a peripheral axon projecting to sites in the skin, as well as acentral-projecting axon which enters the dorsal horn of the spinal cord andcontacts interneurons and motor neurons. Although lesions of both peripheral orcentral projections result in regeneration within the PNS, central-projectingneurites stop growing when they reach the dorsal root entry zone of the spinalcord (Ramon y Cajal 1958; Stenaas et al. 1987). Peripheral neuronstransplanted into the CNS are also incapable of axonal extention within theenvironment of the CNS (Ramon y Cajal 1958).Thus, it would appear that the unfavorable extraneuronal environment ofthe CNS accounts in large part for the poor regenerative capacity of centralneurons. Studies which identify the particular inhibitory factors and the relativecontribution of these factors to the inhibitory nature of the CNS are important inefforts to evoke regeneration of CNS tissue following injury. The developmentalonset of myelination coincides with a loss of regenerative capability of injuredspinal cord (see chapter 2); this suggests that myelin may inhibit the regrowthof injured axons. Immunological myelin-suppression (see chapter 3) serves as atool with which to investigate the effects on myelin on the ability of thedeveloping spinal cord to regenerate following injury. This chapter illustratesthat developmental myelin-suppression in vivo facilitates regeneration oftransected spinal cord in late embryonic chicks. Chick spinal cord transections110during late embryonic development in normally-myelinated animals result in norepair whatsoever. These findings demonstrate that myelin is inhibitory to theregeneration of transected spinal cord in embryonic chick and constitute thefirst demonstration of functional recovery following complete spinal cordtransection.111MATERIALS AND METHODSDevelopmental Myelln-SuppressionSee page 65.Spinal Cord TransectionTransections consisted of a pinch lesion of the mid to high-thoracic spinalcord using sharpened Dumont #5-45 forceps (Fine Science Tools, NorthVancouver, British Columbia #11253-25). A #00 pin marked to the appropriatedepth of the cord for that stage of development, was then passed across theentire width of cord through the lesion to ensure that the spinal cord wascompletely severed (for details, see Keirstead et al. 1992). In addition, embryoswere randomly selected from batches of transected animals and perfusedintracardially with O.1M PBS containing 2500 USP units of heparin per 50 mIsPBS, pH 7.4 (at 37°C) followed by perfusion with 4% paraformaldehyde inO.1M phosphate buffer, pH 7.4 (at 4-10°C). The dissected tissue was thenpostfixed for 24 hours at 4°C and subsequently embedded in paraffin (seebelow). Parasagittal 10 um sections were cut and mounted on gelatin-coatedslides, stained with Toluidine blue, and examined under a light microscope toensure complete transection. Lastly, some hatchling chicks received lumbarinjections of 1 .0 ul of tetramethylrhodamine-labelled dextran amine (RDA; see‘neuroanatomy’ below) at the time of thoracic spinal cord transection. Afterhatching, they were perfused as outlined above and then postfixed for 24 hoursat 4°C. The dissected brainstems were transferred to 30% sucrose in O.1M112PBS, pH 9.0 (4°C) for 24 hours. Each brainstem was sectioned in the transverseplane using a Leitz liquid CO2 freezing microtome and examined for the presenceof retrogradely-labelled brainstem-spinal neurons. The dissected spinal cordswere subsequently embedded in paraffin (see below). Parasagittal 10 urnsections were cut and examined for evidence of the injection site andtransection site.Paraffin EmbeddingSee page 29ImmunohistochemistrySee page 30.NeuroanatomyBirds were anesthetized with an intramuscular injectipn of ketaminehydrochloride (30 mg/kg) plus xylazine (Rompun, 3 mg/kg). After removal of thedorsal 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 indicatedthat this amount of RDA, injected at this level of the cord, remains confined tothe lumbar level (ie. does not diffuse rostrally to or above the site oftransection), and within 24-48 hr is retrogradely transported via brainstemspinal axons to the cell bodies of origin, with no trans-synaptic transport to113brainstem neurons not having spinal projections (Hasan et al. 1991; Keirstead etal. 1992; Hasan et al. 1993). After 48 hours, the P4 birds were given a lethalintramuscular injection of anesthetic (sodium pentobarbital, 75 mg/kg) and thenperfused prior to cardiac arrest and fixed as outlined above. The dissectedbrains were subsequently postfixed for another 24hr and then transferred to30% sucrose in 0.1M PBS, pH 9.0 (4°C). Each brainstem was sectioned in thetransverse plane using a Leitz liquid CO2 freezing microtome. The number andposition of retrograde labelled brainstem-spinal neurons, for each brainstemsection, were then noted and photographed under a microscope equipped forepifluorescence.114RESULTSComplete transections of the El 5 thoracic spinal cord were confirmed byrandomly selecting embryos from experimental batches after surgery andsectioning the tissue for light microscopic histological examination. Completetransections were confirmed in all cases (22 sacrificed immediately aftersurgery, 16 sacrificed on E16). Additionally, 3 myelin-suppressed embryosreceived lumbar injections of 1 .0 ul tetramethylrhodamine-labelled dextran amine(RDA) solution at the time of the thoracic spinal cord transection. Only acompletely transected spinal cord would prevent the retrograde transport of thisneuroanatomical tracer to the brainstem. Subsequent examination of thebrainstem and cervical spinal cord on posthatching day (P) 5 showed acomplete lack of RDA rostral to the transection site (Fig. 4-lA), althoughneuronal and axonal labelling was evident near the area of injection in thelumbar spinal cord (Fig. 4-1B). This confirms that the transection procedurereliably severs the entire thoracic spinal cord.Myelin-suppression was confirmed in those animals randomly selectedfrom batches of myelin-suppressed, El 5 transected embryos (see above).lmmunohistochemical analysis revealed a complete lack of MBPimmunoreactivity in all areas of the spinal cord excluding the most rostralsegments (Fig. 4-2). These findings confirm that immunological myelinsuppression reliably delays the developmental onset of myelination until latestages of embryonic development (i.e. E17; see chapter 3).115Figure 4-1. The transection procedure reliably severs the entire thoracic spinalcord. Control embryos received lumbar injections of tetrarnethylrhodaminelabelled dextran amine (RDA) solution at the time of the thoracic spinal cordtransection. Only a completely transected spinal cord would prevent theretrograde transport of this neuroanatomical tracer to the brainstem. A:Ventromedial reticular formation of the caudal pons in a posthatching day (P) 5chick that received a thoracic transection and a lumbar injection of RDA onembryonic day (E) 15; note the absence of retrogradely-labelled gigantocellularreticulospinal neurons. B: Lumbar spinal cord of the same hatchling chick inparasaggital section; note the neuronal and axonal labelling evident near theprevious injection site. This confirms that the transection procedure reliablysevers the entire thoracic spinal cord. (Bars = 5Oum for A, 100 urn for B).117Figure 4-2. Developmental myelin-suppression in the thoracic spinal cord of theembryonic chick in parasaggital section. Developmental myelin-suppression (andcomplete transections) were confirmed in animals randomly selected fromexperimental batches immediately after surgery. A: Unoperated (normallymyelinated) control embryonic day (E) 15 spinal cord in parasaggital sectionshowing extensive myelin basic protein (MBP) immunoreactivity within whitematter. B: MBP immunofluorescence staining of an E15 spinal cord, randomlyselected from an experimental batch of animals, each of which recieved a singleinjection of polyclonal galactocerebroside (GalC) antibodies plus complementproteins on El 1 and a complete thoracic spinal cord transection on El 5; notethe absence of myelin. In all photographs the outer edge of the spinal cord lieson the right hand side, with the white matter adjacent. (Bars = lOOum).119Neuroanatomical or functional (see Discussion) assessments wereconducted on: a) 21 myelin-suppressed, El 5 transected embryos; b) 8 normallymyelinated (ie. uninjected) E15 transected control animals; c) 6 immunologicalcontrol El 5 transected animals (see Materials and Methods) and d) 11 normallymyelinated and untransected control animals. Neuroanatomical and functionalassessments were often carried out on the same animal.Neuroanatomical regeneration following transection was assessed bycounting the brainstem-spinal neurons labelled by the retrograde transport ofRDA injected into the lumbar spinal cord after hatching (Fig. 4-3). There was asimilar number and distribution of retrogradely labelled reticulospinal neurons inthe 11 normally-myelinated, untransected control animals (Fig. 4-3A) and the 21myelin-suppressed, E15-transected experimental animals (Fig. 4-3B) following apost-hatching injection into the lumbar spinal cord. In the ventromedial reticularformation of the pons, the myelin-suppressed El 5-transected animals averaged1003 retrogradely labelled reticulospinal neurons per animal (range: 920-1292cells). Normally myelinated, untransected control animals averaged 1043retrogradely labelled reticulospinal neurons per animal (range: 692-1311 cells).Comparable numbers and distributions of retrogradely labelled neurons werealso noted for other brainstem-spinal nuclei with projections to the lumbar spinalcord including the vestibular nucleus (Fig. 4-3C and D), locus ceruleus,subceruleus nucleus and raphe nucleus. In contrast, 8 normally myelinated and6 immunological control embryos (see above) transected on E15 showed no120Figure 4-3. Neuroanatomical regeneration of brainstem-spinal projections.Photomicrograghs of retrogradely labelled neurons within the brainstem inposthatching day (P) 4 chicks. Brainstem-spinal neurons were labelled by theretrograde axonal transport of tetramethyirhodamine-labelled dextran amine(RDA) injected into the lumbar spinal cord on P2 and allowed two days fortransport. A: RDA labelled gigantocellular reticulospinal neurons within theventromedial reticular formation of the caudal pons in an unoperated (normallymyelinated) control P4 chick. B: RDA labelled gigantocellular reticulospinalneurons within the ventromedial reticular formation of the caudal pons in a P4experimental hatchling that was subjected to developmental myelin-suppressionon El 1, followed by a complete thoracic spinal cord transection on El 5; notethat the number and distribution of retrogradely labelled neurons is comparableto A. C: RDA labelled vestibulospinal neurons within the lateral vestibularnucleus of the dorsolateral pons in an unoperated (normally-myelinated) controlP4 chick. D: RDA labelled vestibulospinal neurons within the lateral vestibularnucleus of the dorsolateral pons in a P4 experimental hatchling that wassubjected to developmental myelin-suppression on El 1, followed by a completethoracic spinal cord transection on E15; note that the number and distribution ofretrogradely labelled neurons is comparable to C. Comparable neuroanatomicalrepair was evident for other brainstem-spinal projections. E: Ventromedialreticular formation of the caudal pons in a P4 hatchling that was (normallymyelinated and) transected on E15; note the absence of retrogradely labelled121neurons. F: Lateral vestibular nucleus of the dorsolateral pons in a P4 hatchlingthat was (normally-myelinated and) transected on El 5; note the absence ofretrogradely labelled neurons. A similar lack of neuroanatomical repair wasobserved in animals that received immunological control solutions on El 1followed by a complete thoracic spinal cord transection on E15. (Bars = 5Oum).125retrogradely labelled brainstem-spinal neurons within the ventromedial reticularformation of the pons (Fig. 4-3E), the vestibular nucleus (Fig. 4-3F) or any otherbrainstem nucleus with projections to the lumbar spinal cord.Axonal repair/regeneration of descending brainstem-spinal projectionswas also observed in 3 myelin-suppressed animals injected with cascade blue-labeled dextran amine (CBDA) into the lumbar spinal cord at the time of the El 5thoracic transection, and a second retrograde fluorescent tracer (RDA) into thelumbar spinal cord on P4 (Fig. 4-4). The animals were sacrificed 48 hours laterand their brains and spinal cords processed for light microscopic analysis.Although CBDA was present in the lumbar spinal cord (Fig. 4-4A), there was noevidence of CBDA-labelled neurons within the brainstem, confirming that theE15 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 5during myelin-suppression indicated complete recovery of locomotor capabilies(results not shown). Myelin-suppressed, El 5-transected hatchling chicks werecapable of initiating locomotion in response to a positive stimulus (eg. food,water or the presence of other chicks). In addition, the speed and frequency ofmovement as well as the righting capability of all hatchling chicks previouslymyelin-suppressed and El 5 transected were indistinguishable from unoperatedcontrol hatchlings of a similar age. Hatchling chicks that had received an E15126Figure 4-4. Neuroanatomical regeneration of brainstem-spinal projections inmyelin-suppressed animals injected with cascade blue labelled dextran amines(CBDA) at the time of the El 5 thoracic transection. To further ensure completetransections in animals displaying neuroanatomical regeneration, severalembryos received lumbar injections of CBDA at the time of the thoracic spinalcord transection. Only a completely transected spinal cord would prevent theretrograde transport of this neuroanatomical tracer to the brainstem.Neuroanatomical regeneration was evidenced by the retrograde axonal transportof tetramethylrhodamine-labelled dextran amine (RDA) injected into the lumbarspinal cord on P4 and allowed two days for transport. A: Lumbar spinal cord ofa hatchling chick that was subjected to developmental myelin-suppression onEl 1, followed by a complete thoracic spinal cord transection and lumbarinjection of CBDA on E15; note the CBDA neuronal and axonal labelling evidentat the previous (El 5) injection site. No CBDA was present within the brainstem.This confirms that the transection reliably severed the entire thoracic spinalcord. B: Lumbar spinal cord from the same animal; note the RDA neuronal andaxonal labelling evident at the previous (P4) injection site. C: RDA labelledgigantocellular reticulospinal neurons within the ventromedial reticular formationof the caudal pons in the same animal; note that the number and distribution ofretrogradely labelled neurons is comparable to unoperated control labelling (seeFig. 4-3; Bars = 100 urn for A and 8; 5Oum for C)128transection plus no injection or an immunological control injection on E9-E12exhibited no locomotor capabilities whatsoever.129DISCUSSIONThe experiments reported here examined the influence of myelin on theability of chick spinal cord axons to regenerate following complete embryonicspinal cord transection. Embryonic chicks lose the ability to repair their injuredspinal cord on or about embryonic day (E) 13 of the 21 day developmentalperiod in ovo. Spinal cord transections prior to E13 result in partial or completerepair (Shimizu et al. 1990; Hasan et al. 1991). Transections after E13 result inlittle or no repair, rendering the animal incapable of voluntary locomotion afterhatching (Sholomenko and Steeves 1987; Eidelberg 1981; Shimizu et al. 1990;Hasan et al. 1991; Keirstead et al. 1992). This loss of regenerative capability iscoincident with the developmental onset of myelination within the spinal cord(Bensted et al. 1957; Hartman et al. 1979; MackIm and Weill 1985; Keirstead etal. 1992). Chapter 4 of this thesis outlined an immunological method ofdelaying the developmental onset of myelination until E17. The studies reportedhere 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 completeanatomical regeneration. Furthermore, El 5 transections in myelin-suppressedanimals resulted in complete locomotor recovery after hatching; behavioralobservations indicated that voluntary locomotion was completelyindistinguishable from that of unoperated control hatchlings of a similar age.These findings illustrate that myelin is inhibitory to the functional regeneration130of transected spinal cord in embryonic chick (Keirstead et al. 1992).Physiological assessments of post-hatching chicks previously myelinsuppressed and transected on E15 also indicate complete recovery of locomotorcapabilities (Fig. 4-5; Keirstead et al. 1992). These studies were conducted incollaboration with Dr. Gillian D. Muir (a Postdoctoral Fellow working under thesupervision of Dr. John D. Steeves, University of British Columbia, Departmentsof Zoology and Anatomy, Vancouver, B.C., Canada). EMG recordings from legmuscles during post-hatching walking were obtained from normally myelinated,untransected control chicks and myelin-suppressed, El 5 transected chicks. Thepattern of leg muscle activity obtained from myelin-suppressed, El 5-transectedchicks did not differ from those obtained from normally-myelinated,untransected control chicks (Fig. 4-5A and B). As expected during walking, thesame muscle (eg. lateral gastrocnemius muscle, an ankle extensor muscle) inthe right and left leg showed alternating periods of activity. In addition, anantagonist muscle of the right lateral gastrocnemius, the sartorius (a kneeextensor/hip flexor muscle) also exhibited activity that alternated with the rightlateral gastrocnemius. The right iliofibularis (knee flexor/hip extensor) burstconcurrently with the right lateral gastrocnemius and alternated with the rightsartorius. None of the normally-myelinated, El 5-transected chicks were capableof locomotion or even unsupported standing.The relationships between muscle activity (burst duration) and step cycleduration for normally-myelinated, untransected control and myelin-suppressed,131Figure 4-5. Physiological recovery of brainstem-spinal projections. Simultaneouselectromyographic (EMG) recordings from four leg muscles during overgroundwalking by an unoperated (normally-myelinated) control chick (A) and a myelinsuppressed E15-transected chick (B). The myelin-suppressed E15-transectedchick shows the same muscle activity patterns as the control chick. C:Regression of muscle activity (burst) duration versus step cycle duration forlateral gastrocnemius muscle (open squares) and sartorius muscle (filledsquares) during overground walking by a normally-myelinated, untransectedhatchling chick. The burst duration of the lateral gastrocnemius muscleincreases with increasing cycle duration, while the burst duration of thesartorius muscle remains constant as cycle duration increases. D: Regression ofburst duration versus step cycle duration for lateral gastrocnemius muscle (opensquares) and sartorius muscle (filled squares) during overground walking by amyelin-suppressed El 5-transected hatchling chick. This animal displays thesame relationships as the control animal in C. The slopes of correspondingregression lines in C and D are not significantly different. All regressions aresignificant to p<O.05. R2 for lateral gastrocnemius and sartorius are 0.58 and0.04 in (C) and 0.59 and 0.08 in (D).A R. Lat.4jøb 4*4$4R. Sari.i fr4R. Iliofib.L. Lat Gastroc.R. Lat.Gastroc.-R. Sart.*1Øl WA.- -*JL4.R. Iliofib.t14L. Lat. Gastroc.0 0 (D ‘1 p) Ut 0 Ii (D 00 0 (D Ii ‘1 Ut I-,.0 0burstduration(sec.)000burstduration(sec.)0000000M0 0C,U I00 000U I0 w o 0•100U 00 w Lii0I.IiIiiiiIII1111111III0IIIIIIiiiiiIIi11111IIX’134E15-transected chicks were also similar (Fig. 4-5C and D). The lateralgastrocnemius muscle is active during the weightbearing phase (stance phase)of the step cycle. As cycle duration increases (ie. the animal’s velocitydecreases), the duration of the stance phase increases, as does the burstduration 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 theswing phase remains relatively constant, as does the burst duration of thesartorius muscle (Hollyday and Hamburger 1977). This supports the observationthat the suppression of the onset of myelination extends the permissive periodfor 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 myelinsuppressed, E15 transected chicks is not dependent on the functionalrepair/regeneration of brainstem-spinal projections, but due to intrinsic activityof neural networks confined to the lumbar spinal cord (Sholomenko and Steeves1987; Eidelberg 1981; Steeves et al. 1987). If this were the case, however, it isunlikely that the locomotor abilities would have been so equivalent (Sholomenkoand Steeves 1987). In addition, myelin-suppressed, E15-transected hatchlingchicks were capable of initiating locomotion in response to a positive stimulus(eg. food, water or the presence of other chicks). Further evidence comes from135previous demonstrations in late embryos or hatchling chicks that direct focalstimulation of brainstem-spinal neurons within the gigantocellular reticularformation (an identified brainstem locomotor region), only elicits locomotoractivity 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 highquality of functional locomotor recovery in myelin-suppressed, El 5-transectedanimals is due to the functional repair/regeneration of brainstem-spinalconnections.Normally-myelinated or immunological control injected embryos thatreceived thoracic transections on El 5 exhibited no anatomical or functionalspinal cord repair after hatching. Retrograde tract tracing studies revealed noanatomical regrowth of severed axons across the transection site (Fig. 4-3E andF), and behavioral and physiological observations indicated no voluntarylocomotor capabilities after hatching (Fig. 4-5A and C).The lack of spinal cord repair in immunological control injected, E15-transected animals demonstrates that the spinal cord injection itself on E9-E1 2did not ‘prime’ the spinal cord to regenerate. Pre-conditioning injuries have beenshown to facilitate axonal regeneration in several systems (Richardson and Issa1984; Richardson and Verge 1986; Richardson and Verge 1987). E15-transected animals that received E9-E1 2 spinal cord injections of either nonspecific antibody plus complement, GaIC antibody only, complement proteinsonly, vehicle only (0.1M PBS, pH 7.4) or the mouse GaIC antibody plus heat-136inactivated serum showed no anatomical or functional recovery (Fig. 4-3; Fig. 4-5). This demonstrates that both the myelin-specific antibody plus complementare required to facilitate functional repair of injured spinal cord.It is most probable that the functional repair of myelin-suppressed, E15-transected spinal cord was due to true axonal regeneration. Neurogenesis in thedeveloping chick embryo is complete by E5 (McConnell and Sechrist 1980;Sechrist and Bronner-Fraser 1991). Therefore it is unlikely that recovery wasdue to projections from newly differentiated neurons. Chick brainstem-spinalprojections complete their axonal projections to lumbar levels of the spinal cordby E10-E12 (Okado and Oppenheim 1985). By E12, brainstem-spinal projectionsthroughout the spinal cord are equivalent in number and distribution to those ina hatchling chick (Okado and Oppenheim, 1985). Therefore, it is unlikely thatsubsequent development could contribute to recovery from spinal cordtransections during the final week of embryonic development. Direct proof thatthe functional repair of myelin-suppressed, El 5-transected spinal cord was dueto true axonal regeneration could be obtained from double-labelling studies. In adouble-labelling paradigm, the neuronal population with projections to thelumbar spinal cord would be maximally labelled on E12 with a retrograde tracttracer conjugated to a specific fluorochrome. After myelin-suppression, El 5thoracic transection, and a period for repair, a second retrograde tract tracerconjugated to a different fluorochrome would then be injected into the lumbarspinal cord. Only truely regenerated axons would be labelled with both137fluorochromes. Although theoretically sound, this approach could not be appliedin practice by this investigator. The additional operations required by a double-labelling study decreased the viability of experimental animals so as to renderthe study impractical and unethical.During early embryonic development, CNS neural projections have a highcapacity for plastic changes in response to injury (So et al. 1981; Shimizu et al.1990; Treherne et al. 1992). It is possible that developmental myelinsuppression may have enhanced and prolonged the capacity of developingaxonal projections for plastic changes. Plasticity of neural projections is oftenassociated with an increased expression of growth-associated protein GAP-43;GAP-43 expression in growing neurites typically decreases after contact withthe target cell (Meiri et al. 1986; Skene 1989; Baizer and Fishman 1987). Ifmyelination in the rat lumbar spinal cord is prevented by neonatal X-irradiation,the expression of GAP-43 within the spinal cord as assessed byimmunohistochemistry and immunoblotting is extended to later stages ofdevelopment (Kapfhammer and Schwab 1994). These studies suggest thatmyelin may directly or indirectly suppress the capacity of the developing CNSfor plastic changes such as sprouting and fibre growth. Developmental myelinsuppression of the chick embryo may have similarly prolonged the capacity ofdeveloping axonal projections for plastic changes. This possibility could beinvestigated by an immunohistochemical analysis of GAP-43 expression withinthe normal and myelin-suppressed chick spinal cord.138Myelin has been shown to inhibit the cellular attachment of, and neuriteoutgrowth from neuroblastoma cells in vitro (Savio and Schwab 1989; Savioand Schwab 1990). Myelin-associated proteins that inhibit the anatomicalgrowth of axons in vitro (Caroni and Schwab 1988) as well as the regrowth ofaxotomixed corticospinal fibers in vivo (Schnell and Schwab 1990) have beenidentified in rat spinal cord. Myelin also expresses the growth inhibitingmolecule janusin (Wintergerst et al. 1993; Bartsch et al. 1993). Janusin hasbeen shown to be a repulsive substrate for neuronal cell bodies as well asgrowth cones (Pesheva et al. 1989; Taylor et al. 1993). Thus, it would appearthat myelin may inhibit axonal regeneration by suppressing the capacity of thedeveloping CNS for plastic changes, as well as providing an inhibitory substratefor growing neurites.In conclusion, these findings are the first demonstration that suppressionof myelination results in both neuroanatomical repair and functional CNSrecovery after an embryonic spinal cord injury. The present data clearly confirmand extend the proposition that the presence of CNS myelin contributes to theinhibition of neuronal repair after an adult CNS injury (Ramon y Cajal 1959;Caroni and Schwab 1988; Schnell and Schwab 1990). This suggestion is alsoindirectly supported by the demonstration that lamprey, which do not havemyelinated CNS axonal fiber tracts, are capable of functional regeneration aftereither a larval or adult spinal cord injury (McClellan 1990).139CHAPTER 5NEUROANATOMICAL REPAIR AND PHYSIOLOGICAL RECOVERY FOLLOWINGTRANSECTION AND IMMUNOLOGICAL DEMYELINATION OF THE HATCHLINGCHICK SPINAL CORD140INTRODUCTIONMyelin within the embryonic chick spinal cord provides an inhibitoryenvironment for the functional regeneration of axotomized brainstem-spinalprojections concerned with locomotion (Keirstead et al. 1992). Chapter 3 of thisthesis outlines an immunological method of delaying the developmental onset ofmyelination in vivo (developmental myelin-suppression) until later stages ofembryonic development. Spinal cord transections on embryonic day (E) 15 in amyelin-suppressed animal result in complete anatomical regeneration andfunctional repair (see chapter 4). Spinal cord transections on E15 in a normallymyelinated animal result in no repair whatsoever, rendering the animalcompletely incapable of voluntary locomotion after hatching. These studiesindicate that myelin is inhibitory to the functional regeneration of transectedspinal cord in embryonic chick (Keirstead et al. 1992) and suggest thatdemyelination of hatchling spinal cord may facilitate repair following hatchlingspinal cord transections. This chapter discusses the development andcharacterization of a protocol effective in demyelinating the hatchling chickspinal cord in vivo (immunological demyelination). Additional experimentsreported here demonstrate that complete transection and concurrentimmunological demyelination of the hatchting chick spinal cord result in partialneuroanatomical regeneration of brainstem-spinal projections. Collaborativeexperiments reported in the chapter discussion indicate that this partial141neuroanatomical regeneration of demyelinated and transected hatchling spinalcord is followed by functional synapse formation such that stimulation ofbrainstem 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 (DalCantoand Lipton 1980; Herndon et al. 1977), nerve compression (Bunge et al. 1961;Clifford-Jones et al. 1980) and cell-mediated immunological reactions (Lampert1968; Raine and Bornstein 1970). A brief review of these alternativedemyelination methods may serve to illustrate the uniqueness of, andadvantages offered by, the immunological demyelination technique used in theexperiments reported here.X-ray irradiation of living tissue results in mitotic interference andultimately the death of actively dividing cells. Because neogenesis of spinal cordneurons is complete by birth in the rat and mouse, X-ray irradiation duringneonatal or adult life targets the glial population of the spinal cord. Irradiation ofthe rat spinal cord has been reported to result in an inhibition of neonatal myelinformation, as well as demyelination of adult myelinated spinal cord viadestruction of oligodendrocytes (Blakemore 1977). X-ray irradiation also resultsin failure of the astrocyte proliferation necessary to maintain the integrity of theglial limitans (Sims et al. 1985). The absence of oligodendrocytes, combinedwith defects in the glial limitans, leads to Schwann cell myelination of CNS142axons (Sims et al. 1985).Primary demyelination of adult CNS can also be obtained viaadministration 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 thismanner is relatively long-term (greater than 6 months for lysolecithin in rabbits)and may or may not be followed by oligodendrocyte remyelination (Blakemore1978). Remyelination is often accomplished by invading Schwann cellsfollowing drug-induced demyelination (Blakemore 1982). Although effective inevoking oligodendrocyte cell death and primary demyelination, drugadministration is often also accompanied by a loss of astrocytes (Blakemore1992; Blakemore 1978). Cuprizone-induced demyelination, on the other hand, isaccompanied by proliferation of astrocytes characterized by swollen processedand a ‘watery’ cytoplasm (Ludwin 1978). In the case of diptheria toxin,demyelination is accompanied by extensive scarring and Wallerian-typedegeneration of nerve fibres surrounding the injection site (Eames et al. 1977).Models of virally-induced demyelination include Theiler’s murineencephalomyelitis virus (TMEV) infection (Dal Canto and Lipton 1980) and JHMmouse hepatitis virus infection (Herndon et al. 1977). Both of these modelsresult in cytolytic infection of oligodendroglia and subsequent myelin breakdownaccompanied by prominent gliosis and extensive inflammatory cell infiltrates(Dal Canto and Lipton 1980; Herndon et al. 1977). Demyelination induced by143TMEV infection lasts for as long as 8 months and remyelination is predominantlydue to Schwann cells (Dal Canto and Lipton 1980). Demyelination induced byJHM virus is relatively short-lived; remyelination begins within two weeks post-infection 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 predictabilityas to the site of the lesion (Dal Canto and Lipton 1980; Herndon et al. 1977).Other methods of inducing primary demyelination in vivo include nervecompression (Bunge et al. 1961; Clifford-Jones et al. 1980) and experimentalallergic encephalomyelitis (Raine and Bornstein 1970; Lampert 1968). Althougheffective in producing myelin loss, these demyelinating methods are complicatedby extensive proliferation of astrocytes and inflammatory cells as well as axonaldegeneration (Clifford-Jones et al. 1980; Raine and Bornstein 1970).In order to study the specific effects of myelin on the ability of thehatchling chick spinal cord to regenerate following injury, it was necessary toemploy a method of demyelination that does not produce non-specificpathological changes such as a chronic breach of the glial limitans, astrogliosis,intense inflammation or axonal degeneration. Unfortunately, each of thedemyelination methods outlined above suffer significantly from one or more ofthese non-specific complications. This prompted the development of theimmunological method of myelin suppression outlined in this chapter andchapter 3. The experiments presented here illustrate that the injection of144galactocerebroside (GaIC) antibodies or 04 antibodies plus complement proteinsinto 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 todemyelinate guinea pig optic nerve (Sergott et al. 1982; Ozawa et al. 1989), catoptic 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 3illustrate that immunological demyelination is not accompanied by alterations ofthe axonal or astroglial population of the spinal cord. Details regarding theinteraction of complement plus GaIC or 04 antibody with the oligodendrocyteand myelin membranes are discussed in chapter 3 of this thesis. -Immunological demyelination was employed in the present experiments inorder to test the hypothesis that the removal of spinal cord myelin wouldfacilitate neuroanatomical and. functional regeneration by providing a morepermissive extraneuronal environment for regenerating brainstem-spinalprojections within the transected hatchling chick spinal cord. CNS myelin andcultured oligodendrocytes are strong inhibitors of neurite outgrowth in vitro(Schwab and Caroni 1988). Myelin and myelin-associated proteins have alsobeen shown to inhibit the functional regeneration of transected spinal cord inthe embryonic chick (Keirstead et al. 1992) as well as the neuroanatomicalregeneration of corticospinal fibres in the rat (Schnell and Schwab 1990).The hypothesis outlined above assumes that hatchling chick spinal cord145neurons have the intrinsic ability to regenerate, given a permissive extraneuronalenvironment. Although vertebrate CNS axons will not regrow in the environmentof the adult spinal cord (Eidelberg 1981; Sholomenko and Steeves 1987),peripheral nerve grafts into the CNS provide a favorable environment throughwhich CNS axons will regenerate (Ramon y Cajal 1959; David and Aguayo1981; Aguayo et al. 1991). CNS neurons have also been shown to extendfibres in vivo through lesion sites containing implants of fetal CNS tissue(Bjorklund and Stenevi 1979; Kromer et al. 1981; Bjorklund 1991), as well asimplants consisting of fibroblasts genetically modified to express growth factors(Fisher and Gage 1993; Tuszynski et al. 1994). In addition, motor neuronsresiding within the ventral horn of the spinal cord are capable of regeneratingtheir peripheral projecting axon if axotomized within the PNS (Ramon y Cajal1959). These findings indicate that adult CNS neurons retain intrinsic axonalgrowth programs which enable long-distance axonal regeneration in thepresence of a favorable extraneuronal environment.The experiments presented here illustrate that complete transection of thehatchling chick spinal cord in vivo followed immediately by 2-3 weeks ofimmunological demyelination results in partial regeneration of brainstem-spinalprojections. Retrograde tract tracing studies indicate that 5-15% of brainstemspinal projecting neurons (as compared to the maximal number of retrogradelylabelled brainstem-spinal projecting neurons obtained from untransected controlhatchlings) regenerate processes through the previous thoracic transection site146to lumbar levels of the spinal cord. Evidence presented in the chapter discussionillustrates that these regenerated fibres make functional synaptic connectionswith motorneurons of the lumbar spinal cord.147MATERIALS AND METHODSImmunological DemyellnationBirds between the ages of post-hatching day (P) 1 and P9 wereanesthetized with an intramuscular injection of ketamine hydrochloride (30mg/kg) plus xylazine (Rompun, 3 mg/kg). After removal of the dorsal vertebraeoverlying the low thoracic spinal cord, direct spinal cord injections wereperformed using a glass micropipette (tip diameter = 30-40 urn; A-M Systems,Everett, Washington #6045) connected to a Picospritzer II pressure injectionsystem (General Valve Corp., Fairfield, New Jersey). Each animal received atotal volume of 10 ul, over 1-4 penetrations. Alternatively, solution wasdelivered over a longer time period by inserting into the exposed low thoracicspinal 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 dorsalsurface of the neck. The pumps used in these experiments deliver solution at arate of 0.5 ul per hour, or 12 ul per day. Immunological demyelination wasevoked with either an lgG1 rabbit GaIC antibody (Chemicon International Inc.,Temecula, California #AB142) at a dilution of 1:5 with 33% guinea pigcomplement (Gibco BRL, Burlington, Ontario #19195-01 5) in O.1M phosphatebuffered saline (PBS), pH 7.4 or an 1gM polyclonal 04 antibody (a gift fromMelitta Schachner, Wayne State University School of Medicine, U.S.A.) at adilution of 1:5 with 33% guinea pig complement (Gibco BRL, Burlington, Ontario148#19195-01 5) in 0.1M PBS pH 7.4.Immunological control hatchlings were administered either: 1) GalCantibody only, 2) 04 antibody only, 3) guinea pig complement only or, 4) vehicleonly (0.1M PBS, pH 7.4). These immunological control solutions were deliveredby direct spinal cord injection as well as by pump.Animals not undergoing further experimentation were perfusedintracardially with 0.1M PBS containing 2500 USP units of heparin per 50 mIsPBS, pH 7.4 (at 37°C) followed by perfusion with 4% paraformaldehyde in0.1M phosphate buffer, pH 7.4 (at 4-10°C). The dissected tissue was thenpostfixed for 24 hours at 4°C and subsequently embedded in paraffin forimmunohistochemical analysis (see below).Spinal Cord TransectionBirds between the ages of post-hatching day (P) 4 and P9 wereanesthetized with an intramuscular injection of ketamine hydrochloride (30mg/kg) plus xylazine (Rompun, 3 mg/kg). After removal of the dorsal vertebraeoverlying the low thoracic spinal cord, the spinal cord was transected using asharpened corneal blade (Fine Science Tools, North Vancouver, BritishColumbia) with a diameter roughly equivalent to the inside diameter of thehatchling chick spinal cord. The corneal blade was inserted through the spinalcord to the inner ventral surface of the spinal column and gently rotated.To ensure complete transection of the spinal cord, hatchling chicks wererandomly selected immediately after transection and their spinal cords fresh-149dissected and examined under a dissecting microscope. In addition, somehatchling chicks received lumbar injections of 0.5 ul of tetramethyirhodaminelabelled dextran amine (RDA; see ‘neuroanatomy’ below) at the time of thoracicspinal cord transection. 21 days after RDA injection, they were perfusedintracardially with 0.1M PBS containing 2500 USP units of heparin per 50 mIsPBS, pH 7.4 (at 37°C) followed by perfusion with 4% paraformaldehyde in0.1M phosphate buffer, pH 7.4 (at 4-10°C). Others received lumbar injections of0.5 ul of RDA 17 days after the thoracic spinal cord transection. 48 hours afterRDA injection, they were perfused as outlined above. The dissected tissue wasthen postfixed for 24 hours at 4°C. The dissected brainstems were transferredto 30% sucrose in 0.1M PBS, pH 9.0 (4°C) for 24 hours. Each brainstem wassectioned in the transverse plane using a Leitz liquid CO2 freezing microtomeand 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 injectionsite and transection site.Paraffin EmbeddingSee page 29.ImmunohistochemistrySee page 30.NeuroanatomySee page 112.150RESULTSImmunological DemyellnationThe removal of spinal cord myelin from the hatchling chick was achievedby administering complement proteins plus an oligodendrocyte-specific,complement-binding antibody directly into the low thoracic spinal cord betweenposthatching day (P) 1 and P9 (immunological demyelination). The state ofspinal cord myelination within experimental and control animals was assessedwith myelin basic protein (MBP) immunohistochemistry. Polyclonalgalactocerebroside (GaIC) antibody and polyclonal 04 antibody areoligodendrocyte-specific, complement-binding antibodies that, when combinedwith guinea pig complement, caused demyelinating lesions within the spinalcord that extended for approximately six (6) spinal cord segments either side ofthe injection site in the majority of cases (n = 69, 60 with single injections and 9with osmotic mini-pumps). Of the 69 demyelinated lesions examined, 14 wererestricted to one side of the spinal cord only, 28 were limited to 2-4 spinal cordsegments either side of the injection site, and 2 were accompanied by anadditional demyelinated lesion in the cervical region of the spinal cord. Theextent and degree of spinal cord demyelination was similar when thecomplement 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 demyelinatedspinal cords contained ‘clumps’ of MBP immunoreactivity homogeneouslydistributed throughout the myelin lesion (Fig. 5-iC).151Figure 5-1. Immunological demyelination in the thoracic spinal cord of thehatch 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: MBPimmunofluorescence staining of a P7 spinal cord from an animal that received asingle injection of polyclonal galactocerebroside (GaIC) antibodies pluscomplement on P6; note the absence of myelin. C: MBP immunofluorescencestaining of a P7 spinal cord from an animal that received a single injection ofpolyclonal galactocerebroside (GaIC) antibodies plus complement on P6; notethe presence of myelin ‘clumps’. In all photograghs the outer edge of the spinalcord lies on the right hand side, with the white matter adjacent. (Bars =1 OOum).“1153Immunological control hatchlings were administered either: 1) GaICantibody only (n = 12), 2) 04 antibody only (n = 12), 3) guinea pig complementonly (n=12) or, 4) vehicle only (0.1M PBS, pH 7.4; n=12) on P6. From eachgroup of immunological control hatchlings, four animals were sacrificed on eachday, 1, 5 and 1 2 days after treatment. MBP immunohistochemical analysis oftheir spinal cords revealed no evidence of demyelination (Fig. 5-2).Single micropipette injections of complement proteins plusoligodendrocyte-specific antibody into the spinal cord were performed on a totalof 102 hatchlings (91 animals with GaIC antibodies and 11 animals with 04antibodies). See table 1. There was no evidence of immunological demyelination6 or 9 hours after injection (Fig. 5-3A), however, decreased MBP -immunoreactivity was evident in 3 of 5 animals sacrificed 12 hours afterinjection (Fig. 5-3B). Immunological demyelination was evident 24 and 48 hoursafter injection (Fig. 5-3C) and in the majority of animals sacrificed 3 days afterinjection. No signs of immunological demyelination was detected in animalssacrificed 4 and 5 days after injection (Fig. 5-3D).Long-term infusion (with 7- and 14-day Aizet osmotic mini-pumps) ofcomplement plus GaIC antibody into the spinal cord was performed on a total of23 hatchlings. Immunological demyelination was clearly evident in 5 animalssacrificed 5 days after installation of a 7-day pump. Immunologicaldemyelination was also evident in 4 animals sacrificed 12 days after installationof a 14-day pump (Fig. 5-4A). No evidence of immunological demyelination was154Figure 5-2. Pattern of myelin basic protein (MBP) immunoreactivity inimmunological control thoracic spinal cords in parasaggital section. A: MBPimmunofluorescence staining of a posthatching day (P) 7 spinal cord from ananimal that received a single injection of polyclonal galactocerebroside (GaIC)antibodies only on P6; note that myelin is unperturbed. B: MBPimmunofluorescence staining of a posthatching day (P) 7 spinal cord from ananimal that received a single injection of complement only on P6; note thatmyelin is unperturbed. In all photograghs the outer edge of the spinal cord lieson the right hand side, with the white matter adjacent. (Bars = lOOum).Table 5-1: Summary of results from single injection of complement proteinsplus GaIC or 04 antibodies directly into the hatchling chick spinal cordSacrifice Time Post- Total Number Number Normally-Injection Demyelinated Myelinated6hours 4 0 49hours 4 0 4l2hours 5 3 21 day 36 30 (8 with 04 Ab) 62days 11 8 33 days 31 19 (3 with 04 Ab) 124days 7 0 75days 4 0 4157Figure 5-3. Time course of immunological demyelination and remyelinationfollowing a single injection of polyclonal galactocerebroside (GaIC) antibodiesplus complement. A: Myelin basic protein (MBP) immunofluorescence staining ofa hatchling chick spinal cord in parasaggital section 9 hours after the injectionof GaIC antibodies plus complement; note the presence of myelin. B: MBPimmunofluorescence staining of a hatchling chick spinal cord in parasaggitalsection 12 hours after the injection of GaIC antibodies plus complement; notethat myelin is decreased. C: MBP immunofluorescence staining of a hatchlingchick spinal cord in parasaggital section 24 hours after the injection of GaICantibodies plus complement; note the absence of myelin. D: MBPimmunofluorescence staining of a hatchling chick spinal cord in parasaggitalsection 5 days after the injection of GaIC antibodies plus complement; note thepresence of myelin. In all photograghs the outer edge of the spinal cord lies onthe right hand side, with the white matter adjacent. (Bars = lOOum).160Figure 5-4. Immunological demyelination and remyelination with a 14 dayosmotic mini-pump containing polyclonat galactocerebroside (GaIC) antibodiesplus complement. A: Myelin basic protein (MBP) immunofluorescence staining ofa hatchling chick thoracic spinal cord in parasaggital section 12 days after theinstallation of an osmotic mini-pump containing GaIC antibodies pluscomplement; note the absence of myelin. B: MBP immunofluorescence stainingof a hatchling chick thoracic spinal cord in parasaggital section 21 days afterthe installation of an osmotic mini-pump containing GaIC antibodies pluscomplement; note the presence of myelin. In all photograghs the outer edge ofthe spinal cord lies on the right hand side, with the white matter adjacent. (Bars= lOOum).162detected in 6 animals sacrificed 21 days (Fig. 5-4B), and 8 animals sacrificed 24days, after installation of a 14-day pump.The astrocyte population of the spinal cord did not appear to be disturbedby immunological demyelination, as assessed by glial fibrillary acidic protein(GFAP) immunohistochemistry (Fig. 5-5). The astrocyte number and distributionin immunologically demyelinated spinal cords (Fig. 5-5B) was similar to that ofunoperated control tissue of the same age (n=8; Fig. 5-5A).Immunohistochemical analysis of spinal cords from animals sacrificed 24 hoursafter the injection of complement proteins and GaIC antibodies on P2 (n=3) andP5 (n=3), revealed no evidence of astogliosis. Additionally, individualastrocytes in demyelinated spinal cords did not appear to express higher levelsof GFAP than individual astrocytes from unoperated control tissue of the sameage.Direct pressure injection of control and experimental solutions into thethoracic spinal cord did not result in significant damage to the spinal cordtissue. The injected solution did not detectably displace spinal cord tissue orresult in an area of necrosis at the injection site. Likewise, canula insertion intothe spinal cord was not followed by necrosis or cavity formation. Three weeksafter pump installation, the hole in the spinal cord initially created by the canuladid not exceed the diameter of the canula itself.TransectionTransection of the hatchling chick spinal cord did not result in the163Figure 5-5. Pattern of glial fibrillary acidic protein (GFAP) immunoreactivityduring immunological demyelination. A: Unoperated (normally-myelinated)control posthatching day (P) 6 spinal cord showing extensive GFAPimmunoreactivity within the white matter. B: GFAP immunofluorescencestaining of a P6 spinal cord from a hatchiing chick that received a singleinjection of polyclonal galactocerebroside (GaIC) antibodies plus complement onP5; note that GFAP immunoreactivity is comparable to A, indicating thatimmunological demyelination does not induce an astrogliotic response. In allphotograghs the outer edge of the spinal cord lies on the right hand side, withthe white matter adjacent. (Bars = lOOum).165formation of a large cavity between the proximal and distal segments of thetransected spinal cord (Fig. 5-6). In all of the 43 spinal cords examined 1 5-24days after complete transection, the proximal and distal segments wereintimately juxtaposed at the transection site. Autofluorescent inflammatoryinfiltrates at the transection site were minimal.To ensure complete transection of the spinal cord, 1 2 hatchling chickswere randomly selected immediately after spinal cord transection, freshdissected, and examined under a dissecting microscope. In all cases a completespinal cord transection was confirmed.In addition, 6 animals received a lumbar injection of RDA at the time ofthoracic spinal cord transection. Only a complete spinal cord transection wouldprevent the retrograde axonal transport of the tracer to the cell bodies of originin the brainstem (Fig. 5-7). Twenty-one (21) days after RDA injection, they weresacrificed and their brains examined for the presence of retrogradely-labelledbrainstem-spinal neurons. In no case was there evidence of retrograde transportof RDA to the brainstem (Fig, 5-7A). In all cases analysis of the spinal cordsrevealed a discrete area of RDA-Iabelled cells within the lumbar spinal cord andno evidence of RDA transport to any region of the spinal cord rostral to theprevious transection site (Fig. 5-7B).Other normally-myelinated control animals (n=3) received lumbarinjections of 0.5 ul of RDA 17 days after the thoracic spinal cord transection.Again, only a complete spinal cord transection would prevent the retrograde166Figure 5-6. Spinal cord transection site in parasaggital section 20 days aftercomplete thoracic transection and 14-day immunological demyelination. Notethat the proximal and distal segments of the spinal cord are intimatelyjuxtaposed at the transection site. Cavity formation was never observed inspinal cords examined 15-24 days post-transection and autofluorescentinflammatory infiltrates were minimal. (Bars = 5Oum).168Figure 5-7. The transection procedure reliably severs the entire thoracic spinalcord. Control embryos received lumbar injections of tetramethylrhodaminelabelled dextran amine (RDA) solution at the time of the thoracic spinal cordtransection. Only a completely transected spinal cord would prevent theretrograde transport of this neuroanatomical tracer to the brainstem. A:Ventromedial reticular formation of the caudal pons in a posthatching day (P) 26chick 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; notethe neuronal and axonal labelling evident near the previous injection site. Thisconfirms that the transection procedure reliably severs the entire thoracicspinalcord. Similar results were observed when RDA was injected 17 days aftercomplete thoracic spinal cord transection of a normally-myelinated (control)hatchling chick. (Bars = 5Oum for A, 100 urn for B).170axonal transport of the tracer to the cell bodies of origin in the brainstem. 48hours after RDA injection, they were sacrificed and their brains examined for thepresence of retrogradely-labelled brainstem-spinal neurons. In no case was thereevidence of retrograde transport of RDA to the brainstem. Analysis of the spinalcords revealed in all cases a discrete area of RDA-labelled cells within thelumbar spinal cord and no evidence of RDA transport to any region of the spinalcord rostral to the previous transection site.Demyellnation and TransectionSpinal cord transections and osmotic mini-pump installations (containingdemyelinating or control solutions) were performed an a total of 48 hatchlingchicks aged P4-P9. In all cases, demyelinating or control solutions were -administered using a 14-day osmotic mini-pump connected via catheter to acanula inserted 2 spinal cord segments caudal to the transection site.Neuroanatomical assessments of spinal cord regeneration were conducted on all48 of these animals. Physiological assessments of spinal cord regeneration (inaddition to the neuroanatomical assessments) were conducted on 8 of theseanimals (see chapter discussion). In all cases, the spinal cord transectionpreceded the osmotic mini-pump installation by approximately 15 minutes (thetime required for the surgery itself).Neuroanatomical regeneration was assessed 15-24 days after spinal cordtransection and immunological demyelination by counting the brainstem-spinalneurons labelled by the retrograde transport of RDA injected into the lumbar171spinal cord 24 hours prior to sacrifice (Fig. 5-8). In transected, demyelinatedhatchling chicks the number of retrogradely-labelled neurons within the reticularformation averaged 148 cells (range= 71-202; n=21; Fig. 5-8B) which isapproximately 14% of the number of retrogradely-labelled neurons within thereticular formation of RDA-injected, unoperated controi hatchlings of the sameage (average= 1043; range= 692-1311; n= 11; Fig. 5-8A). Comparablenumbers and distributions of retrogradely-labelled neurons were also noted forother brainstem-spinal nuclei with projections to the lumbar spinal cord includingthe vestibular nucleus (Fig. 5-8C and D), locus ceruleus, subceruleus nucleusand raphe nucleus.In contrast, transected hatchlings that were administered controlsolutions over a 2-week period showed no signs of neuroanatomicalregeneration 15-24 days post-transection (Fig. 5-8E and F). No retrogradelylabelled brainstem-spinal projecting neurons were observed in transectedhatchling 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) overa 2-week period.Prior to sacrifice, hatchling chicks that had been transected anddemyelinated for 2 weeks did not show any behavioral signs of locomotorrecovery. From the day of spinal cord transection, all animals remainedincapable of standing or supporting their own weight. Recumbancy was sternalin most cases. Alternating leg movements were often observed. A decrease in172Figure 5-8. Neuroanatomical regeneration of brainstem-spinal projections.Photomicrograghs of retrogradely labelled neurons within the brainstem inposthatching day (P) 28 chicks. Brainstem-spinal neurons were labelled by theretrograde axonal transport of tetramethylrhodamine-labelled dextran amine(RDA) injected into the lumbar spinal cord on P26 and allowed two days fortransport. A: RDA labelled gigantocellular reticulospinal neurons within theventromedial reticular formation of the caudal pons in an unoperated (normallymyelinated) control P28 chick. B: RDA labelled gigantocellular reticulospinalneurons within the ventromedial reticular formation of the caudal pons in a P28experimental hatchling that was subjected to immunological demyelination andcomplete thoracic spinal cord transection on P5; note that the number anddistribution of retrogradely labelled neurons is substantially less than A. C: RDAlabelled vestibulospinal neurons within the lateral vestibular nucleus of thedorsolateral pons in an unoperated (normally-myelinated) control P28 chick. D:RDA labelled vestibulospinal neurons within the lateral vestibular nucleus of thedorsolateral pons in a P28 experimental hatchling that was subjected toimmunological demyelination and complete thoracic spinal cord transection onP5; note that the number and distribution of retrogradely labelled neurons issubstantially less than C. Comparable neuroanatomical repair was evident forother brainstem-spinal projections. E: Ventromedial reticular formation of thecaudal pons in a P28 hatchling that was transected on P5 and receivedgalactocerebroside (GaIC) antibodies only over a 14 day period; note the173absence of retrogradely labelled neurons. F: Lateral vestibular nucleus of thedorsolateral pons in a P28 hatchling that was transected on P5 and receivedgalactocerebroside (GaIC) antibodies only over a 14 day period; note theabsence of retrogradely labelled neurons. A similar lack of neuroanatomicalrepair was observed in animals that received other immunological controlsolutions over a 14 day period and a complete thoracic spinal cord transectionon P5. (Bars = 5Oum).177the 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 (andnot neogenesis and\or subsequent development), a double labelling paradigmwas adopted (Fig. 5-9). On P6 a maximal number of brainstem-spinal projectionswas labelled with a lumbar spinal cord injection of RDA. After 24 hours to allowfor retrograde transport, the thoracic spinal cords were completely transectedand an osmotic mini-pump containing GaIC antibodies and complement proteinsinstalled 2 segments caudal to the transection site. A second retrograde tracttracer (cascade blue labelled dextran amine; CBDA) was injected into the lumbarspinal cord 20 days later. Analysis of the brainstem 24 hours later revealeddouble-labelled neurons within the major locomotor regions of the brainstemwith 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 byviewing the brain sections with a non-specific filter. When viewed with afluoresceine filter, no cells were visible, indicating that the RDA and CBDAlabelling was indeed specific, and not due to autofluorescence (Fig. 5-9C). Lessthan 10 cells per nucleus were double-labelled. Previous studies have indicatedthat, even in an untransected chick, the maximal number of double-labelledneurons obtainable from the intraspinal injection of 2 different retrograde tracttracers is 30% of the number labelled by either tracer alone (Hasan et at. 1993).178Figure 5-9. Double labelling of brainstem-spinal projections. Photomicrographsof retrogradely double-labelled reticulospinal neurons within the ventromedialreticular formation of the caudal pons in the chicken. Brainstem-spinal neuronswere first labelled by retrograde axonal transport of rhodamine-labelled dextranamine (RDA) injected into the lumbar spinal cord on P6. The thoracic spinal cordwas completely transected on P8. The infusion of complement plusgalactocerebroside (GaIC) antibodies into the thoracic cord (2 segments caudalto the transection site) was begun immediately thereafter via an osmotic pumpfor the next 14 days. On P28, the second retrograde tracer, cascade blue-labelled dextran amine (CBDA), was injected into the lumbar spinal cord and theanimal was sacrificed two days later on P30. A: Gigantocellular reticulospinalneurons labelled prior to spinal transection with the first retrograde tracer, RDA.B: Giganotcellular reticulospinal neurons subsequently labelled with the secondtracer, CBDA, after immunological demyelination and spinal transection. NOTE:some of the same brainstern-spinal neurons are double-labelled with both RDAand CBDA. C: Fluorescence photomicrograph of the same photographic fieldusing a (green) fluorosceine filter. This indicates that the double-labelling ofreticulospinal neurons (RDA + CBDA) was not an autofluorescent artifact. (Bars= 50 urn).A180DISCUSSIONThe results presented here demonstrate that the transient removal ofmyelin from the hatchling chick spinal cord by immunological means facilitatespartial neuroanatomical regeneration of axotomized brainstem-spinal projectionsin vivo. Collaborative efforts described below further indicate that this partialneuroanatomical 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 ofaxotomized brainstem-spinal projections in the post-hatching chick.The injection of galactocerebroside (GalC) antibodies or 04 antibodies andcomplement proteins directly into the spinal cord of the P1-P9 hatchling chickresults in immunological demyelination which extends several segments eitherside of the injection site (Fig. 5-1; Fig. 5-3; Fig. 5-4). The astrocyte populationof the spinal cord is undisturbed by the demyelination procedure (Fig. 5-5).Immunological demyelination takes place within 24 hours (Fig. 5-3) and persistsfor as long as the antibodies and complement proteins are administered (thelongest trial period was 14 days; Fig. 5-3; Fig. 5-4). Remyelination of the spinalcord takes place within 4-7 days of the cessation of treatment (Fig. 5-3; Fig. 5-4). Neither antibody alone, complement proteins alone, nor vehicle (PBS) alonecaused demyelination when injected into the spinal cord, indicating that boththe oligodendrocyte-specific antibody and complement proteins are necessary in181order to evoke immunological demyelination (Fig. 5-2).GaIC antibodies and complement proteins were delivered to the hatchlingchick spinal cord over a 14-day period in an attempt to rescue previouslyaxotomized brainstem-spinal projections. Osmotic mini-pumps containing GaICantibodies and complement proteins were installed shortly after spinal cordtransection, and neuroanatomical regeneration of brainstem-spinal projectionswas assessed 15-24 days later by counting the brainstem neurons retrogradelylabelled by an injection of tetramethyirhodamine-labelled dextran amine (RDA)into the lumbar spinal cord (Fig. 5-8). In transected, demyelinated animals thenumber of retrogradely-labelled brainstem-spinal neurons was approximately 5-15% of the number of retrogradely-labelled brainstem-spinal neurons in RbAinjected, untransected control hatchlings of the same age. Transected animalsthat received no treatment or control solutions (GaIC antibody only, complementproteins only or vehicle only) over a 2-week period showed no signs ofneuroanatomical regeneration.True axonal regeneration contributed to the neuroanatomical recovery oftransected, demyelinated hatchling chicks as evidenced by double-labelling ofbrainstem-spinal projecting neurons (Fig. 5-9). Prior to transection and initiationof immunological demyelination, the existing population of brainstem-spinalprojecting neurons were maximally labelled with a lumbar injection of RDA. Asecond retrograde tract tracer (cascade blue labelled dextran amine; CBDA) wasinjected into the lumbar spinal cord 20 days later. Analysis of the brainstem 48182hours later revealed double-labelled neurons within the major locomotor regionsof the brainstem with projections to the lumbar spinal cord. This demonstratesthat axons labelled with the first tracer (RDA) regenerated through thetransection site to pick up the second tracer (CBDA). This finding is supportedby studies which indicate that neurogenesis of brainstem-spinal projectingneurons in the chick is complete by embryonic day (E) 5 (McConnell andSechrist, 1980; Sechrist and Bronner-Fraser, 1991) and the development ofbrainstem-spinal projections is complete to all levels of the spinal cord by ElO orEli (Okado and Oppenheim 1985).In all cases, RDA was injected approximately 8mm caudal to the previoustransection site. Prior studies have indicated that RDA remains confined to thearea of injection (ie. does not diffuse rostrally to or above the site oftransection; Hasan et al. 1991; Keirstead et al. 1992; Hasan et al. 1993).Transected and demyelinated .hatchling chicks lived, on average, 21 days priorto neuroanatomical assessments of regeneration, which indicates a rate ofaxonal regeneration of approximately 0.4mm per day. The rate of axonalregeneration reported here is much slower than those reported in other studies,which document rates of axonal outgrowth of 1-2mm per day (Ramon y Cajal1959; Schnell and Schwab 1990). This discrepancy may be accounted for byan initial period of axonal retraction immediately following spinal cordtransection, which the present techniques are incapable of discerning. Therelatively severe nature of the lesion used in the present studies may be183associated with a greater degree of secondary degenerative events which mayextend the period of axonal retraction and/or impede the growth of regeneratingaxons. Alternatively, the rates of axonal outgrowth in the chick CNS may beinherently slower than those of the rat and mouse.The neuroanatomical regeneration of transected, demyelinated hatchlingssummarized above did not result in any behavioral signs of locomotor recovery.After spinal cord transection, all animals remained incapable of standing orsupporting their own weight. Although alternating leg movements were oftenobserved, this does not indicate supraspinal-mediated voluntary control oflocomotion. When supported on a treadmill, transected animals are capable oflocomotion, mediated by the intinsic activity of neural networks confined to thelumbar spinal cord (Sholomenko and Steeves 1987; Eidelberg 1981; Steeves etal. 1987). The lack of behavioral recovery in transected, demyelinatedhatchlings may have been due to insufficient leg musculature. A decrease in thesize of the leg muscles during the recovery period post-transection indicatedextensive muscular atrophy in all cases. Alternatively, the lack of behavioralrecovery in transected, demyelinated hatchlings may have been due to a lack ofsynaptogenesis of regenerated brainstem-spinal axons with spinal cordmotoneurons or interneurons, ultimately innervating the leg muscles. However,the experiments described below negate this possibility.Physiological assessments of locomotor capabilities of transected anddemyelinated or control-treated (PBS only or GalC only) hatchling chicks were184undertaken in collaboration with Dr. Gerry Sholomenko (a postdoctoral fellowunder 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 theinitiation of immunological demyelination, brainstem stimulation (3OHz/0.5msecmonophasic pulses) of decerebrate, hatchling chicks evoked motor activity bothabove (pectoralis; PECT) and below the site of transection (n = 3).Electromyograms from the sartorius muscles (SART; major hip flexor) illustratealternating periods of activity in the right and left legs (Fig. 5-10). Followingelectromyographic recording, the animals were paralysed and unidirectionallyventilated to prevent movement artifact. Electroneurograms from an episode ofbrainstem 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 electromyographicactivity above the transection site but no electromyographic orelectroneurographic activity was present be!ow the transection site. All of theseanimals were subsequently included in the neuroanatomical studies outlinedabove. These findings indicate that neuroanatomical regeneration of brainstemspinal projections following spinal cord transection in immunologicallydemyelinated hatchlings is accompanied by functional synaptogenesis withspinal cord neurons which directly or indirectly innervate the muscles of thelegs.The proper connectivity of growing neurites with their targets presumably185Figure 5-10. Electromyograms (EMG) from the sartorius (SART; major hip flexor)and pectoralis (PECT; major wing depressor) muscles of a decerebrate brainstemstimulated 27 day chick subjected to immunological demyelination andtransection on posthatching day (P) 9. Brainstem stimulation at 5OuA(3OHz/O.5msec monophasic pulses) evoked motor activity both above andbelow the transection site. In this case the wings were held to preventmovement artifact in the leg myograms.LllIkJ•111 J,I1J__________1SECL.SARTI11111IIIiIIIiiIIR.SARTiiirr1I1’FhIIliiII,—a-I-.LLLI1 11hgLiftJIIIaliLh“ia111pI1III1tjIL.PECTIII-1 hIALIiI.IIIII-I[.1T’iA[--1187Figure 5-11. Electroneurograms (ENG) from an episode of brainstem stimulated(300uA) activity in the same chick as Fig. 5-10. The animal was paralyzed andunidirectionally ventilated to prevent movement artifact. At the start ofstimulation one of the left toes was held for ten seconds and released. The toptwo traces show the start of the episode with a slow time base to demonstratethe onset of rhythmic pectoralis (PECT) activity and mainly tonic sartorius(SART) activity. The expanded bottom two traces show the presence ofrhythmic SART activity superimposed upon the increase in tonic activity belowthe transection site.LiJIU Ii ItII1’j1 iJ JiiI ii iiJr jr IIiiIl)JL I’ jLLid j’rj[IIPECTAPECT‘r1I.SARTi .1. j1 iIiII‘I•I I ‘II I’ ‘1 1’‘r iiB1.1 liii ii ,I ii liiiSARTI II••l II‘‘‘I •1I! L 1i I IIII••III189arises as a result of transient spaciotemporal patterns of guidance factors duringdevelopment, or to the presence of complementary guidance factors which arepermanent features of the growing neurites, the pathway(s) and the targettissue(s). The neuroanatomical regeneration and functional synaptogenesisobserved following transection and demyelination of the hatchling chick (ie.mature) spinal cord would support the later scenario. These two scenarios arenot mutually exclusive. Neuroanatomical regeneration and functionalsynaptogenesis with appropriate targets has also been observed followingaxotomy of the fibre connections between the entorhinal area and thehippocampal complex (Li et al. 1994). In horizontal slices of rat postnatal day 9and 10 tissue, severed fibre projections between the entorhinal cortex and thehippocampal complex regenerated in both directions and re-established correctlaminar, pathway and target specificity. This study lends further support to thesuggestion 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 isextremely fast (immunological demyelination takes place within 24 hours oftreatment; see Table 1; Fig. 5-3). The antibody-mediated, complementdependent nature of immunological demyelination may account for its rapidonset. Complement fixation by cell-surface binding antibodies has been shownto compromise the ionic homeostasis of many different cell types in culturewithin minutes or hours of treatment (Mayer 1972; Morgan 1989). GalC190mediated, complement-dependent injury of oligodendrocyte myelin in the guineapig optic nerve in vivo (Sergott et al. 1984) is detectable within 1-2 hours oftreatment.Remyelination following immunological demyelination is also extremelyfast, occurring within one week of the cessation of treatment (Fig. 5-3; Fig. 5-4). The speedy onset of remyelination following immunological demyelinationitself suggests that oligodendrocyte cell bodies survive the immunologicaldemyelination procedure, and are a likely origin of subsequent myelin.Complement membrane attack of neutrophils in culture is accompanied by ainhibition of energy-requiring processes, followed by complete biochemical andfunctional cell recovery when the cells are transferred to complement-freemedium (Morgan 1988). In oligodendrocytes, complement membrane attack invitro is also followed by a complete restoration of cellular energy stores(Scolding et al. 1989).More direct proof of oligodendrocyte cell body survival followingimmunological demyelination was obtained from collaborative studiesundertaken with Dr. Alan Peterson (Mount Royal Hospital, Monteal, Quebec).Immunological demyelination as described above was performed on MBP-LacZtransgenic mice (described in Foran and Peterson 1993; Fig. 5-12). Theoligodendrocyte cell bodies of this transgenic mouse line contain the LacZ geneproduct, fl-galactosidase, which can be detected with the histological stain BlueX-Gal (Foran and Peterson 1993). The injection of GaIC antibodies and191Figure 5-12. Immunological demyelination of the MBP-Lac Z, dlO-8O transgenicmouse 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 positiveoligodendrocyte distribution on the surface of the spinal cord; note the focaldecrease in fl-galactosidase positive oligodendrocytes at the site of injection.This suggests that oligodendrocyte cell bodies are destroyed at the site ofinjection. B: Photomicrograph of toluidine blue stained, ,8-galactosidase reactedtransverse 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 aroundthe area of injection. This section was taken from the same spinal cord shownin whole mount above (A). C: Photomicrograph of toluidine blue stained, figalactosidase reacted transverse section of an unoperated control spinal cord;note the abundance of myelinated axons (arrows) in the vicinity ofoligodendrocytes (stars).C/ /193complement proteins into the spinal cord of postnatal day 12 transgenic miceresulted 24 hours later in oligodendrocyte cell death which was restricted to theimmediate injection site (most probably due to the injection itself; Fig. 5-1 1A).Toluidine blue staining of lum transverse sections of these spinal cords 2segments rostral to the injection site indicated a dramatic decrease in thenumber of myelinated axons, however, Bluo-Gal stained oligodendrocyte cellbodies were clearly present (n=3; Fig. 5-11B). Toluidine blue staining of lumtransverse sections of unoperated control spinal cords reveal denselycompacted myelin profiles in the vicinity of Bluo-Gal stained oligodendrocyte cellbodies (Fig. 5-1 1C). These preliminary studies suggest that oligodendrocyte cellbodies survive immunological demyelination and are a likely source of -subsequent myelin.It is also possible that de-differentiation of astrocytes or astrocyteprecursors may contribute to spinal cord myelination following immunologicaldemyelination. In demyelinating lesions of the cat optic nerve, reactive glial cellswhich have begun to differentiate along the astrocyte lineage (GFAPI mayreverse their direction of differentiation, evidenced by the coexpression of bothastrocyte- and oligodendrocyte-specific markers (GFAP, GaIC; Kim 1985;Carroll et al. 1987). Switching glial cultures from one type of culture media toanother favoring the alternative direction of differentiation results in thetransient coexpression of both astrocyte and oligodendrocyte phenotypes(Ingraham and McCarthy 1989). Cells of the 02A lineage may actually exhibit194greater plasticity in demyelinating/remyelinating conditions. Cells coexpressingastrocyte and oligodendrocyte phenotypes can be isolated from the spinal cordsof young adult mice demyelinated by corona virus infection (Armstrong et al.1990). In addition, cells coexpressing astrocyte and oligodendrocytephenotypes have been reported in vivo in a number of demyelinating situations(Bunge et al. 1961; Carroll et al. 1987; Godfraind et al. 1989). Although theresults presented in this chapter suggest that remyelination followingimmunological demyelination is facilitated by surviving oligodendrocyte cellbodies, it is possible that dedifferentiation of astrocytes may additionallycontribute to the recovery of spinal cord myelinating oligodendrocytes. Pendingdouble labelling studies of immunologically demyelinated spinal cords using bothastrocyte- and ol igodend rocyte-specific antibodies, the possibility of astrocytededifferentiation and redifferentiation into myelinating oligodendrocytes can notbe excluded.Following complete transection, the proximal and distal segments of thesevered hatchling chick spinal cord remained intimately juxtaposed (Fig. 5-6).Fifteen to 21 days after spinal cord transection, light microscopic analysis ofsectioned spinal cords indicated a lack of cavity formation at the transection siteand an absence of autofluorescent inflammatory infiltrates separating theproximal and distal segments. In contrast, mouse spinal cord transection resultsin cavity formation at the lesion site (Schnell and Schwab 1990) that is oftenassociated with a robust inflammatory response. The lack of secondary damage195in the hatchling chick spinal cord after transection may be due to the uniquemethod of transection. Alternatively, this discrepancy may be due to differencesbetween species in the immunological response to spinal cord transection andthe corresponding breach of the blood-brain barrier.In conclusion, the findings presented here demonstrate that the injectionof oligodendrocyte-specific antibodies plus complement proteins into thehatchling chick spinal cord results in transient immunological demyelination thatpersists for as long as these compounds are administered. Furthermore, thesefindings demonstrate that complete transection of the hatchling chick spinalcord in vivo followed immediately by 2-3 weeks of immunological demyelinationresults in neuroanatomical regeneration of brainstem-spinal projections that isaccompanied by functional synapse formation. These findings constitute thefirst demonstration of functional regeneration following injury to the maturespinal cord and clearly demonstrate that hatchling chick spinal cord myelin isinhibitory to the regeneration of axotomized brainstem-spinal connectionsinvolved in locomotion.196CHAPTER 6GENERAL DISCUSSION197The studies outlined in the preceding chapters began with the observationthat the developmental onset of myelination in the embryonic chick spinal cordis 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 bycentral nervous system (CNS) oligodendrocytes and oligodendrocyte-producedmyelin that inhibit neurite outgrowth in vitro (Pesheva et al. 1993; McKerracherand David, unpublished observations) and in vivo (Schnell and Schwab 1990).These findings suggested that myelination within the embryonic chick spinalcord may inhibit regeneration following injury, and prompted the development ofthe developmental myelin-suppression procedure used in the subsequent studiesdescribed here.To assess a potential inhibitory role for myelin in the functionalregeneration of severed brainstem-spinal projections in the embryonic chick, Ideveloped a method of delaying the developmental onset of spinal cordmyelination until embryonic day (E) 17 of the 21 day developmental period inovo (chapter 3). Myelination within the embryonic chick spinal cord normallybegins on E13 (Bensted et al. 1957; Hartman et al. 1979; MackIm and Weill1985; Keirstead et al. 1992). In order to evoke developmental myelinsuppression, oligodendrocyte cell surface-binding antibodies were injecteddirectly into the thoracic spinal cord, along with a source of complementproteins. Galactocerebroside (GaIC) and 04 antibodies were utilized due to their198specificity for oligodendrocytes and myelin, as well as their ability to fixcomplement (Ranscht et al. 1982; Sommer and Schachner 1982).Immunological control solutions (GaIC antibodies only, complement proteinsonly, vehicle only, non-specific antibody plus complement or GalC antibodiesplus heat-inactivated serum) had no discernable effects on spinal cord myelindevelopment, indicating that both the oligodendrocyte-specific antibodies andcomplement proteins are necessary to evoke myelin-suppression. Developmentalmyelin-suppression specifically targeted spinal cord myelin, apparently leavingthe oligodendrocyte cell bodies intact, and did not appear to elicit an astroglialresponse or alter the dendritic architecture of the neuronal population of thespinal cord. Remyelination following myelin-suppression was shown to be fullycompensatory. Transections of the myelin-suppressed spinal cord during thepreviously defined restrictive period for spinal cord repair then serve as a directtest of the inhibitory properties of CNS myelin to regenerating axons.Transections as late as El 5 in myelin-suppressed embryos resulted incomplete neuroanatomical and physiological repair, enabling functionallocomotor capabilities after hatching comparable to an untransected controlchick (chapter 4). Transections on E15 in normally-myelinated, or immunologicalcontrol embryos resulted in no anatomical or physiological repair, rendering suchan animal completely incapable of voluntary locomotion after hatching. Thesefindings indicated that myelin within the spinal cord of an embryonic chickinhibits functional regeneration following spinal cord injury. In addition, these199studies suggested that demyelination of the (myelinated) hatchling chick spinalcord may also be an effective means of promoting regeneration following spinalcord injury.A method of demyelinating the hatchling chick spinal cord was developedby adapting the method of developmental myelin-suppression so as toeffectively demyelinate the hatchling spinal cord over a two week period(immunological demyelination; chapter 5). Osmotic mini-pumps containingcomplement proteins plus polyclonal GaIC or 04 antibodies were implantedunder the skin and attached to a canula which directly infused the low thoracicspinal cord. Immunological control solutions (GaIC antibodies only, complementproteins only or vehicle only) had no discernable effects on spinal cord myelin,indicating that both the oligodendrocyte-specific antibodies and complementproteins are necessary to evoke demyelination. Immunological demyelination inthis manner did not disturb the astrocyte population of the spinal cord, nor did itproduce any signs of necrosis at the infusion site. Remyelination of the spinalcord, following immunological demyelination, took place within one (1) week ofcessation of treatment (exhaustion of the pump reservoir) and was fullycompensatory.GaIC antibodies and complement proteins were delivered to the hatchlingchick spinal cord in an attempt to rescue previously axotomized brainstem-spinalprojections (chapter5). Immunological demyelination was initiated shortly aftercomplete thoracic transection of the spinal cord, and neuroanatomical200regeneration of brainstem-spinal projections was assessed 1 5-24 days later bycounting the brainstem neurons retrogradely-labelled by an injection oftetramethylrhodamine-labelled dextran amine (RDA) into the lumbar spinal cord.In transected, immunologically demyelinated animals the number of retrogradelylabelled brainstem-spinal neurons was approximately 5-15% of the number ofretrogradely-labelled brainstem-spinal neurons in RDA-injected, untransectedcontrol hatchlings of the same age. Transected animals that received notreatment or control solutions (GalC antibody only, complement proteins only orvehicle only) over a 2-week period showed no signs of neuroanatomicalregeneration. Double-labelling studies indicated that true axonal regenerationcontributed to the neuroanatomical recovery of transected and immunologicallydemyelinated hatchling chicks.Collaborative studies undertaken with Dr. Gerry Sholomenko (apostdoctoral fellow under the supervision on Dr. Kerry Delany, Simon FraserUniversity, Vancouver, B.C.) indicated that the neuroanatomical regeneration oftransected, immunologically demyelinated hatchling chicks was followed byfunctional synaptogenesis (chapter 5). Three weeks after spinal cord transectionand subsequent initiation of immunological demyelination, brainstem stimulationof decerebrate hatchlings evoked motor activity both above (pectoralis muscles)and below (sartorius muscles) the site of transection. Electromyographicrecordings from the sartorius muscles illustrated alternating periods of activity inthe right and left legs. Subsequent electroneurographic recordings also showed201the presence of rhythmic sartorius and pectoralis activity. In transected animalstreated with immunological control solutions over a 2-week period, brainstemstimulation resulted in electromyographic activity above the transection site butno electromyographic or electroneurographic activity was present below thetransection site. These findings indicate that neuroanatomical regeneration ofbrainstem-spinal projections following spinal cord transection in immunologicallydemyelinated hatchlings is accompanied by functional synaptogenesis withspinal cord neurons which directly or indirectly innervate the muscles of thelegs.Immunological myelin-suppression was also effective in removing (alreadypresent) myelin from the neonatal mouse spinal cord (chapter 5). These resultsindicate that immunological myelin-suppression is not species restricted. Itremains to be determined whether this intervention will improve the repair andrecovery of function after injury to the neonatal mouse spinal cord.Axonal regeneration following spinal cord transection in both lateembryonic and hatchling chicks was restricted to those animals subjected to theimmunological suppression of CNS myelin. However, the degree of functionalrecovery accompanying neuroanatomical regeneration differed substantially inembryonic and hatchling chicks. It is conceivable that the dramatic functionalrecovery noted in the myelin-suppressed, El 5 transected embryos, as comparedto the transected, demyelinated hatchlings, may be due to; 1) an enhancedintrinsic ability for axonal extention, 2) a relative abundance of growth-202promoting factors, and/or, 3) a relative paucity of growth-inhibitory factors.Several studies have indicated that neurons in culture display an inherentdecrease in speed of neurite extention, axonal regeneration as well as adecrease in terminal sprouting with age (Argiro et al. 1984; Collins and Lee1982; Black and Lasek 1979; Pestronck et al. 1980). Age-related differences inaxonal growth potential are most obvious in culture systems that simulate agrowth-inhibitory environment. One such system is the in vitro correlate of theglial scar, the three dimensional astrocyte culture system (Fawcett et al. 1989).In this culture system, embryonic retinal ganglion cells and sensory neurons arecapable of extending axons through the astrocyte matrix, whereas projectionsfrom postnatal retinal ganglion cells and sensory neurons fail to penetrate theastrocyte matrix (Fawcett et al. 1989a). Other studies have indicated that thedevelopmental programme of gene expression is not recapitulated duringregeneration attempts in the adult. Microtubule-associated protein (MAP) la,one of the many developmentally regulated MAPs which control the assemblyand stabilization of microtubules behind the axon growth cone, is not reexpressed during regeneration (Woodhams et al. 1989). Oligodendrocytes arealso an unfavorable substrate for growing postnatal axons, which cease growthor are repelled upon contact (Schwab 1990; Fawcett et al. 1989b). In contrast,embryonic retinal ganglion cells and dorsal root ganglion cells are apparentlyinsensitive to the inhibitory properties of oligodendrocytes, growing into contactwith, and extend neurites over oligodendrocytes (Ard et al. 1991). Thus, as a203result of an apparent insensitivity to glial-derived inhibitory molecules,embryonic neurons may be intrinsically more capable of regenerating severedaxons.Developmental changes in the abundance of growth-promoting factorshave also been reported. Extracellular matrix (ECM) molecules such as laminin,fibronectin and proteoglycans, essential for cell attachment, migration andprocess extention, are widely expressed in the developing central nervoussystem (CNS) and are found in a more restricted distribution in the adult(Sheppard et al. 1991; Carbonetto 1984; Steindler et al. 1990). Cell adhesionmolecules, although present in both the embryonic and adult CNS, decreasetheir neurite-outgrowth promoting ability during development and are alsodistributed in a more restrictive fashion in the adult (Doherty et al. 1990b). Asone might imagine, integrin receptors for many ECM molecules are alsofunctionally down-regulated during development (Reichardt and Tomaselli1991).Growth factors are perhaps the most influential neurite growth-promotingfactors available to the developing CNS. Growth factors regulate the size ofdeveloping neuronal populations (Harper and Thoenen 1981), promotematuration and cell survival (Hamilton and Levi-Montalcini 1949), prevent celldeath (Arenas and Persson 1994) and enhance sprouting (Schnell et al. 1994).Many growth factors and their receptors are developmentally regulated (Knuselet al. 1994), suggesting that injured adult neurons may not receive the benefits204that growth factors provide to developing, and perhaps injured, embryonicneurons. Attempts to increase the degree of functional recovery of transected,demyelinated hatchling chicks may be expedited by identifying and providingthe specific growth factor(s) that brainstem-spinal projecting neurons areresponsive to.The limited functional recovery of transected, demyelinated hatchlingchicks (compared to El 5-transected, developmentally myelin-suppressedembryos) may also be due to neurite growth-inhibitory factors present in themature CNS, but absent in the developing system. Spinal cord transections inthe hatchling chick result in a pronounced astroglial response that is confined toone spinal cord segment either side of the injury (Fig. 6-1). The proliferation andhypertrophy of astrocytes in and around a site of CNS injury has beensuggested to provide a physical barrier to axonal growth (Reier and Houle 1988;Hertz et al 1990). Furthermore, astrocytes express several anti-adhesivemolecules including tenascin (Brodkey et al. 1993) and chondroitin sulfate,dermatan sulfate and keratan sulfate proteoglycans (Herndon et al. 1990). Incontrast, spinal cord transections in the embryonic chick are not accompaniedby an astrogliotic response (Shimizu et al 1990), despite the presence ofastrocytes in the late embryonic spinal cord (chapter 2). A lack of astrogliosisfollowing embryonic CNS injury has also been noted in other systems (Berry et205Figure 6-1. Astrogliosis following thoracic transection of the hatchling chickspinal cord. Photomicrographs of glial fibrillary acidic protein (GFAP)immunoreactivity in parasaggital section. A: Gray matter two segments rostralto the transection site shown in B; note the lack of astrogliosis. B:Photomontage through the transection site; note that astrogliosis is restricted toapproximately one segment either side of the transection. (Bars lOOum).207al. 1968; Maxwell et al. 1990). This implies a developmentally-regulated changein the response of astrocytes to CNS injury. Methods of reducing or eliminatingastrogliosis following adult spinal cord injury would be useful for investigatingthe relative contribution of astrogliosis to the lack of spinal cord repair intransected hatchling chicks.Despite age-related differences in the inherent capability of axons toregenerate, as well as developmental changes in the abundance and distributionof growth-promoting and growth-inhibiting factors (reviewed above), myelinclearly plays a major role in determining the regenerative success of axotomizedembryonic and hatchling chick brainstem-spinal projecting neurons. Only thoseanimals subjected to immunological myelin-suppression showed any signs ofneuroanatomical regeneration and physiological recovery following spinal cordinjury. Functional recovery has also been observed following CNS injury toinvertebrates as well as several lower vertebrate systems such as the lampreyspinal cord, which is unmyelinated and is capable of functional regenerationfollowing injury to either the larval or adult animal (McClellan 1990).Considering the inhibitory nature of CNS myelin for regenerating axons, aphilosophical question arises: Why, over the course of evolution, has a systemdeveloped that prohibits regeneration following CNS injury?It is likely that the inhibitory nature of CNS myelin for neurite outgrowthevolved for reasons other than to inhibit neural regeneration following injury.One might imagine natural selection pressures contributing to the development208of CNS protective structures, such as the shock-absorbing cerebrospinal fluidlayer or the protective enclosure provided by the cranium and vertebral column.Individuals with less well developed CNS protective structures would be farmore likely to suffer serious CNS injury, which would in turn decrease oreliminate their chances of contributing to the gene pool. However, the CNSitself may be immune to natural selection pressures which would favor CNSregeneration. Injury to the CNS is almost exclusively associated with extensivedamage to extraneural protective structures and is commonly associated with aloss of sympathetic and parasympathetic control (including control ofreproductive organs) or an immediate cessation of vital functions. If the wheelsof evolution, genetic recombination and mutation, did generate mechanismswhich could facilitate CNS regeneration, this phenotype may not be selected foras a result of these various extraneural complications. Therefore, it is arguablethat the CNS is immune to natural selection pressures concerned withregeneration.The inhibitory nature of CNS myelin for neurite outgrowth may haveevolved for developmental reasons that incidently affect regenerating neurons.Myelination may serve to suppress abberent sprouting in nerve fibre tracts. Thedevelopmental onset of CNS myelination in higher vertebrates generally takesplace after the developmental period of axonal process outgrowth and targetrecognition. In the embryonic chicken, for example, the developmental onset ofspinal cord myelination takes place on embryonic day (E) 13 (Keirstead et al.2091992; Bensted et al. 1957; Hartman et al. 1979; MackIln and Weill 1985);brainstem-spinal neurons complete their projections to all levels of the spinalcord by E12 (Okado and Oppenheim 1985). Oligodendrocytes appear in thehamster optic tract and rostral superior colliculus by postnatal day (PN) 5 andspread throughout most of the stratum opticum by PN9, after retinocollicularaxons have innnervated the superior colliculus (Schneider et al. 1990; Jhaveri etal. 1992). In the rat spinal cord, the corticospinal tract descends during the firstpostnatal week and begins to be myelinated at PN1O-1 1 (Schreyer and Jones1982). Furthermore, neutralization of myelin-associated neurite growth inhibitorsin the rat optic nerve (Colello et al. 1991) and hamster optic tectum(Kapfhammer et al. 1992) leads to increased sprouting. Given the permissiveextraneuronal environment for developing neurites, the nonpermissive propertiesof CNS myelin may serve to inhibit aberrant collateral branching and terminalarborization 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 tractsmay 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 isseen in the developing corticospinal tract (CST) of the rat. The CST descendsthrough the dorsal funniculus of the spinal cord during the first postnatal week,and is intimately surrounded by the myelinating fasciculus cuneatus and gracilis210(Schreyer and Jones 1982). Removal of spinal cord oligodendrocytes and myelinwith X-ray irradiation, or application of activity-blocking antibodies to identifiedmyelin-associated neurite growth inhibitors results in abberent growth of CSTaxons outside of the anatomical region that the CST normally occupies (Schwaband Schnell 1991). These findings imply that late developing fibres may beindirectly guided to their targets by the non-permissive nature of adjacentmyelinated tracts.The postulated developmental roles for neurite growth inhibition by CNSmyelin (outlined above) clearly affect regenerative efforts by injured axonswithin myelinated regions of the CNS. Injured central fibres undergo a period ofextensive sprouting that recapitulates neurite outgrowth in development (Ramony Cajal 1959). Unlike developing axons, however, regenerating axons exist in anextremely hostile extraneuronal environment, created in part by the presence ofmyelin. The studies outlined in this thesis demonstrate that the regenerativesuccess of transected hatchling and late-embryonic chick spinal cord isdetermined in part by the presence or absence of myelin. This suggestion issupported by the demonstration that oligodendrocytes are responsible forrestricting innervation of the superior colliculus following postnatal injury(Kapfhammer et al. 1992). In a normal hamster, retinocollicular axons arrive atthe superior colluculus prior to myelination, and innervate the superficial gray aswell as the deeper stratum opticum. If the outer layers of one superior colliculusare destroyed, the lesioned retinal fibres will cross the tectal midline and211innervate the contralateral superior colliculus. The recrossing retinal fibres willterminate throughout the entire mediolateral extent of the superior colliculus ifthe eye that normally innervates this side is removed. However, the terminalsformed by the recrossing fibres are restricted to the superficial gray, which isunmyelinated at the time when the recrossing terminals arrive. The deeperstratum opticum of the superior colliculus is actively myelinating at this time andremains uninnervated by recrossing fibres. However, in the presence ofantibodies that neutralize the myelin-associated neurite inhibitory activity,recrossing retinal fibres terminate within the stratum opticum as well as thesuperficial gray. These studies indicate that myelin restricts plasticity within theinjured CNS. The inhibitory nature of CNS myelin for regenerating axons may bea consequence of its developmental roles.Despite the hostile extraneuronal environment for regenerating CNSaxons, immunological myelin-suppression in the late-embryonic or hatchlingchick spinal cord facilitates partial neuroanatomical regeneration of brainstemspinal projections that is accompanied by functional synaptogenesis (chapters 4and 5). These studies indicate that CNS myelin is inhibitory to the regenerationof transected spinal cord in late-embryonic and hatchling chick. Furthermore,the different degrees of regeneration observed after injury in the myelinsuppressed embryonic and hatchling chick spinal cords suggest that othertherapies may be required in more mature animals, therapies that provideregenerating brainstem-spinal projections with a more ‘embryonic-like’212environment. 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