Review Setting the Stage for Functional Repair of Spinal Cord Injuries

Review

Setting the stage for functional repair of spinal cord injuries: a cast of thousands

LM Ramer1, MS Ramer1 and JD Steeves*,1 1ICORD (International Collaboration on Repair Discoveries), The University of British Columbia, Vancouver, BC, Canada

Here we review mechanisms and molecules that necessitate protection and oppose axonal growth in the injured spinal cord, representing not only a cast ofvillains but also a company of therapeutic targets, many ofwhich have yet to be fullyexploited. We next discuss recent progress in the fields ofbridging, overcoming conduction block and rehabilitation after spinal cord injury (SCI), where several treatments in each category have entered the spotlight, and some are being tested clinically. Finally, studies that combine treatments targeting different aspects ofSCI are reviewed. Although experiments applying some treatments in combination have been completed, auditions for each part in the much-sought combination therapy are ongoing, and performers must demonstrate robust anatomical regeneration and/or significant return of function in animal models before being considered for a lead role. Spinal Cord (2005) 43, 134–161. doi:10.1038/sj.sc.3101715; Published online 25 January 2005

Keywords: spinal cord injury; neuroprotection; transplantation; rehabilitation; plasticity; regeneration

Introduction For clinician and scientist alike, the injured spinal cord them. It is widely acknowledged that a combination of is a formidable scene: a large cast of endogenous cells treatments will be required to address the complex issues and molecules act in concert to prevent or restrict ofSCI; 1 however, designing such a treatment strategy is functional connectivity. The players and their roles in incredibly daunting. The first step is to identify the most limiting spinal cord repair continue to be elucidated, and powerful and/or versatile performers to fill the roles of each discovery unveils the character ofa molecular protection, stimulating growth, bridging, conduction antagonist that hinders return of function after spinal and rehabilitation: each treatment must audition in cord injury (SCI). As the villains reveal themselves, our isolation before it can take the stage as part of a repertoire ofpossible targets forintervention grows, and combination therapy. The second step is equally potential treatments fall into one or more of five ambitious: once the protagonists ofSCI are identified, categories: (1) protection, to prevent death ofneural they must appear in the appropriate spatial andtemporal cells undamaged by the initial injury; (2) stimulating combination. Like a performance, the development of a axonal growth, either by enhancing the intrinsic regen- reliable therapy for SCI must be directed with intimate erative capacity ofspinal and supraspinal neurons or by knowledge ofeach character, leading to the perfect blocking or removing endogenous inhibitors to repair; balance among the players. (3) bridging, to provide a permissive substrate for elongating axons and to replace lost tissue; (4) enhancing Neuroprotection in the setting of SCI axonal transmission, to alleviate conduction block in spared or regenerated axons and (5) rehabilitation,to Although traumatic SCI is often the result of a single enhance functional plasticity within surviving circuits mechanical insult, the functional outcome is determined and consolidate anatomical repair. by a cascade ofbehind-the-scenes events described As the cast ofvillains in SCI is vast and collaborative, collectively as ‘secondary injury’. Displacement or so too must be the chorus ofheroes that rise to meet penetration ofthe spinal cord both inflicts direct tissue damage (ie primary injury) and initiates destructive processes that expand the injury site. This phenomenon *Correspondence: JD Steeves, ICORD (International Collaboration on Repair Discoveries), The University ofBritish Columbia, Rm. 2469 of secondary injury, which extends for days after Biosciences Bldg., 6270 University Blvd., Vancouver, BC, Canada V6T the initial trauma, presents the first opportunity for 1Z4 intervention after SCI. Events that fall under the Orchestrating spinal cord repair LM Ramer et al 135 umbrella ofsecondary injury include vascular changes agent. Following SCI, systemic minocycline administra- (edema, ischemia, and hypoxia), excitotoxic biochemical tion has also been documented to inhibit microglial events (formation of free radicals and nitric oxide, activation, to promote oligodendrocyte survival, to protease release), and cellular responses (invasion by reduce lesion-induced cavity formation, and to prevent immune cells, activation ofglial cells, and death of the retraction or dieback ofinjured dorsal column, neurons and glia) (reviewed in Hausmann2). How rubrospinal, and corticospinal axons.18–20 The axonal significantly each component ofsecondary injury con- protection afforded by this treatment may be ofinterest, tributes to loss offunction remains unclear: what is since the rats also exhibit improved functional recov- evident is that the resulting pathology might be ery,18,19,21 although the restricted subtotal nature ofthe ameliorated by eliminating one or more ofthese players dorsal column lesion would permit neural plasticity in the early stages ofSCI. within preserved pathways to contribute to recovery. Clinical trials ofneuroprotective agents, delivered The mechanism by which sensory dorsal column axons acutely following SCI, have been carried out with dieback following injury may involve macrophage- equivocal success. The two most widely discussed are mediated degeneration ofproximal myelin sheaths, the National Acute Spinal Cord Injury Study (NASCIS) since in myelin-deficient rats macrophage/microglial trial ofhigh-dose systemic methylprednisolone (MP) 3 activation with zymosan does not induce axonal and the Sygens trial ofGM-1 gangliosides. 4 GM-1 retraction.20 Interestingly, suppression ofp75, a putative gangliosides appeared to enhance the rate ofrecovery signaling receptor for myelin inhibition, also reduced from SCI, but did not significantly improve the ultimate axonal retraction/dieback from the lesion site in p75 functional outcome, whereas MP was reported to knockout mice (p75À/À).22 This effect ofp75 deletion improve functional outcome when administered within was evident in pictures ofthe lesion site, although the 8 h ofSCI. 5 Although MP has since been widely used as authors ofthe study did not discuss reduced retraction/ a neuroprotective therapy, several more recent studies dieback as a result ofthis study. have reported equivocal benefits and concerns about the The loss ofneurons and glia at the site ofSCI is well- potential toxicity ofthe required high doses and documented. However, at more remote distances, the observed medical complications (reviewed by Kwon extent ofneuronal death following SCI has probably et al6). Administration ofthis steroid is no longer a been overestimated. Even 1 year after a cervical SCI, standard treatment, nor even a guideline for treatment, the cell bodies ofsevered descending rubrospinal ofacute SCI in Canada 7 and many other countries have axons, which were previously thought to have perished, adopted similar policies. regained normal neuronal morphology when BDNF In the midst ofcontroversy surrounding the use of was administered in the vicinity ofthe red nucleus. 23 currently available pharmacologic interventions, the Rather than being generated de novo, these rubrospinal search for an effective neuroprotective agent is ongoing neurons had atrophied to the point where they were in animal models ofSCI. Neuroprotective strategies easily missed during previous histological screens, have been reviewed at length previously.8,9 Here we leading to an underestimation oftheir numbers and focus on recent work from our group and elsewhere resulting in the premature declaration oftheir death. dealing with some ofthe neuronal consequences of Recently, adeno-associated viral (AAV) vector- secondary injury, including axonal retraction/dieback mediated BDNF gene transfer into the red nucleus and neuronal atrophy/death. similarly counteracted atrophy ofchronically lesioned One potential mechanism for secondary neuronal rubrospinal neurons.24 It is not known to what extent injury which is undergoing thorough investigation in a severe atrophy (rather than neuronal loss) is a general number oflaboratories involves endogenous (micro- response ofCNS neurons to chronic injury, but glial) or invading (hematogenous) phagocytic cells. corticospinal neurons also exhibit atrophy after spinal While the potential therapeutic use ofpreviously axotomy.25 IfSCI-induced atrophy is common among myelin-activated macrophages has received much atten- CNS neurons, it may mean that rather than attempting tion oflate (reviewed in; 10 see ‘Myelin and myelin to replace neurons through transplantation ofembryo- signaling’, below), several lines ofevidence demonstrate nic tissue or multipotent cells,26 therapies directed at that the microglial/macrophage response to SCI is reviving these ‘hibernating’ neurons may be more responsible for demyelination, oligodendrocyte death, appropriate. It has been observed that the direct CNS and damage to intact axons.11–14 The nontraumatic application of neurotrophic factors following SCI can injection ofthe micgroglia/macrophage activator zymo- prevent the atrophy ofrubrospinal, 27,28 cortico- san into the brain and spinal cord results in cavitation, spinal,25,29 and ascending propriospinal neurons,30 as well demyelination, and axonal injury.15,16 The molecules as stimulate the expression ofaxonal growth-associated released by mature phagocytes, including superoxide, genes. Many ofthe same factors can also prevent hydrogen peroxide, hypochlorous acid, as well as corticospinal and ascending sensory axonal dieback.31 neurotoxic chemokines and cytokines may be partially responsible for axonal damage and retraction, and Stimulating axonal growth in the injured spinal cord neuronal death.17 The second-generation tetracycline derivative, mino- At the site ofSCI, axons attempting to regrow and/or cycline, has been shown to be an antiinflammatory re-establish functional connections get a chilly recep-

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tion, not only because ofnecrotic and apopotic forms type mice.41 Recent characterization ofplasticity follow- ofsecondary cell death and the activation ofimmune ing SCI in three lines ofmice lacking one or more Nogo cells (discussed in the previous section above), but also molecules has been similarly perplexing. While corti- because a troupe ofinhibitory molecules confront cospinal axon sprouting following spinal hemisection growth cones at and around the site ofSCI. These was evident and apparently robust in some mice,42,43 molecules can be broadly classified into two groups: almost no augmentation ofregeneration or sprouting myelin-associated molecules produced by oligodendro- was seen in others.44 Immunizing mice with MAG-plus- cytes, and extracellular matrix (ECM) molecules, many NogoA enhances regeneration and/or sprouting of ofwhich are produced by astrocytes in response to corticospinal axons following spinal hemisection; how- injury. Nerve grafting experiments and other manipula- ever, myelin immunization has a greater overall effect tions (discussed in the section on Bridging the site ofSCI than MAG/NogoA immunization.45,46 Manipulations below) indicate that damaged CNS axons can regenerate ofOMgp have yet to be carried out, but one might when their local environment is replaced with a hypothesize that, like the focused targeting of MAG or substrate that is less inhibitory (or more permissive). Nogo alone, specifically reducing OMgp activity would Even so, it has been demonstrated repeatedly that the have less effect on axonal regeneration than targeting capacity for axonal regeneration is hindered by an multiple myelin-derived inhibitors. impoverished neuronal growth program being generated Fortuitously, recent data indicate that this trio of by severed neurons. Cajal was perhaps the first to myelin-derived inhibitors – NogoA, MAG and OMgp – recognize that ‘yfor these [growth cones] to become signal through a common receptor (the Nogo receptor, consolidated and to progress, the action ofthe trophic NgR).47–50 As an apparent point ofconvergence in center or nerve cell is indispensable from every point inhibitory signaling, NgR has taken the stage as a target ofview.’ 32 In this section, we will consider both the ofstrategies aimed at increasing plasticity in the injured molecular antagonists to regeneration after SCI, and the spinal cord. A competitive antagonist ofNgR, NEP1- protagonistic manipulations that might coax CNS axons 40, blocks the inhibitory action ofmyelin in vitro51 and into a state ofgrowth in their native environment, enhances both growth ofsupraspinal axons and func- through direct targeting ofthe responsible inhibitory tional recovery when administered intrathecally or factors within CNS tissue, the axonal responses to those systemically after thoracic hemisection.51,52 Another inhibitors, or through enhancing the response ofthe cell antagonist, NgREcto, is a soluble, truncated form of body to injury. NgR, which has been shown to alleviate inhibition of both Nogo and myelin in vitro (Fournier et al, 2002). Recent work from our group examines the effects of Myelin andmyelin signaling: an inhibitory chorus line NgR activity using a different form of soluble NgR In the normal CNS, myelin plays a critical role in in vivo;53 in these experiments, we found that NgR solidifying connections established in development. antagonism augmented rhizotomy-induced sprouting of After SCI, myelin-derived molecules take the stage in both descending monoaminergic axons and peptidgeric a new light, as critical inhibitors ofaxonal growth/ primary afferents in the cervical dorsal horn. The regeneration. The role ofmyelin in suppressing plasticity discovery ofan anti-NgR monoclonal antibody, capable after SCI was first illustrated in landmark experiments ofinhibiting binding ofNogo, MAG, and OMgp, and employing the Nogo-blocking antibody (IN-1):33,34 ofblocking inhibition ofmyelin in vitro, has now been antagonizing Nogo, the now notorious inhibitory reported.54 constituent ofcentral myelin, permitted extensive Downstream ofNgR are several other molecules that growth ofcut corticospinal axons in young rats. In fact, might also serve as targets for intervention after SCI. Nogo – now known to exist in three isoforms35,36 –is The Nogo receptor is thought to transmit myelin- but one ofa number ofmyelin-derived inhibitory inhibitory signaling through its interaction with the proteins. Others identified thus far include myelin- low-affinity neurotrophin receptor p75.55,56 Recently, associated glycoprotein (MAG)37,38 and oligodendro- LINGO-1, a neural transmembrane protein, has been cyte myelin glycoprotein (OMgp).39 revealed as an essential component ofthe functional The role ofMAG as an impediment to axonal NgR/p75 complex.57 Ligand-bound NgR associates regeneration is exemplified by in vitro studies in which with p75 and LINGO-1 to activate the GTPase RhoA, its immunodepletion from myelin extracts attenuated which transduces myelin-derived inhibition to the inhibitory activity.37 It has since been shown that cytoskeleton.58–60 Since p75 binds neurotrophins, its elevating cAMP can stimulate adult neurons to extend contribution to axonal growth is complex (see below), axons on MAG substrates,40 an effect related to the whereas the activation ofRhoA leads to the recruitment growth-enhancing effects ofconditioning lesions and ofRho kinase and subsequent remodeling ofthe actin neurotrophic factor application (see below). Studies in cytoskeleton to inhibit regeneration. Targeting the Rho MAG knockout mice have been equivocal, however, pathway with the bacterial enzyme C3 transferase or since myelin preparations from these mice are still with specific inhibitors ofRho kinase has led to reports effective in blocking regeneration, and since severed ofboth corticospinal regeneration and functional optic nerve and corticospinal tract (CST) axons recovery after rat SCI.61,62 Interestingly, it seems that regenerate no better in MAG knockouts than in wild- astrocyte or meningeal cell-derived ECM molecules may

Spinal Cord Orchestrating spinal cord repair LM Ramer et al 137 also signal through the Rho pathway,63–65 indicating a Astrocytes andthe glial scar: another inhibitory ensemble convergence ofmultiple inhibitory signaling pathways, An excellent setting in which to study the astroglial as well as suggesting that multiple tiers ofinhibition can contributions to inhibition ofregeneration is that ofthe be overcome by focusing on a single intracellular dorsal root entry zone (DREZ), the point at which pathway (for a review, see Kwon et al66). sensory axons enter the spinal cord. First, dorsal root It is likely that other myelin-associated inhibitors injury does not compromise the blood–brain barrier, remain to be elucidated. A broader, approach, then, is to and spinal cyst cavity formation does not occur; second, temporarily and focally remove myelin and/or myelin astrocytes ofthe glia limitans forma precisely demar- debris from the site of SCI. We have used a comple- cated border between peripheral and central nervous ment-fixing antibody to galactocerebroside (GalC), tissue; and third, astrocytic and degenerative changes coadministered intraspinally with serum complement, (microglial activation and myelin breakdown) are to achieve transient focal demyelination at the site of spatially and temporally separated from one another. SCI.67–69 Within the CNS, GalC is restricted to the cell Astrogliosis begins within days ofa dorsal rhizotomy, membrane ofoligodendrocytes and myelin; specifically, whereas frank Wallerian degeneration does not begin it is present in the outer layer ofthe myelin membrane. until 1 week later.73,74 It is clear that activated Binding ofthe GalC antibody triggers activation of astrocytes, whether part ofa scar following SCI or as the complement cascade, which results in disruption a component ofthe reactive DREZ following rhizo- ofmyelin membranes and recruitment ofendogenous tomy, deter regeneration. Following SCI, axons retract microglia or invading macrophages to phagocytically from the lesion site and are surrounded by CNS myelin. clear CNS myelin within the injured region. Using this They approach, but do not come into contact with the approach, the attack on myelin is volume- and newly formed glia limitans at the innermost margin of concentration-dependent, can be readily tracked with the glial scar. This was demonstrated in studies by Silver noninvasive magnetic resonance imaging, ceases when and colleagues,75,76 in which eGFP-expressing axons the infusion is stopped, and is reversible, as remyelina- from grafted sensory neurons elongated within the tion occurs when treatment is withdrawn. Myelin degenerating dorsal columns, but halted within a halo disruption with the GalC antibody permits regeneration ofchondroitin sulfateproteoglycan (CSPG) immuno- ofbrainstem-spinal axons when administered afteracute reactivity surrounding a small, previously generated, hemisection in the adult rat.68 More recently, we have wound. Sensory axons injured within the dorsal root found that immunological myelin disruption stimulates also regenerate up to the DREZ. Once there, their both regeneration ofsupraspinal axons and improved growth is arrested by the astrocytes ofthe glia limitans, recovery oflocomotion aftersevere thoracic contusion against which they often form terminal-like axon injuries (where controls are permanently paralyzed), structures,77 or from which they turn back, and are even when treatment is delayed for 1 or 2 months after redirected peripherally.78 injury (JDS, unpublished observations). Based on observations such as these, it has been It has been suggested that another method ofreducing hypothesized that there exists an astrocytic ‘physiologi- myelin-derived inhibition at the site ofSCI is to cal stop signal’ at the DREZ which is responsible for the transplant autologous macrophages at the lesion site axonal arrest at the DREZ.79 While the nature ofthe to phagocytically clear myelin debris. In rat, macro- signal(s) remains unknown, a number ofECM proteins, phages activated by prior exposure to degenerating induced by dorsal rhizotomy, are well-situated to peripheral nerve segments stimulated both regeneration prohibit penetration by sensory axons ofCNS tissue, and functional recovery when grafted at the site of including CSPGs, the CSPG NG2 made by oligoden- complete thoracic transection;70 more recently, macro- drocyte precursors, as well as Tenascin-C and Tenascin- phages coincubated with skin improved functional R.78 The astrocytic character ofthe inhibition strongly outcome when grafted at the site of severe thoracic suggests secreted factors, and these are most likely to be contusion.71 Activated autologous macrophages have components ofthe ECM laid down as a result of entered clinical trials as ProCord, a therapy adminis- astrocyte activation. A recent DNA microarray carried tered by ProNeuron, based in Israel. Although patients out in our group to identify rapidly upregulated (3 days have already received ProCord, no published reports post-rhizotomy) astrocytic mRNAs has revealed a large oftheir progress are available. As all patients number ofcandidate stop signals; 80 here we review received ProCord within 14 days ofSCI, the effect of recent data on a selection ofthese molecules. stimulated macrophage transplantation on their clinical outcome may prove difficult to interpret: in spite ofthis potential limitation, a Phase II multicenter trial has been Chondroitin sulfate proteoglycans A number ofCSPGs initiated (http://www.proneuron.com/ClinicalStudies/ are upregulated soon after dorsal rhizotomy, and are index.html). Despite their foray into clinical also candidate inhibitors ofaxonal regeneration at the testing, macrophage transplantation remains a contro- site ofSCI (reviewed in Morgenstern et al81 and Rhodes versial therapy for SCI, largely because the activation and Fawcett82). These include neurocan and versican, ofmacrophages afterSCI may also have other both ofwhich are made by reactive astrocytes and actions within the CNS, which have yet to be charac- inhibitory to neurite outgrowth.83–86 While the growth- terized.72 inhibitory effects ofversican are well established, 87 its

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pattern ofexpression following SCI is controversial, 84,86 with fibroblast growth factor (FGF) receptors:106 and thus its role in the failure of regeneration remains HSPGs increase the affinity ofFGFs fortheir receptors. unclear. Neurocan is more consistently upregulated FGFs (particularly FGF-2) increase astrogliosis in after SCI, and thus this CSPG is a better candidate to vitro107 and in vivo,108 and they also increase the mediate scar-related inhibition. While more slowly expression ofsyndecan-1 in oligodendrocytes and upregulated (only by 6 days post-rhizotomy), chondroi- astrocytes.109 To date, few HSPGs have been identified tin synthase is likely to also play an important role in at the DREZ, with the exception ofglycipan, which glial scar formation since it is involved in CSPG is expressed in the E13-E16 DREZ,110 but not in the synthesis.88 The CSPG NG2 has been implicated in adult.111 The rapid upregulation ofsyndecan-1 revealed regenerative failure, based on its localization in the CNS by the DNA microarray data is suggestive ofa role for part ofthe DREZ; 78 in fact, its upregulation occurs too HSPGs in mediating inhibition at the DREZ and thus slowly (after 1 week) to compellingly implicate it in the probably also in the damaged spinal cord, although rapid conversion ofthe DREZ frompermissive to HSPGs have not yet been targeted in animals models of inhibitory (within 3 days) following rhizotomy. SCI. Inhibitory effects ofCSPG accumulation afterSCI have been partially alleviated by enzymatic degradation. Chondroitinase ABC (ChABC), a bacterial enzyme that Keratan sulfate proteoglycans (KSPGs) Lumican is cleaves glycosaminoglycan (GAG) side chains from the one ofthe fewcloned KSPGs. 112 The induction of protein core to attenuate CSPG inhibitory activity,89–91 lumican in our DNA array data was surprising, given promotes functional recovery when administered that its expression to date has been confined to cartilage acutely following lesions of the cervical dorsal spinal and cornea.113 It is also noteworthy that lumican has cord in adult rats.92 Recently, ChABC was applied after been shown, using the Affymetrix DNA microarray, to thoracic hemisection, at the distal end ofa Schwann cell- be massively upregulated by pyramidotomy.114 This seeded polyacrylonitrile/polyvinylchloride (PAN/PVC) could mean either that lumican is de novo upregulated polymer channel, where it was reported to enhance in reactive astrocytes or other CNS glia after injury, axonal growth across the caudal graft–host interface.93 or equally plausibly, that there is sufficient sequence Although proven to increase plasticity in both spinal homology between lumican and other yet-to-be-cloned cord and brain,94–97 ChABC does not completely KSPGs to generate a false-positive result. In either case, remove GAG sidechains, and the ‘carbohydrate- the potential role ofKSPGs in mediating axonal stubbed’ protein core that persists after ChABC inhibition warrants discussion. Ofthe KSPGs so far degradation ofCSPGs does exert some inhibitory identified in nervous tissue, claustrin,113 and ABA- influence on growth ofspinal axons. 98 A DNA enzyme KAN115 are the best candidates for inhibiting axonal against the GAG-chain initiating enzyme, xylosyltrans- growth between CNS compartments. Claustrin was ferase-1 (XT-1), may remove GAG chains more originally identified as a midline barrier molecule,116,117 completely, and was recently applied to spinal stab and the pattern ofABAKAN expression (separating lesions in adult rat:99 in this study, enzymatic treatment thalamic nuclei, barrel fields and in astrocytes surround- diminished GAG chain presence around the lesion and ing cortical stab lesions) is highly suggestive ofa strong facilitated growth of GFP-positive axons from grafted role in boundary formation.115,118,119 A recent report of sensory neurons beyond the lesion site. KSPG induction following SCI has indicated that astrocytes are not robust producers ofsome KSPGs; 120 however, the 5D4 antibody used has been reported to Heparan sulfate proteoglycans (HSPGs) Syndecan-1 is react with molecules other than claustrin and ABA- a member ofthe HSPG family identified in our KAN,121 and so these remain potential candidates for microarray data. Four different syndecan genes have regeneration failure in the CNS. been identified, and all are upregulated with a rapid time course following brain injury.100 During development, the HSPGs are differentially expressed in lateral and ECM modifying proteins A number ofmolecules medial astrocytes, with higher expression toward the involved in ECM synthesis and stabilization are also midline thought to inhibit crossing ofdeveloping axonal implicated by microarray data. Lysyl oxidase, while projections.101–103 While the more medial astrocytes probably not astrocyte expressed, is rapidly upregulated tend to inhibit axonal outgrowth, their treatment with following CNS trauma,122 and is known to catalyze the heparatinase caused them to be more permissive to cross linking ofECM proteins. 123,124 Osteopontin axonal elongation and rendered their surfaces (as (secreted phosphoprotein 1) is expressed by neurons, measured with atomic force microscopy) more like macrophages/microglia, and astrocytes following the growth-promoting lateral astrocytes.104 A role for stroke125,126 and ventral root avulsion injury.127 Its role HSPGs in preventing inappropriate axon crossing at the in glial scar formation is suggested by its adhesive optic chiasm, and in retaining retinal axons in the frog nature, as well as by its ability to promote astrocyte tectum has also been demonstrated.105 migration.128 Thrombomodulin is upregulated in acti- HSPGs also may act to increase inhibition by vated astrocytes and may act to stabilize a glial scar promoting astrocyte reactivity through an interaction following an initial astrocytic proliferative response

Spinal Cord Orchestrating spinal cord repair LM Ramer et al 139 induced by rhizotomy or direct CNS trauma.129 Two the same group has shown that by augmenting inhibitors ofmatrix metalloproteinases (MMPs) were intracellular cAMP in DRG neurons in vivo, central also upregulated: tissue inhibitor ofmatrix metallopro- sprouting/regeneration is enhanced following dorsal teinase 1 (TIMP1) and alpha-2-macroglobulin (A2M). column lesions.145 Intriguingly, the downstream effec- Both TIMP1 and A2M counteract the ECM-degrading tors appear to be polyamines synthesized in part by properties ofMMPs, leading to ECM reinforce- Arginase 1.140 Both Arginase 1 and cAMP levels are ment;130,131 however, A2M has also been shown to have high in young (neonatal) DRG neurons, correlating both astrocyte proliferation and neurite outgrowth- well with their ability to elongate in the presence of promoting properties.132 MAG.146,147 Rolipram, a compound previously tested in In contrast to the stabilizing effects oflysyl oxidase, clinical trials for treatment of depression,148 is a osteopontin, thrombomodulin, and MMP inhibitors, a phosphodiesterase inhibitor, and thus causes cAMP to number ofastrocytic proteins were upregulated which accumulate by preventing its enzymatic degradation. are known to have antiscarring properties. These include Rolipram has recently been administered systemically decorin, which when delivered to the site ofa cortical after SCI in rats, with encouraging results;149,150 the stab wound, inhibits glial scar formation,133 and SC1, an central role ofRolipram in recent reports ofcombina- antiadhesive glycoprotein synthesized and secreted by tion approaches stimulating regeneration and functional astrocytes.134,135 It is likely that both stabilizing and de- recovery are discussed below (see ‘Casting the combina- stabilizing processes are operational in the first days tion’). following injury as the glial scar takes shape. Most studies examining the growth-enhancing effects ofneurotrophic factors applied to the site ofSCI have tested neurotrophins administered in conjunction with The cell body response and neurotrophic factors: other manipulations. For example, nerve growth factor upstaging intrinsic inhibition (NGF) can enhance axonal growth through fetal spinal Perhaps the most convincing studies demonstrating the cord transplants or peripheral nerve grafts.151–153 requirement ofan appropriate cell body response for Additionally, BDNF and NT-3 enhanced growth of axonal regeneration were carried out by Richardson and supraspinal and propriospinal axons into Schwann cell- Issa,136 in which a prior lesion ofthe peripherally seeded guidance channels in vivo,154 and boosted growth projecting axon ofa dorsal root ganglion (DRG) neuron ofdescending axons into fetaltransplants. 155 Recently, enhanced penetration into the CNS ofa grafted NT-3 was reported to promote regeneration ofdorsal centrally projecting branch. These experiments have column sensory axons into and beyond a graft of bone since been repeated.137,138 Ofthe various effects ofsuch marrow stromal cells, but only when these neurons were ‘conditioning’ lesions, two ofthe most important appear preconditioned by cAMP injection into the DRG.156 to be the upregulation ofregeneration-associated Fewer studies have shown that treatment with neuro- genes (RAGs) such as GAP-43 and CAP-23 (reviewed trophic factors on their own can induce axonal in Bulsara et al139), and the upregulation ofcAMP regeneration within the damaged spinal cord; however, (reviewed in Qiu et al140). While the expression ofGAP- intrathecally delivered NT-3 can induce regeneration of 43 does not necessarily imply that a neuron is injured dorsal column axons.157 Neurotrophins have regenerating,141 it does correlate well with the regen- also been delivered to the site ofSCI via genetically erative potential ofa neuron or neuronal population. modified cells, which could conceivably play dual roles Even though GAP-43 is not upregulated following a as both suppliers oftrophic molecules and substrates dorsal rhizotomy or dorsal column crush lesion, a prior for axonal growth. Fibroblasts, SCs, neural multipotent peripheral nerve injury both increases GAP-43142 and cells and olfactory ensheathing cells (OECs) have been improves the sprouting and/or regenerative response to modified to express various neurotrophins by ex vivo a subsequent central lesion.137 gene transfer; results of these important experiments Neurotrophic factors are also well-recognized for have been reviewed recently.158 their ability to induce RAG expression and to promote Since DRG neurons have been the best-characterized the elongation ofaxons. Following a dorsal rhizotomy, population in terms ofresponsiveness to neurotrophic neurotrophin-3 (NT-3) application promotes functional factors, we will use them as a primary example. centripetal regeneration as well as GAP-43 upregula- Significant inroads have been made into promoting re- tion.74,143,144 Following dorsolateral funiculus lesions of entry ofsensory axons into the adult spinal cord the spinal cord, brain-derived neurotrophic factor following dorsal root injury with these molecules application at the level ofthe red nucleus increases (reviewed in Ramer et al159). Intrathecal NGF promoted GAP-43 expression and improves regeneration of the ingrowth ofsmall-calibre axons that express CGRP, rubrospinal axons into a peripheral nerve graft.27 but not large-calibre axons that express NF200. By Like RAG expression, cAMP also appears to play an contrast, NT-3 promoted ingrowth ofNF200-expressing 40 important role in regenerative potential. Cai et al axons, but not axons that express the P2X3 receptor (ie demonstrated that ifsensory or cerebellar neurons were glial cell line-derived neurotrophic factor (GDNF)- pretreated with neurotrophic factors, there was a sensitive axons).160 GDNF promoted the regeneration resulting increase in intracellular cAMP, which allowed oflarge- and small-caliber axons, in line with the axons to elongate on inhibitory substrates. Since then, expression patterns ofthe requisite receptors. 161,162

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Encouragingly, regrowth was accompanied by synaptic inhibited, promoting growth cone motility and neurite reconnection, again in a selective fashion, such that outgrowth.58 On the other hand, several studies have NGF and GDNF treatments resulted in reactivation of pointed to a negative role ofp75 in neurotrophin dorsal horn neurons by slowly conducting (smaller signaling and neurite outgrowth: activating p75 in PC- diameter) sensory axons, and NT-3 and GDNF treat- 12 cells with BDNF inhibited trkA activation through ments facilitated the recovery of postsynaptic potentials the ceramide pathway;170 in sensory neuronal compart- that are evoked by rapidly conducting (larger diameter) mented cultures, binding ofBDNF or a p75-specific sensory fibers. NGF- and GDNF-treated animals antibody inhibited NGF-mediated trkA phosphoryla- regained the ability to sense pain, indicating that tion and slowed neurite outgrowth;171 and in vitro and in appropriate spinal circuits were being activated by vivo, BDNF-mediated p75 activation inhibited sympa- sensory input. NT-3 promoted the appropriate recovery thetic neurite outgrowth and target innervation.172 ofproprioception, 144 in-line with the distribution of Additionally, in p75À/À mice in vivo, some brain trkC on proprioceptive sensory neurons. While much structures are normally hyperinnervated,173 and regen- remains unanswered regarding the responsible mechan- eration following peripheral nerve injury is more robust isms, effects other than those directly on neurons (such in p75 knockouts than in wild-type mice.174,175 In vivo, as nonspecific effects on glial cells) can largely be p75À/À sympathetic axons invade injured DRGs (where excluded as fundamental due to the specificity of the glial expression ofNGF and NT-3 are increased 176) regenerative response and functional target reconnection. more profusely than wild-type axons following a spinal Despite the robust regeneration obtained using nerve lesion.177 Other startling in vivo data argue neurotrophic factors in animal models, the use of strongly for a negative contribution of p75 to axonal neurotrophic molecules as potential therapeutic agents growth in the CNS: in mice which overexpress NGF in is not without some pitfalls. The most obvious example CNS astrocytes, there is a massive developmental here is NGF, which while promoting the robust invasion ofCNS white matter tracts by sympathetic regeneration ofsmall-diameter peptidergic afferents into axons, and this invasion was greatly augmented when the spinal cord following rhizotomy also produces the NGF overexpressors were crossed with p75 knock- severe hyperalgesia.163 One ofthe apparent issues is out mice.178 that NGF-treated axons far overshoot their tar- More recently, it has been suggested that p75 gets164,165 to terminate deep in the dorsal horn. Tang mediates inhibitory signaling ofmyelin-derived inhibi- et al166 have taken advantage ofa developmental tory proteins (see above).58–60 Upon binding to the mechanism ofaxon repulsion in the spinal grey matter common receptor for these proteins (the Nogo recep- by transfecting ventral cells with semaphorin 3A. This tor), p75 is activated, leading to RhoA activation and acts to prevent nociceptive axons from penetrating too inhibition ofactin polymerization at the growth cone. deeply into the cord, and to partially inhibit the On balance, these findings indicate a clear rationale for development ofabnormal pain. 166 Direct spinal cord blocking p75 function in order to extend neurotrophin- trauma leads to the NGF-mediated sprouting ofCGRP- mediated regeneration into or within the CNS following positive fibers, which may be partly responsible for the injury. Indeed, our group has recently found that central development ofautonomic dysreflexia (AD), a severe regeneration following dorsal rhizotomy is enhanced in and life-threatening condition characterized by large p75À/À mice, as is intraspinal sprouting ofdescending and inappropriate elevations in blood pressure:167,168 in monoaminergic (serotonergic, noradrenergic, dopami- this situation, sequestering NGF improves outcomes in nergic) fibers.179 A recent publication showed that p75 rats and mice. By refining our ability to manipulate antagonism did not promote the regeneration ofinjured guidance cues, it may eventually be possible to extract dorsal column ascending (sensory) axons, but it from NGF its desired effects. For now, however, it noticeably prevented axonal retraction from the lesion remains an impractical member ofthe neurotrophin site.22 It is interesting to note that ascending dorsal family for clinical administration after SCI. column axons also appear to be refractory to anti- NogoA treatment,180 indicating that the selection ofa neuronal population for the study of regeneration is of p75: a wolf in sheep’s clothing or a sheep in wolf’s critical importance in the development ofpotential clothing? therapies. It also argues for the investigation of multiple Despite the well-characterized interactions oftrks with CNS pathways and at least the confirmation ofany p75 in neurotrophin signaling169 it remains unclear how preclinical finding in another population, especially if p75 contributes to (or subtracts from) neurite out- the intervention is conceived as having a potential global growth. In vitro studies using ciliary neurons have therapeutic benefit for stimulating functional regenera- indicated that p75 activation by neurotrophins can tion in patients. increase trk-mediated neurite outgrowth: it has been suggested that by constitutively activating RhoA (a small GTPase involved in the control ofactin filament A role for sparedaxons in functional recovery assembly and focal adhesions), p75 impedes neurite Most SCIs are incomplete, and repair strategies should elongation in the absence ofneurotrophin. With the include the optimization ofspared systems. In the addition ofligand (such as NGF), RhoA activation is absence ofregenerative therapy, rodents exhibit func-

Spinal Cord Orchestrating spinal cord repair LM Ramer et al 141 tional recovery ofcomplex motor tasks afterSCI, and Classical experiments established that injured spinal detailed analysis is often required to reveal deficits.181,182 axons can grow into a graft of peripheral nerve after Axonal sprouting was inferred by Liu and Chambers,183 complete transection ofthe spinal cord: 32,199 these and has since been demonstrated repeatedly.114,184–189 seminal works initiated the search for the most suitable This response ofundamaged axons has been credited biological bridging agent. Peripheral nerves are still used not only with producing motor improvements (reviewed experimentally to examine axonal growth in response to in Muir and Steeves190 Dunlop and Steeves,191 and other manipulations, and can support some return of ‘Rehearsing roles’, below), but also with the instigation function after complete thoracic transection (in rat) with ofchronic pain and AD. 192 concomitant administration ofgrowth factors. 200–202 The ability ofintact CNS axons to sprout remains Peripheral nerves have been implanted at the site ofSCI largely unknown, but appears to depend on phenotype in patients in Taiwan, China, Peru, and Brazil and a and the degree ofcord trauma. NGF-sensitive axons report ofsuch surgery recently became available. 203 In sprout more than GDNF-sensitive axons following this case, a patient with chronic paraplegia resulting partial cord deafferentation.193 Intact descending sys- from thoracic SCI was treated 4 years following injury tems (serotonergic and noradrenergic) respond differ- with autologous sural nerve grafts and acidic FGF. ently to dorsal rhizotomy.194 Intrinsic dorsal spinal cord In the 2 years following surgery, the patient exhibited neurons will sprout into dorsal roots, but only when motor and sensory improvements, indicated by the there has been (at least) some mild direct trauma to the American Spinal Injury Association (ASIA) rating score spinal cord, as occurs following dorsal root implanta- (which changed from ASIA C to ASIA D) and pin-prick tion or insertion ofa fine needle into the cord, but not tests. However, data from a small study involving eight after dorsal root injury alone.195,196 That mild trauma to patients with functionally complete SCI who received the cord promotes sprouting ofintact axons suggests the similar treatment, presented at the International Clinical involvement ofneurotrophic factors and cytokines Trials Workshop on SCI earlier this year, was less produced as a result ofinflammation. There have been encouraging.204 Autografts of peripheral nerve have also a limited number ofdemonstrations ofneurotrophic been used to bypass the lesion site: sural nerves grafted factor-promoted sprouting of intact spinal axons. While between ventral spinal cord above a thoracic lesion and NGF has been shown to promote intraspinal sprouting ventral roots below restored some voluntary movement ofnociceptive primary afferents, 164,165 the usefulness of in one person with chronic SCI.205 Today, nerve bridges NGF in SCI is severely limited by its painful side effects have largely given way to the development ofcellular (discussed above).163 Exogenous NT-3 promotes sprout- transplants for a number of reasons: single cells can be ing ofintact CST axons developmentally 197 and follow- better-defined than the assortment ofcells present in a ing rubrospinal tract ablation in adults.184 Like whole nerve, and can be characterized in vitro; cells can neurotrophic factors, neutralizing inhibitory myelin be injected as a suspension, to fill all aspects ofa cyst and ECM molecules can promote sprouting ofsome cavity at a lesion site, whereas implantation ofa nerve intact systems: blocking Nogo-A promotes sprouting of segment may require resection; and finally, cells can be adult CST axons,198 and digestion ofglial scar- genetically modified to produce and release factors at associated CSPGs promotes behavioral recovery follow- the lesion site (see ‘Stimulating axonal growth in the ing dorsal column injury in the absence ofrobust injured spinal cord’ above). regeneration ofinjured axons. 92

In the spotlight: Schwann cells andOECs Bridging the site of SCI Schwann cells (SCs) were the first to be cast as bridging agents for SCI. The rationale for injecting SCs into the Neuroprotective treatments might soon increase the injured spinal cord is clear, as SCs play a critical role in continuity across the lesion site (see ‘Neuroprotection in establishing the permissive nature for axonal regrowth the setting ofSCI’ above), but this advancement may within the peripheral nerve environment: when mitosis not eliminate the need for bridging transplants. In ofhost SCs is inhibited after sciatic nerve injury, axonal addition to providing a permissive substrate for axonal growth into acellular autographs is slowed.206,207 After elongation, the ideal bridge may confer continued peripheral nerve injury, SCs act as ushers for growing growth support, remodel the lesion site to allow axons axons by proliferating, decreasing their expression of to pass through, lend protection from inhibitory various myelin proteins, and increasing the expression of molecules, and/or even stimulate remyelination. Here, neurotrophic factors and cell adhesion molecules.208,209 we review recent data on axonal growth and behavioural Finally, Schwann cells can be obtained from adult recovery stimulated by cellular bridges tested in rats and peripheral nerve and rapidly expanded for autologous humans. The use ofcellular transplants to deliver transplantation: human SCs have been successfully neurotrophic factors (see ‘Stimulating axonal growth isolated, examined in vitro, and implanted into the in the injured spinal cord’), to remove inhibitory debris injured rat spinal cord.210–212 at the lesion site (see ‘Stimulating axonal growth in the Schwann cell transplantation at the site ofSCI in the injured spinal cord’), or to stimulate remyelination (see adult rat enjoys a prolific history and has been reviewed ‘Overcoming conduction block’) is discussed elsewhere. previously.213 Many experiments have tested a SC-filled

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PAN/PVC polymer channel, grafted at the site of acute growth and functional recovery.234–238 These data have thoracic transection in the adult rat.154,211,212,214–219 generated sufficient enthusiasm in the SCI community to Without genetic modification or addition ofneuro- instigate several clinical trials testing OECs worldwide. trophic factors, SC-filled grafts fuse with the cut stumps The first report ofa large trial in China was recently ofthe spinal cord, support ingrowth ofpropriospinal, published.239 Although OEC transplantation was re- sensory, and brainstem-spinal axons, and may underlie ported to generally improve both sensory and motor modest recovery offunction, such as rhythmic step- function, clinical follow-up was limited, confounding ping.213 However, supraspinal axons that enter SC interpretation ofthese results. As well, OECs in this trial bridges typically do not exit caudally to re-enter the were harvested and expanded from the fetal olfactory spinal cord. The inability ofdescending axons to re- bulb, an ethically problematic cell source. In Lisbon, enter host tissue after complete transection may result Carlos Lima has grafted pieces of autologous olfactory from myelin inihibition and/or an accumulation of mucosa at the site ofSCI in 20 patients, without CSPGs at the distal graft-host interface,220 an obstacle intervening in vitro manipulation or isolation ofOECs; which might be overcome with concomitant application some return ofboth sensory and motor functionhas ofanti-inhibitory compounds and/or neurotrophic been reported after these surgeries.204 Recently, OECs factors93 (see ‘Stimulating axonal growth in the injured have been successfully isolated and expanded from the spinal cord’ above). adult human olfactory mucosa, accessible by nasal In the early 1990s, another candidate for cellular biopsy, potentially permitting autologous transplanta- bridging took the stage. OECs are glia that support tion:240 these cells have entered a small Phase I trial in growth ofolfactory neurons – a routinely replenished Brisbane, Australia. population ofneurons – fromPNS-to-CNS throughout As OECs are being clinically tested, work in animal adult life. The rationale for implanting OECs into the models continues, both to identify potential mechanisms injured spinal cord is less obvious, since when the axons ofOEC-mediated recovery and to optimize methods of ofolfactory neurons are injured, they do not regrow OEC delivery. Recent data suggest that the mechanism their axons like peripheral nerves, but rather rapidly offunctional return may be more complex than degenerate and die.221–223 New neurons reconstitute regrowth oflesioned axons. In fact,OEC transplanta- the injured peripheral olfactory system,224 but do not tion protects spinal tissue from secondary damage and restore original connectivity, and this is reinforced prevents cavitation,241–246 enhances vascularization of clinically: permanent anosmia or dysosmia are relatively the lesion site235,246 and promotes branching ofneigh- common after blunt head injury, when movement of the boring axons spared by the primary injury,247 all of brain relative to the skull shears olfactory axons at the which might subserve improved functional outcome. cribriform plate.225 The rationale for using OECs, then, While earlier transplant experiments sought to isolate is not that they support axonal regeneration in situ, but OECs without other cellular components ofthe olfac- that they exist in a unique capacity to permit axonal tory nerve, recent work suggests that the recovery of growth across a PNS–CNS interface in the adult. function is enhanced by including other cell types, such Since olfactory axons grow across a PNS–CNS as olfactory nerve fibroblasts (ONFs), in the graft.248 interface, OECs (from the rat olfactory bulb) were first Grafts of OECs and ONFs were recently shown to injected into the rat spinal cord after dorsal root injury, reconstitute ipsilateral breathing rhythm in rats with to examine their ability to bridge the DREZ.226 In this cervical hemisections,249 although functional recovery study, afferent ingrowth was reported to extend into the after spinal hemisection must be interpreted with dorsal horn, in the areas ofappropriate targets, and caution, as it has been well-documented that both subsequent electrophysiological experiments suggested hemisected people and animals experience substantial that these axons achieved functional reconnection.227–229 and spontaneous recovery via preserved (undamaged) However, recent data from our group and others projections. Finally, the timing ofOEC transplantation indicate that OEC transplantation does not support may be important. Recent data indicates that OEC regeneration ofsensory axons across the injured transplantation has functional benefits even when DREZ.230–232 Using OECs from the GFP mouse, we delayed (for 1 week to 2 months after injury in the have observed that OEC injection into the spinal cord rat)250,251 and one study suggests that delayed trans- mechanically disrupts the DREZ, permitting sensory plantation is actually more beneficial than acute axon ingrowth but not functional regeneration involving delivery.244 To address some ofthese important issues, the re-acquisition ofappropriate targets on the other and to more confidently predict the effects ofOEC side ofthe injured DREZ. 232 transplantation in humans, OEC transplantation is After SCI, OECs were also applied to remodel a being investigated in non-human primates with com- PNS–CNS interface, namely, that established by graft- plete spinal cord transection (A. Ramon-Cueto, pers. ing Schwann cells into CNS tissue. When OECs were comm.). injected into the proximal and distal stumps of transected spinal cord spanning a SC bridge, regenerating axons crossed the graft–host interfaces and re- SCs and OECs: a duel or a duet? entered the spinal cord.233 Other experiments indicated Despite the relatively rapid progression from animal that OECs alone were sufficient to support both axonal models to clinical trials, OECs have not clearly out-

Spinal Cord Orchestrating spinal cord repair LM Ramer et al 143 performed SCs as cellular bridging agents. The two cell grafts of fetal tissue, but emerge distally in a more types are comparable in some respects, but not in limited fashion, a limitation that may be overcome others.252 Like SCs, OECs express adhesion molecules by concomitant administration ofneurotrophic fac- and neurotrophic factors and can support growth by tors.155,276 Transplantation offetalspinal tissue has many types ofneurons. 152 Unlike SCs, OECs do not been tested clinically in the United States in patients proliferate or migrate in response to injury of their with progressive post-traumatic syringomyelia, and associated axons.253 Both types ofcells have been preliminary reports suggest that this treatment is genetically modified prior to transplantation to deliver safe,277,278 although complete reports have not been growth factors at the lesion site.245,254,255 OECs have published. Since the use offetal tissue entails ethical been reported to integrate more successfully with challenges, it may need to be proven superior to other astrocytes,256 and to induce less glial scarring257 than biological bridges in order to assume the role ofa SCs, but may themselves express inhibitory molecules preferred clinical bridging intervention. (such as Nogo or proteoglycans, discussed in ‘Stimulat- Recently, various populations ofmultipotent and ing axonal growth in the injured spinal cord’ above) progenitor cells have gained increasing attention as after transplantation.258 Few experiments have directly potential grafting agents for SCI. Spinal and supraspinal compared SC- and OEC-transplantation in the same axons grow into grafts of neural multipotent cells, injury. When SCs and OECs were injected (separately indicating that they do function as a permissive and together) into acute thoracic contusion injuries in bridge,279–281 but the possibility that multipotent/pro- rat, both cells induced tissue sparing and angiogenesis genitor cells might differentiate into spinal neurons to at the lesion; however, functional outcome was only replace lost spinal circuitry is perhaps more intriguing. improved in animals that received SCs.243 SCs were Both embryonic multipotent cells and neural progenitor also recently reported to support regeneration ofrat cells can differentiate into neurons and extend axons retinal ganglion axons, while OECs applied in the same into host tissue,26,282 raising the possibility that multi- manner did not.259 In a recent combination study, SCs potent cell grafts could also function as a relay center bridged a thoracic contusion site to support growth and within the injured spinal cord. To date, however, remyelination in rolipram and cAMP-treated animals neuronal differentiation ofmultipotent cells graftedat (see ‘Casting the combination’, below).260 the site ofSCI has been limited, and the vast majority On a final note, OECs and SCs may share the stage: ofgraftedtoti- or mulitpotent progenitors become recent work from our group demonstrates that OEC glia.283,284 Interestingly, functional recovery after thor- transplantation enhances invasion ofhost SCs into the acic hemisection was improved when neural multipotent injured spinal cord.246 This finding is especially sig- cells were delivered in a degradable polymer scaffold, nificant in light ofrecent data indicating that myelin despite the fact that grafted cells did not differentiate within OEC grafts is SC-, not OEC-derived261 (discussed into neurons.285 In this study, improved functional in the subsequent section below). Whether the observed outcome was associated with tissue sparing and reduced axonal growth and/or functional recovery after SCI can glial scarring, suggesting that multipotent cells may be attributed to the presence ofgraftedOECs or might exert protective effects. Improved functional recovery also be due to infiltrating host SCs remains to be seen. from multipotent-cell-treated SCI associated with oligo- SC infiltration/penetration ofthe CNS has been linked dendrocyte differentiation and remyelination ofhost to regeneration ofspinal 262,263 and vagal afferents.264 axons is discussed as a potential remyelination strategy (in next section). Multipotent cell-induced recovery in animal models In the wings: fetal tissue, multipotent/progenitor cells has prompted clinical testing in Russia, where 15 andsynthetic implants patients received injections offetalbrain and hemapoie- Spinal transection in the vertebrate embryo, prior to the tic (liver) cells; 11 patients received concomitant grafts onset ofmyelination, results in functional regeneration ofOECs and fetalspinal cord. 286 In this study, six and repair.265–268 Akin to the use ofperipheral nerve patients that exhibited complete sensory and motor grafts, grafts of fetal brain and spinal cord have been paralysis prior to transplantation regained the ability to applied to the site ofSCI to provide axons in the adult walk with or without assistance. However, five patients spinal cord with the permissive environment ofthe in this group received transplants within 4 months of embryo269–271 (reviewed in Anderson et al272 and SCI, and four received OECs and fetal spinal cord in Lakatos and Franklin273). In addition to acting as a addition to multipotent cells, so the results ofthis study permissive substrate for elongating host axons, grafted are difficult to interpret. While no complications related embryonic neurons can both receive synaptic input from to multipotent cell transplantation have been reported, and extend axons to host spinal neurons, thereby acting the incomplete understanding ofmultipotent cell regula- as a relay across the injured spinal cord.274 Fetal tissue tion, proliferation, and differentiation both in situ and can be used to replace some ofthe neurons lost in SCI: upon transplantation may have important implications for example, pieces of ventral embryonic spinal cord can for imminent clinical translation of this technology.287,288 give rise to neurons that reinnervate muscular targets Despite its promise in animal models, cellular and reverse muscle atrophy in rat.275 Analagous to the transplantation presents several challenges for clinical peripheral nerve environment, axons grow readily into translation. Timing oftransplantation, cell source, and

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cellular maintenance/manipulation are important inher- reviewed recently;302–304 therefore, we limit this discus- ent variables that may preclude comparison ofresults sion to recent data that implicates SCs as a source of from clinical trials at different centers. Additionally, peripherally derived myelin after both SC- and OEC- immunological compounds may be associated with graft transplantation into spinal lesions. rejection. Synthetic implants under development offer OEC myelination was first reported in vitro. When rat the attractive potential ofbeing identical and reprodu- OECs were cocultured with rat DRG neurons or cible in composition, immunologically inert, and even olfactory receptor neurons, myelin sheaths formed that absorbable. Ifcellular transplantation afterSCI does were indistinguishable from those formed by SCs under become a clinical reality, synthetic implants may be used similar conditions.305,306 In a recent study, however, to contain/deliver the cells at the injury site and/or to OECs cultured from adult rats did not retain the ability guide axons growing through these biosynthetic to myelinate embryonic DRG neurons; instead, myelin grafts.213 Recently, biomaterials have been designed to in DRG cultures was formed by Schwann cells that deliver neurotrophic factors, and may provide an persisted in culture despite antimitotic treatment with attractive alternative to the intrathecal pump, as delivery fluorodeoxyuridine (FUDR).307 These data, demon- is reproducible and immobilized concentration gradients strating the propensity ofSCs to myelinate axons even can be created.289–291 In two recent experiments, in the presence ofOECs, have potentially important synthetic implants were applied to the site ofcomplete implications for interpreting the results of OEC trans- thoracic transection in rat; in these studies, BDNF-filled plantation experiments. foam292 and synthetic hydrogel guidance channels293 Focal areas ofdemyelination in the adult spinal cord integrated with host tissue and supported axonal can be created by injections ofethidium bromide or ingrowth, although axons did not regenerate beyond lysolecithin, both ofwhich are toxic to oligodendro- the implant. While we want to stress the potential cytes. Typically, the demyelinating lesion is created in importance ofsynthetic implants in stimulating regen- tissue that has been X-irradiated several days previously, eration and recovery after SCI, we are constrained by which prevents host oligodendrocytes from repopulating space, and direct the interested reader to reviews of the lesion site.308 Using this model, peripheral myelin developments in this field.294–296 has been observed in the demyelinated dorsal columns ofthe adult rat subsequent to transplantation ofrat, 309–314 Overcoming conduction block human, and canine OECs and recently, in the dorsal columns ofthe monkey followingporcine OEC Although the CNS has the capacity to replace oligoden- transplantation.315 In these experiments, peripheral drocytes lost in spinal trauma, and remyelination of myelin was morphologically and/or antigenically indis- 297,298 intact demyelinated axons can be spontaneous, tinguishable from that formed by SCs myelinating remyelination in the wake ofSCI is not robust, and previously demyelinated CNS axons.316,317 the resulting conduction block ofaction potentials due When OECs and SCs were grafted at the same type to persistent demyelination likely contributes to loss of ofcontusion injury, more myelinated axons were found 299 function in SCI. To supplement intrinsic CNS within SC-containing grafts than within OEC-only remyelination, myelinogenic and progenitor cells have grafts.243 Importantly, peripherally myelinated axons been grafted at the site of SCI in rats, and such cells were also found in nontransplanted lesions, demonstrat- have been tested in both focal demyelinating lesions ing that host SCs could access the untreated contusion (inducing oligodendrocyte death without axonal trau- site, in limited numbers, and produce myelin.243 ma) and traumatic lesions (resulting in axon damage). Remyelination has also been achieved by stimulating Remyelination may differ among intact versus regener- Schwann cell immigration via cellular delivery of ating axons; nonetheless, models offocaldeymelination GDNF at the site ofthoracic transection. 318 Recently, have provided important information regarding the Boyd et al261 used virally labeled OECs to demonstrate extent and mechanisms ofspinal remyelination stimu- that peripheral myelin that formed within the OEC- lated by cell transplants. Here, we review recent data transplanted site ofSCI was derived entirely from in this field, and also consider another approach to invading host SCs and not from grafted OECs. Taken alleviating conduction block via systemic drug treat- together, these provocative data highlight the role of ment, which is currently being tested in a clinical trial. endogenous SCs in accomplishing remyelination of spinal axons. While it has been reported that SC Stage left: Schwann cells andOECs remyelination ofspinal axons is a transient phenomen- In addition to the fame they have acquired in supporting on, with SC myelin progressively replaced by oligoden- axonal elongation (see ‘Bridging the site ofSCI’), SCs drocyte myelin during recovery,319 the most recent data and OECs have also been acclaimed for their ability to indicates that SC myelin is stable and persists in the remyelinate axons after SCI. Endogenous SCs can spinal cord. invade spinal lesions and form functional myelin,300,301 providing the impetus for transplantation of SCs (and Stage right: progenitor cells by association, OECs) to alleviate SCI-induced conduc- In response to demyelination ofspinal axons, a tion block. Experiments reporting SC- and OEC- resident population ofendogenous oligodendrocyte provoked remyelination after spinal insult have been precursors proliferate and differentiate into mature

Spinal Cord Orchestrating spinal cord repair LM Ramer et al 145 oligodendrocytes to initiate remyelination.320–322 Pro- short- and long-term changes within existing genitor cells have been grafted at the site of SCI to circuits.334,335 The extent to which such plasticity is enhance this repair process. While grafts of SCs or accomplished as a result ofaxonal sprouting or short- OECs trigger formation of peripheral myelin, progenitor distance regeneration versus compensatory changes cell transplantation aims to reconstitute central myelin within existing circuits remains to be determined.190,336 at the site ofSCI. Neural progenitors fromboth mouse Nevertheless, the available studies point to an innate embryonic multipotent cells and embryonic rat spinal and remarkable ability for the damaged adult CNS cord have been injected at the site ofcontusion injury in to undergo spontaneous or activity-dependent plastic rat, where both types ofgraftedcells differentiated into changes. A choreographed recovery after SCI is oligodendrocytes (as well as astrocytes and neurons) and dependent on a diverse number ofplayers, contributing enhanced functional recovery,323,282 although remyeli- their parts in many subtle, but important, ways. Recent nation was not examined in either study. Stem-like cells, research has been able to identify a diverse number of derived from bone marrow, have been reported to poorly understood mechanisms that can contribute to a myelinate spinal axons when introduced at the site offocal remodeling of the CNS after injury. The challenge for demyelination.324,325 Intriguingly, these cells also stimu- rehabilitation after SCI is to ensure that these altera- lated remyelination – and themselves formed myelin – tions are beneficial rather than detrimental and max- when injected into the bloodstream.325,326 Ifsuch cells can imized to their fullest extent. reliably overcome the blood–brain barrier, migrate to For example, damage to the periphery (eg, by digit demyelinated CNS regions and differentiate appropriately, amputation, retinal lesions) results in latent responses with their proliferation being controlled, then an intrave- being recorded almost immediately (within minutes) nous delivery route presents an attractive alternative to from cortical regions that had previously received input direct parenchymal injection into the damaged CNS. from the damaged regions.335 In other words, loss of input from the periphery can result in the rapid appearance ofexpanded receptive fields ofthe remaining Now showing: treatment with 4-aminopyridine cortical neurons. This phenomenon has been referred to While cellular transplantation might rebuild stable as synapse ‘unmasking’, a process proposed by Bas- myelin to permanently restore conductivity, transplan- baum and Wall to underlie receptive field expansion tation procedures are invasive and immunologically following partial spinal deafferentation.337 Later, an problematic. Pharmacological treatment, although it anatomical correlate ofsynapse unmasking was de- must be chronically sustained, may represent a more scribed by Goshgarian et al, which occurred within 4 h imminent solution to the problem ofconduction block. ofSCI, and involved astrocytic process retraction and a Fampridine, or 4-aminopyridine (4-AP), is a potassium subsequent increase in the area ofsynaptic apposition. channel blocker that restores conduction in de- or The rapidity ofthe changed response patterns revealed a dysmyelinated axons.327,328 When 4-AP is added to an previously under appreciated feature of CNS organiza- in vitro preparation ofinjured spinal cord, conduction tion, namely that each region receives input from a across the site ofSCI is improved. 329–331 In people with much wider peripheral area than can normally be incomplete SCI a sustained-release (oral) formula of detected electrophysiologically.334,335 Furthermore, the 4-AP (Fampridine) was reported to improve sensory data suggest that much ofthe input to the receptive field and motor function and reduce spasticity during must normally be subthreshold for detection or actively treatment.332 As a result ofthese encouraging data, suppressed (inhibited) and that damage brings about an Acorda Therapeutics (New York, USA) initiated two almost immediate expansion ofthe receptive field. large, multicenter clinical trials testing 4-AP in chronic In addition to short-term plastic activity changes, SCI (8) and published results ofthese trials may soon long-term changes also occur and appear to be under- become available.204 Animal experimentation with 4-AP pinned by anatomical sprouting or rewiring ofsynaptic is ongoing, in an effort to clarify the optimal parameters connections (see above338) Small binocular retinal of4-AP treatment. Although pharmacological interven- lesions initially silence the corresponding portion of tion is logistically simpler than cell transplantation, the visual cortex. However, within weeks to months, the issues ofeffective dose and delivery may be complex; for cortex becomes responsive to inputs from adjacent example, recent data revealed that 4-AP more potently portions ofthe uninjured retina. Anatomical tracing overcomes conduction deficit induced by stretching studies revealed extensive axonal sprouting from the forces than conduction deficit induced by compres- adjacent cortex, which was interpreted as arising from sion.333 These experiments underscore the importance of intrinsic neurons that had formed new stable connec- animal models in optimizing treatments for SCI, even tions within the denervated region. Likewise, recent and especially after clinical trials are underway. reports using trans-synaptic tracing techniques, electro- physiology and behavioural assessments have demon- Rehearsing roles: rehabilitation and CNS plasticity strated a significant degree ofplasticity involving compensatory sprouting from corticospinal projections Even though there is little evidence for spontaneous with concomitant functional rewiring after a dorsal de novo regeneration ofdamaged CNS axons over hemisection injury ofthe adult rat thoracic cord. 339 It is substantial distances, there is overwhelming evidence for important to recognize that even ifregeneration of

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injured axons is successful, or if synaptic spaces can isolated spinal cord to generate stepping movements become newly occupied by intact axons, the target in after complete spinal cord transections, which were both cases is profoundly altered by injury. As a undertaken at low levels, namely T12–13. It has now consequence, functional reovery will inevitably require been firmly established that, with interactive training, some form of retraining. adult spinal transected cats can rapidly develop locomotion. Initial studies showed that animals developed Practice makes perfect stepping patterns on a treadmill iftheir weight was 345 Given the innate capacity for the damaged nervous partially supported and their balance stabilized. system to undergo plastic changes, the question arises Strong pinching ofthe perianal and abdominal regions as to whether plasticity within undamaged CNS circuits was only necessary in the first few days of step training or regenerating pathways can be harnessed to improve and could be reduced to very light stimulation over the functional outcome. Reports point to the importance of following 3 weeks. All aspects of locomotor behavior appropriate training to enhance recovery after damage. improved over the subsequent months, and animals After sciatic nerve crush in rats, both sensory and motor were able to support their body weight, often stepping recovery is improved by placing water bottles at heights on the treadmill for up to 10 min before losing their such that animals have to extend both hind paws balance. However, without daily step training, perfor- maximally to drink; animals with water bottles on the mance regressed with animals dragging their hindlimbs, floor do not show improved recovery.340 There are, although locomotor performance could be re-estab- however, offsetting data in the same report suggesting lished within a few days by step training. that overactivity or stressful regimes such as forced The improved performance compared to spontaneous running or swimming can impede rather than enhance recovery was attributed to neurons being activated in functional recovery. a more appropriate fashion by training. Recent data Generation ofmovement requires the coordination obtained using a rodent exercise device indicated that a ofa vast network ofneuronal subsets, resulting in the daily exercise program ofmoving paralyzed hindlimbs coordinated activation ofthe limbs. There is ample through the motions ofwalking prevented atrophy of 346 evidence that the intrinsic (or isolated) spinal cord spinal motor neurons. Furthermore, it was suggested circuitry has the capacity to directly generate many of that neural activity facilitated the return of function in the complex aspects oflocomotion. Complex motor existing sensorimotor pathways rather than the genera- patterns are not just programmed by supraspinal tion ofnew pathways. Although some spontaneous systems such as the cortex, basal ganglia, and cerebel- recovery was seen when animals were not step trained lum, with the spinal cord contributing only passively or but were tested briefly on the treadmill, these animals reflexively by relaying sensory and motor information to showed only short bouts ofweight-bearing stepping and 347 the brain or muscles.341 stumbled frequently. Locomotion, whether walking, running, flying, or Ifthe isolated spinal cord can learn specific tasks, how swimming, is ultimately produced by spinal neurons long can it remember them? Cats that underwent that are collectively termed central pattern generators treadmill step training for 12 weeks still performed (CPGs). Normally, CPGs are activated by supraspinal maximally on all criteria when tested on the treadmill 348 control from the brainstem and thalamus and are also for up to 6 weeks after cessation of training. continuously modified by peripheral cutaneous and However, performance declined considerably if training proprioceptive input. However, it has long been known was withheld for 12 weeks. Nevertheless, retraining after that the spinal cord can act independently to produce 12 weeks ofwithdrawal resulted in a more rapid locomotor movements. Indeed, CPGs appear to be a improvement in stepping than that seen during the broad vertebrate characteristic because removal of initial training period. The results suggest that the supraspinal input in lampreys, birds, nonprimates, and isolated spinal cord can learn, and it will forget a task primates (including humans) by complete or nearly without maintained use or practice; however, the complete spinal cord transection results in the retention isolated spinal cord retains trace memories that can be oflocomotor movements. 342–344 In these instances, rapidly recovered upon renewed training. locomotor movements can be initiated by a variety of For the majority ofindividuals with functionally stimuli such as certain postures, electric shocks, or complete injuries, there is a small band ofspared tissue exercise. Given that the spinal cord has the capacity to that bridges the gap. Thus, clinically (functionally) encode and execute both stepping movements and higher- complete lesions are not necessarily ones where anato- order functions associated with locomotion, the evidence mically the spinal cord has been completely severed. is now accumulating that these abilities can be harnessed Studies have shown that some locomotor recovery can to generate beneficial function after spinal injury. occur in animals where only a small subset ofaxons, spanning a lacerating lesion, is spared.349,350 In addition, after an initial contusion injury that spares some axons Training andneural activity to harness spinal cord around a central cavity and that mimics the type of plasticity closed SCI most commonly seen in humans, rats recover The cat has proved to be an extremely valuable model some locomotion.351 The surprise was that ifthe spinal for assessing the effect of training on the ability of the cord was then completely cut, rats were still able to

Spinal Cord Orchestrating spinal cord repair LM Ramer et al 147 maintain some locomotor function, something that further improvements. However, all individuals with rarely happened after spinal cord transection alone. complete injuries were unable to maintain stepping Furthermore, animals recovered a greater range of movements after training stopped; thus, the stepping motor movements and did so more rapidly than after that had been learned during training could not be transection alone. The interpretation was that even translated into overground walking and furthermore small percentages ofspared axons are capable of appeared to be lost in the absence ofcontinued practice ‘instructing’ the spinal cord below the lesion and that (‘use it or lose it’). the instructions are retained. Treadmill training has been used with considerable success in people with SCI, classified as functionally Rehabilitation rigor incomplete,352 that is, with retention ofsome sensory or Rehabilitation research often lacks a foundation of motor function below the level of injury (ASIA scale experimentally derived data. There have been few B–D). Persons were selected ifthey had some voluntary multicenter trials including a statistically significant activity in leg muscles, had mobile joints, had no number ofparticipants with double-blind assessments spasticity and ifthey lacked complications such as and randomized controls. Rehabilitation has been ulceration or infection. Treadmill training involved susceptible to the practice ofprocedures developed on sessions of30–60 min, 5 days a week for3 weeks to 5 the basis ofpersonal experiences ofthe therapist, in part months, starting with low treadmill speeds. The aim was because there are few catastrophic consequences if a to encourage movements that mimic natural walking as therapeutic strategy fails. All too often, it has also been much as possible and involved providing maximal difficult to objectively measure a beneficial outcome or sensory feedback from the muscles, joints, and skin. standardize an effective treatment strategy across multi- Initially, partial body-weight support was provided by ple rehabilitation centers that is independent ofthe a harness, and the legs were moved and the feet were important, but confounding trial variable, ‘chemistry’ placed on the treadmill by physiotherapists. Persons that intrinsically develops within a therapist–client were encouraged to put their full body weight onto the relationship. extended (say, right) leg during the stand phase and then However, this is changing rapidly. For example, there shift their body weight onto the left leg just before is currently a multicenter NIH-sponsored trial, using swinging the right leg forward. As performance im- blinded assessments ofpeople with incomplete SCIs, proved, persons were encouraged to swing their arms in investigating the benefits ofweight-supported treadmill the way they would ifwalking naturally. Persons were training versus more conventional forms of active also encouraged to attempt overground walking as soon physical therapy. Other active forms of rehabilitation as possible. The study included comparison with persons such as constraint-use therapy and functional electrical who had previously received conventional physiother- stimulation are also undergoing more rigorous evalua- apy only. Treadmill training, rather than conventional tions. physiotherapy, resulted in remarkable improvements. In summary, neural activity shapes the developing Of44 patients that were wheelchair-bound, over two- and adult CNS. Ifpeople with SCI can regain some thirds learned to walk independently, at least for short function through the myriad plasticity changes in distances, with only one person not showing any whatever small number ofundamaged pathways persist improvement; about halfofthese patients were capable after injury, then the possibility of combining active ofwalking up stairs. Seven paraplegic persons classified rehabilitation therapy with surgical and therapeutic as complete, that is, no function below the level of interventions promises realistic opportunities for im- injury, were also included in the study and were selected proved functional recovery after SCI in the near future. because some aspects ofstepping could be evoked by placing them on the treadmill. However, despite daily Casting the combination training, no improvements were seen, and they were not able to generate full stepping cycles. Follow-up studies Several recent studies highlight both the potential in persons with incomplete injuries were encouraging success ofcombination therapy, and the complexity and showed that improvements made during a few inherent in its development. These studies combine weeks to months oftreadmill training were maintained growth-promoting and bridging strategies in three for 6 months to 6 1/2 years after training ceased.352,353 different models ofSCI in the adult rat. They are The long-term effects oftreadmill training have notable not only because the results are encouraging, recently been compared for individuals that are func- but because they represent collaborative efforts between tionally incomplete and complete.354 After training experts in different fields, the type ofcooperation (15 min daily, 5 days per week for several months), required to develop a successful combination therapy persons with incomplete injuries showed significant for SCI. improvements and could use their new locomotor In one study, sensory neurons were primed for growth capabilities for overground walking for at least short in some animals by injecting dibutyryl-cAMP (db- distances. The benefits that had been acquired during cAMP) into the lumbar DRGs 5 days prior to a cervical training could thus be maintained by independent lesion ofthe dorsal columns. 156 Immediately following movement, although there did not appear to be any the injury, all animals received implants ofautologous

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Table 1 Combination therapies applied to SCI in adults

Demyeli- Anti- K+ Other Myelin nation/ scarfor- Conditio- Neuro- Synthetic Multi- channel Methyl- neuro- antago- myelin mation/ ning trophic cAMP Nerve Schwann bridges/ Prenatal potent antago- Rehabili- prednisolone protective* nism clearancea deposition lesion factorsb elevation graft cells OECs scaﬀolds tissue cells nism tationc rhsrtn pnlcr repair cord spinal Orchestrating

Methyl-prednisolone 355 À359,361 À364 365 355 360,366,367 212,218,260 236,260 212,218 368 Other neuroprotective* 355 À359,361 À364 369 À372 373 355 369 374 Myelin antagonism 211,375 180 211 Demyelination/myelin 365 376,377 clearancea Anti-scar formation/ 373 378 93 379

deposition Ramer LM Conditioning lesion 355 355 380,381 136,380 À383 Neurotrophic factorsb 360,366,367 369 211,375 380,381 156 23,27,153, 154,211, 245,255 151,292, 151,155, 156,280, 200,201,203, 254,392 À397 398 À403 276,400, 406 380,381, 404,405 tal et 384 À391 cAMP elevation 156 407 150 149 156 Nerve graft 374 180 378 136,380 À383 23,27, 407 153,200, 201,203, 380,381, 384 À391 Schwann cells 212,218,260 211 376,377 93 154,211,254, 150 243,260 212,214 À217, 392 À397 219,408 À414 OECs 236,260 245,255 243,260 Synthetic bridges/ 212,218 379 151,292, 212,214 À217, 151,392, 285 scaﬀolds 398 À403 219, 400,415 408 À414 Prenatal tissue 151,155, 149 151,392, 416,417 276,400, 400,415 404,405 Multipotent cells 156,280,406 156 285 K+ channel antagonism 368 Rehabilitationc 416,417

*Other neuroprotective treatments include therapies aimed at reducing tissue damage or loss related to inflammation and/or excitotoxicity; hypothermia is considered a neuroprotective treatment aDemyelination includes T-cell-based vaccination, but not X-irradiation or ethidium bromide-induced lesions bNeurotrophic factors include all treatments thought to exert or stimulate trophic effects on spinal and/or supraspinal neurons cRehabilitation does not include studies using treadmill training solely as a means ofassessing locomotor recovery Orchestrating spinal cord repair LM Ramer et al 149 bone marrow stromal cells (BMSCs), and some animals ongoing for more than a decade. Table 1 summarizes received injections ofNT-3 at the lesion site. At 1 week many (but by no means all) ofthe attempts at following injury, some animals received injections of combination therapy applied in adult spinal cord. In NT-3 rostral to the lesion. In order to dissect the effects addition to the sheer number ofstudies combining ofeach treatment, this study included five experimental different treatments, the distribution ofcombinations groups. While sensory axon growth into the BMSC graft are ofinterest. For example, cell transplants and was improved by any combination ofcAMP and/or NT- neurotrophic factors have been combined many times, 3 injections, significant growth beyond the graft was while combinations testing rehabilitative treatments are only observed in animals that received both cAMP comparatively scant. This is unexpected, given that most injections prior to surgery and NT-3 injections at and SCI patients have access to some form of rehabilitation beyond the lesion following injury. No significant and that any treatment that moves to clinical trial is recovery offunction was observed in any ofthe animals, likely to be tested on a background ofsome formof likely because regenerating sensory axons did not reach rehabiliation. A review ofcombination therapies also their targets in the nucleus gracilus. Although stimulat- serves as a warning that unforseen interactions between ing regeneration through preconditioning is not clini- treatments may preclude the application ofsome in cally applicable, this study illustrates that growth- combination. For example, a recent study revealed that promoting interventions aimed at different parts ofthe acute delivery ofmethylprednisolone and interleukin-10 neuron – the axon and soma – can have additive worsened the behavioral outcome ofSCI in animals that effects. received SC transplants.260 In another study exploiting the growth-promoting The complexity ofcombination therapy will only effects ofcAMP, a cervical hemisection was treated with escalate in prospective clinical trials where randomized embryonic spinal tissue, and (in experimental animals) control groups and blind assessment will be required to rolipram.149 The graft was implanted immediately test safety and efficacy of candidate therapies. Some following injury, and rolipram was administered 2 components ofSCI therapy may entail less risk, and weeks later, for a duration of 10 days. Rolipram-treated thus move to clinical trial sooner, than others. For animals exhibited both increased axonal growth into the example, pharmacological treatments may entail less embryonic transplant and evidence ofimproved forepaw risk than surgical interventions, although both may be placement compared to vehicle-treated controls. While required to achieve recovery after SCI. Such risk–benefit this experiment tested the efficacy ofdifferent concen- considerations will vary between patients according to trations ofrolipram, it did not examine the effects of the type, level, and extent ofinjury to further complicate rolipram treatment alone, without concomitant grafting, approval for and analysis of clinical trials. These and or ofadministering rolipram immediately following other issues surrounding clinical trial design, including SCI. an urgent need for communication between regulatory The effectiveness ofacute rolipram was examined in a agencies and the SCI research community, were raised at third study, in which a moderate thoracic contusion the International Clinical Trials Workshop on SCI held injury was treated with various combinations ofa SC in Vancouver in 2004.204 graft, a single injection of db-cAMP near the graft, and Despite these intrinsic difficulties, the show must go systemic administration ofrolipram over 2 weeks. 150 on, and clinical trials must be guided by animal The timing ofintervention is ofinterest in this experiments that rigorously test neuroprotective, experiment, as rolipram treatment was initiated either growth-promoting, bridging, remyelinating and rehabi- at the time ofinjury or 1 week following injury, while SC litative therapies for SCI in isolation and in combina- grafts and db-cAMP injection were administered only 1 tion. The successful combination therapy will consider week following injury. Even without testing all combi- the interactions between players to ensure that they take nations ofacute versus delayed interventions, this study the stage at the correct place and time to maximize involved seven different experimental groups. Rats that efficacy and minimize risk, and will reveal that there are, received rolipram immediately following injury plus SC truly, no small parts. grafts and db-cAMP exhibited increased sparing and growth ofsupraspinal axons, as well as improved locomotor recovery, compared to rats that received References only SC transplants; delaying rolipram administration 1 Bunge MB. Bridging areas ofinjury in the spinal cord. generally reduced treatment efficacy. By most outcome Neuroscientist 2001; 7: 325–339. measures reported, the combination ofSC graftplus 2 Hausmann ON. Post-traumatic inflammation following acute rolipram also enhanced axonal sparing/growth spinal cord injury. Spinal Cord 2003; 41: 369–378. and functional recovery. Interestingly, acute rolipram 3 Bracken MB et al. Methylprednisolone or tirilazad alone was sufficient to augment both sparing/ mesylate administration after acute spinal cord injury: 1-year follow up. Results of the third National Acute growth and functional recovery relative to untreated Spinal Cord Injury randomized controlled trial. J controls. Neurosurg 1998; 89: 699–706. While combination therapy has achieved star status in 4 Geisler FH, Coleman WP, Grieco G, Poonian D. The recent years, attempts to synergistically combine treat- Sygen multicenter acute spinal cord injury study. Spine ments aimed at different components ofSCI have been 2001; 26: S87–S98.

Spinal Cord Orchestrating spinal cord repair LM Ramer et al 150

5 Bracken MB et al. Methylprednisolone or naloxone 22 Song XY, Zhong JH, Wang X, Zhou XF. Suppression of treatment after acute spinal cord injury: 1-year follow- p75NTR does not promote regeneration ofinjured spinal up data. Results ofthe second National Acute Spinal cord in mice. J Neurosci 2004; 24: 542–546. Cord Injury Study. J Neurosurg 1992; 76: 23–31. 23 Kwon BK et al. Survival and regeneration ofrubrospinal 6 Kwon BK, Tetzlaff W, Grauer JN, Beiner J, Vaccaro AR. neurons 1 year after spinal cord injury. Proc Natl AcadSci Pathophysiology and pharmacologic treatment ofacute USA 2002; 99: 3246–3251. spinal cord injury. Spine J 2004; 4: 451–464. 24 Ruitenberg MJ et al. Adeno-associated viral vector- 7 Hugenholtz H. Methylprednisolone for acute spinal cord mediated gene transfer of brain-derived neurotrophic injury: not a standard ofcare. CMAJ 2003; 168: factor reverses atrophy of rubrospinal neurons following 1145–1146. both acute and chronic spinal cord injury. Neurobiol Dis 8 Blight AR. Miracles and molecules – progress in spinal 2004; 15: 394–406. cord repair. Nat Neurosci 2002; 5(Suppl): 1051–1054. 25 Giehl KM, Tetzlaff W. BDNF and NT-3, but not NGF, 9 Bethea JR, Dietrich WD. Targeting the host inflamma- prevent axotomy-induced death ofrat corticospinal tory response in traumatic spinal cord injury. Curr Opin neurons in vivo. Eur J Neurosci 1996; 8: 1167–1175. Neurol 2002; 15: 355–360. 26 McDonald JW, Howard MJ. Repairing the damaged 10 Schwartz M, Lazarov-Spiegler O, Rapalino O, Agranov I, spinal cord: a summary ofour early success with Velan G, Hadani M. Potential repair ofrat spinal cord embryonic stem cell transplantation and remyelination. injuries using stimulated homologous macrophages. Prog Brain Res 2002; 137: 299–309. Neurosurgery 1999; 44: 1041–1045. 27 Kobayashi NR, Fan DP, Giehl KM, Bedard AM, 11 Blight AR. Delayed demyelination and macrophage Wiegand SJ, Tetzlaff W. BDNF and NT-4/5 prevent invasion: a candidate for secondary cell damage in spinal atrophy ofrat rubrospinal neurons aftercervical axot- cord injury. Cent Nerv Syst Trauma 1985; 2: 299–315. omy, stimulate GAP-43 and Talpha1-tubulin mRNA 12 Shuman SL, Bresnahan JC, Beattie MS. Apoptosis of expression, microglia and oligodendrocytes after spinal cord contu- and promote axonal regeneration. J Neurosci 1997; 17: sion in rats. J Neurosci Res 1997; 50: 798–808. 9583–9595. 13 Popovich PG. Immunological regulation ofneuronal 28 Tobias CA et al. Delayed grafting of BDNF and NT-3 degeneration and regeneration in the injured spinal cord. producing fibroblasts into the injured spinal cord stimu- Prog Brain Res 2000; 128: 43–58. lates sprouting, partially rescues axotomized red nucleus 14 Jones TB et al. Passive or active immunization with neurons from loss and atrophy, and provides limited myelin basic protein impairs neurological function and regeneration. Exp Neurol 2003; 184: 97–113. exacerbates neuropathology after spinal cord injury in 29 Lu P, Blesch A, Tuszynski MH. Neurotrophism without rats. neurotropism: BDNF promotes survival but not growth J Neurosci 2004; 24: 3752–3761. oflesioned corticospinal neurons. J Comp Neurol 2001; 15 Fitch MT, Doller C, Combs CK, Landreth GE, Silver J. 436: 456–470. Cellular and molecular mechanisms ofglial scarring and 30 Bradbury EJ, King VR, Simmons LJ, Priestley JV, progressive cavitation: in vivo and in vitro analysis of McMahon SB. NT-3, but not BDNF, prevents atrophy inflammation-induced secondary injury after CNS trau- and death ofaxotomized spinal cord projection neurons. ma. J Neurosci 1999; 19: 8182–8198. Eur J Neurosci 1998; 10: 3058–3068. 16 Popovich PG, Guan Z, McGaughy V, Fisher L, Hickey 31 Sayer FT, Oudega M, Hagg T. Neurotrophins reduce WF, Basso DM. The neuropathological and behavioral degeneration ofinjured ascending sensory and corticosp- consequences ofintraspinal microglial/macrophage acti- inal motor axons in adult rat spinal cord. Exp Neurol vation. J Neuropathol Exp Neurol 2002; 61: 623–633. 2002; 175: 282–296. 17 Popovich PG, Guan Z, Wei P, Huitinga I, van Rooijen N, 32 Cajal SR. Cajal’s Degeneration & Regeneration of the Stokes BT. Depletion ofhematogenous macrophages Nervous System. Oxford University Press: Oxford 1991. promotes partial hindlimb recovery and neuroanatomical 33 Caroni P, Schwab ME. Antibody against myelin-asso- repair after experimental spinal cord injury. Exp Neurol ciated inhibitor ofneurite growth neutralizes nonpermis- 1999; 158: 351–365. sive substrate properties ofCNS white matter. Neuron 18 Stirling DP et al. Minocycline treatment reduces delayed 1988; 1: 85–96. oligodendrocyte death, attenuates axonal dieback, and 34 Schnell L, Schwab ME. Axonal regeneration in the rat improves functional outcome after spinal cord injury. spinal cord produced by an antibody against myelin- J Neurosci 2004; 24: 2182–2190. associated neurite growth inhibitors. Nature 1990; 343: 19 Wells JE, Hurlbert RJ, Fehlings MG, Yong VW. 269–272. Neuroprotection by minocycline facilitates significant 35 Chen MS et al. Nogo-A is a myelin-associated neurite recovery from spinal cord injury in mice. Brain 2003; outgrowth inhibitor and an antigen for monoclonal 126: 1628–1637. antibody IN-1. Nature 2000; 403: 434–439. 20 McPhail LT, Stirling DP, Tetzlaff W, Kwiecien JM, 36 GrandPre T, Nakamura F, Vartanian T, Strittmatter SM. Ramer MS. The contribution ofactivated phagocytes Identification ofthe Nogo inhibitor ofaxon regeneration and myelin degeneration to axonal retraction/dieback as a Reticulon protein. Nature 2000; 403: 439–444. following spinal cord injury. Eur J Neurosci 2004; 20: 37 McKerracher L, David S, Jackson DL, Kottis V, Dunn 1984–1994. RJ, Braun PE. Identification ofmyelin-associated glyco- 21 Teng YD et al. Minocycline inhibits contusion-triggered protein as a major myelin-derived inhibitor ofneurite mitochondrial cytochrome c release and mitigates func- growth. Neuron 1994; 13: 805–811. tional deficits after spinal cord injury. Proc Natl AcadSci 38 Mukhopadhyay G, Doherty P, Walsh FS, Crocker PR, USA 2004; 101: 3071–3076. Filbin MT. A novel role for myelin-associated glycopro-

Spinal Cord Orchestrating spinal cord repair LM Ramer et al 151

tein as an inhibitor ofaxonal regeneration. Neuron 1994; 57 Mi S et al. LINGO-1 is a component ofthe Nogo-66 13: 757–767. receptor/p75 signaling complex. Nat Neurosci 2004; 7: 39 Kottis V et al. Oligodendrocyte-myelin glycoprotein 221–228. (OMgp) is an inhibitor ofneurite outgrowth. J Neurochem 58 Yamashita T, Tucker KL, Barde YA. Neurotrophin 2002; 82: 1566–1569. binding to the p75 receptor modulates Rho activity and 40 Cai D, Shen Y, De Bellard M, Tang S, Filbin MT. Prior axonal outgrowth. Neuron 1999; 24: 585–593. exposure to neurotrophins blocks inhibition ofaxonal 59 Yamashita T, Higuchi H, Tohyama M. The p75 receptor regeneration by MAG and myelin via a cAMP-dependent transduces the signal from myelin-associated glycoprotein mechanism. Neuron 1999; 22: 89–101. to Rho. J Cell Biol 2002; 157: 565–570. 41 Bartsch U et al. Lack ofevidence that myelin-associated 60 Yamashita T, Tohyama M. The p75 receptor acts as a glycoprotein is a major inhibitor ofaxonal regeneration in displacement factor that releases Rho from Rho-GDI. the CNS. Neuron 1995; 15: 1375–1381. Nat Neurosci 2003; 6: 461–467. 42 Kim JE, Li S, GrandPre T, Qiu D, Strittmatter SM. Axon 61 Dergham P, Ellezam B, Essagian C, Avedissian H, Lubell regeneration in young adult mice lacking Nogo-A/B. WD, McKerracher L. Rho signaling pathway targeted Neuron 2003; 38: 187–199. to promote spinal cord repair. J Neurosci 2002; 22: 43 Simonen M et al. Systemic deletion ofthe myelin- 6570–6577. associated outgrowth inhibitor Nogo-A improves regen- 62 Fournier AE, Takizawa BT, Strittmatter SM. Rho kinase erative and plastic responses after spinal cord injury. inhibition enhances axonal regeneration in the injured Neuron 2003; 38: 201–211. CNS. J Neurosci 2003; 23: 1416–1423. 44 Zheng B, Ho C, Li S, Keirstead H, Steward O, Tessier- 63 Monnier PP, Sierra A, Schwab JM, Henke-Fahle S, Lavigne M. Lack ofenhanced spinal regeneration in Mueller BK. The Rho/ROCK pathway mediates neurite Nogo-deficient mice. Neuron 2003; 38: 213–224. growth-inhibitory activity associated with the chondroitin 45 Huang DW, McKerracher L, Braun PE, David S. A sulfate proteoglycans of the CNS glial scar. Mol Cell therapeutic vaccine approach to stimulate axon regenera- Neurosci 2003; 22: 319–330. tion in the adult mammalian spinal cord. Neuron 1999; 24: 64 Borisoff JF et al. Suppression ofRho-kinase activity 639–647. promotes axonal growth on inhibitory CNS substrates. Mol Cell Neurosci 2003; 22: 405–416. 46 Sicotte M, Tsatas O, Jeong SY, Cai CQ, He Z, David S. et al. Immunization with myelin or recombinant Nogo-66/ 65 Shearer MC The astrocyte/meningeal cell interface is MAG in alum promotes axon regeneration and sprouting a barrier to neurite outgrowth which can be overcome by after corticospinal tract lesions in the spinal cord. Mol manipulation ofinhibitory molecules or axonal signalling pathways. Mol Cell Neurosci 2003; 24: 913–925. Cell Neurosci 2003; 23: 251–263. 66 Kwon BK, Borisoff JF, Tetzlaff W. Molecular targets for 47 Fournier AE, GrandPre T, Strittmatter SM. Identification therapeutic intervention after spinal cord injury. Mol ofa receptor mediating Nogo-66 inhibition ofaxonal Intervent 2002; 2: 244–258. regeneration. Nature 2001; 409: 341–346. 67 Keirstead HS, Dyer JK, Sholomenko GN, McGraw J, et al. 48 Wang KC Oligodendrocyte-myelin glycoprotein is a Delaney KR, Steeves JD. Axonal regeneration and Nogo receptor ligand that inhibits neurite outgrowth. physiological activity following transection and immuno- Nature 2002; 417: 941–944. logical disruption ofmyelin within the hatchling chick 49 Liu BP, Fournier A, GrandPre T, Strittmatter SM. spinal cord. J Neurosci 1995; 15: 6963–6974. Myelin-associated glycoprotein as a functional ligand 68 Dyer JK, Bourque JA, Steeves JD. Regeneration of for the Nogo-66 receptor. Science 2002; 297: 1190–1193. brainstem-spinal axons after lesion and immunological 50 Domeniconi M et al. Myelin-associated glycoprotein disruption ofmyelin in adult rat. Exp Neurol 1998; 154: interacts with the Nogo66 receptor to inhibit neurite 12–22. outgrowth. Neuron 2002; 35: 283–290. 69 Hiebert GW, Dyer JK, Tetzlaff W, Steeves JD. Immuno- 51 GrandPre T, Li S, Strittmatter SM. Nogo-66 receptor logical myelin disruption does not alter expression of antagonist peptide promotes axonal regeneration. Nature regeneration-associated genes in intact or axotomized 2002; 417: 547–551. rubrospinal neurons. Exp Neurol 2000; 163: 149–156. 52 Li S, Strittmatter SM. Delayed systemic Nogo-66 receptor 70 Rapalino O et al. Implantation ofstimulated homologous antagonist promotes recovery from spinal cord injury. macrophages results in partial recovery ofparaplegic rats. J Neurosci 2003; 23: 4219–4227. Nat Med 1998; 4: 814–821. 53 MacDermid VE, McPhail LT, Tsang B, Rosenthal A, 71 Bomstein Y et al. Features ofskin-coincubated macro- Davies A, Ramer MS. A soluble form of the Nogo phages that promote recovery from spinal cord injury. receptor differentially affects plasticity ofspinally-project- J Neuroimmunol 2003; 142: 10–16. ing axons. Eur J Neurosci 2004; 20: 2567–2579. 72 Popovich PG, Jones TB. Manipulating neuroinflamma- 54 Li W et al. A neutralizing anti-Nogo66 receptor mono- tory reactions in the injured spinal cord: back to basics. clonal antibody reverses inhibition ofneurite outgrowth Trends Pharmacol Sci 2003; 24: 13–17. by central nervous system myelin. J Biol Chem 2004; 279: 73 Liu L, Persson JK, Svensson M, Aldskogius H. Glial cell 43780–43788. responses, complement, and clusterin in the central 55 Wang KC, Kim JA, Sivasankaran R, Segal R, He Z. P75 nervous system following dorsal root transection. Glia interacts with the Nogo receptor as a co-receptor for 1998; 23: 221–238. Nogo, MAG and OMgp. Nature 2002; 420: 74–78. 74 Ramer MS, Duraisingam I, Priestley JV, McMahon SB. 56 Wong ST, Henley JR, Kanning KC, Huang KH, Bothwell Two-tiered inhibition ofaxon regeneration at the dorsal M, Poo MM. A p75(NTR) and Nogo receptor complex root entry zone. J Neurosci 2001; 21: 2651–2660. mediates repulsive signaling by myelin-associated glyco- 75 Davies SJ, Fitch MT, Memberg SP, Hall AK, Raisman G, protein. Nat Neurosci 2002; 5: 1302–1308. Silver J. Regeneration ofadult axons in white matter

Spinal Cord Orchestrating spinal cord repair LM Ramer et al 152

tracts ofthe central nervous system. Nature 1997; 390: 94 Moon LD, Asher RA, Rhodes KE, Fawcett JW. 680–683. Regeneration ofCNS axons back to their target following 76 Davies SJ, Goucher DR, Doller C, Silver J. Robust treatment ofadult rat brain with chondroitinase ABC. regeneration ofadult sensory axons in degenerating white Nat Neurosci 2001; 4: 465–466. matter ofthe adult rat spinal cord. J Neurosci 1999; 19: 95 Pizzorusso T, Medini P, Berardi N, Chierzi S, Fawcett 5810–5822. JW, Maffei L. Reactivation ofocular dominance plasticity 77 Carlstedt T. Regenerating axons form nerve terminals at in the adult visual cortex. Science 2002; 298: 1248–1251. astrocytes. Brain Res 1985; 347: 188–191. 96 Yick LW, Cheung PT, So KF, Wu W. Axonal regenera- 78 Zhang Y et al. Correlation between putative inhibitory tion ofClarke’s neurons beyond the spinal cord injury molecules at the dorsal root entry zone and failure of scar after treatment with chondroitinase ABC. Exp dorsal root axonal regeneration. Mol Cell Neurosci 2001; Neurol 2003; 182: 160–168. 17: 444–459. 97 Tropea D, Caleo M, Maffei L. Synergistic effects ofbrain- 79 Liuzzi FJ, Lasek RJ. Astrocytes block axonal regenera- derived neurotrophic factor and chondroitinase ABC on tion in mammals by activating the physiological stop retinal fiber sprouting after denervation of the superior pathway. Science 1987; 237: 642–645. colliculus in adult rats. J Neurosci 2003; 23: 7034–7044. 80 McPhail LT, Plunet W, Das P, Ramer MS. The astrocytic 98 Lemons ML, Sandy JD, Anderson DK, Howland DR. barrier to regeneration at the dorsal root entry zone is Intact aggrecan and chondroitin sulfate-depleted aggrecan induced by rhizotomy. Eur J Neurosci 2005, in press. core glycoprotein inhibit axon growth in the adult rat 81 Morgenstern DA, Asher RA, Fawcett JW. Chondroitin spinal cord. Exp Neurol 2003; 184: 981–990. sulphate proteoglycans in the CNS injury response. Prog 99 Grimpe B, Silver J. A novel DNA enzyme reduces Brain Res 2002; 137: 313–332. glycosaminoglycan chains in the glial scar and allows microtransplanted dorsal root ganglia axons to regenerate 82 Rhodes KE, Fawcett JW. Chondroitin sulphate proteo- beyond lesions in the spinal cord. J Neurosci 2004; glycans: preventing plasticity or protecting the CNS? 24: 1393–1397. J Anat 2004; 204: 33–48. 100 Iseki K et al. Increased syndecan expression by pleio- 83 Fawcett JW, Asher RA. The glial scar and central nervous trophin and FGF receptor-expressing astrocytes in system repair. Brain Res Bull 1999; 49: 377–391. injured brain tissue. Glia 2002; 39: 1–9. 84 Jones LL, Margolis RU, Tuszynski MH. The chondroitin 101 Garcia-Abreu J, Silva LC, Tovar FF, Onofr GR, sulfate proteoglycans neurocan, brevican, phosphacan, Cavalcante LA, Moura NV. Compartmental distri- and versican are differentially regulated following spinal bution ofsulfatedglycosaminoglycans in lateral and Exp Neurol cord injury. 2003; 182: 399–411. medial midbrain astroglial cultures. Glia 1996; 17: 85 Tang X, Davies JE, Davies SJ. Changes in distribution, 339–344. cell associations, and protein expression levels ofNG2, 102 Garcia-Abreu J et al. Contribution ofheparan sulfateto neurocan, phosphacan, brevican, versican V2, and tenas- the non-permissive role ofthe midline glia to the growth cin-C during acute to chronic maturation ofspinal cord ofmidbrain neurites. Glia 2000; 29: 260–272. scar tissue. J Neurosci Res 2003; 71: 427–444. 103 Cavalcante LA, Garcia-Abreu J, Moura NV, Silva LC, 86 Properzi F, Asher RA, Fawcett JW. Chondroitin sulphate Weissmuller G. Modulators ofaxonal growth and proteoglycans in the central nervous system: changes guidance at the brain midline with special reference to and synthesis after injury. Biochem Soc Trans 2003; 31: glial heparan sulfate proteoglycans. An AcadBras Cienc 335–336. 2002; 74: 691–716. 87 Schmalfeldt M, Bandtlow CE, Dours-Zimmermann MT, 104 Weissmuller G, Garcia-Abreu J, Mascarello BP, Moura Winterhalter KH, Zimmermann DR. Brain derived NV, Cavalcante LA. Glial cells with differential neurite versican V2 is a potent inhibitor ofaxonal growth. J Cell growth-modulating properties probed by atomic force Sci 2000; 113(Part 5): 807–816. microscopy. Neurosci Res 2000; 38: 217–220. 88 Sugahara K, Mikami T, Uyama T, Mizuguchi S, Nomura 105 Walz A, McFarlane S, Brickman YG, Nurcombe V, K, Kitagawa H. Recent advances in the structural biology Bartlett PF, Holt CE. Essential role ofheparan sulfatesin of chondroitin sulfate and dermatan sulfate. Curr Opin axon navigation and targeting in the developing visual Struct Biol 2003; 13: 612–620. system. Development 1997; 124: 2421–2430. 89 Dow KE, Ethell DW, Steeves JD, Riopelle RJ. Molecular 106 Carey DJ. Syndecans: multifunctional cell-surface co- correlates ofspinal cord repair in the embryonic chick: receptors. Biochem J 1997; 327(Part 1): 1–16. heparan sulfate and chondroitin sulfate proteoglycans. 107 Vijayan VK, Lee YL, Eng LF. Immunohistochemical Exp Neurol 1994; 128: 233–238. localization ofbasic fibroblast growth factorin cultured 90 McKeon RJ, Hoke A, Silver J. Injury-induced proteogly- rat astrocytes and oligodendrocytes. Int J Dev Neurosci cans inhibit the potential for laminin-mediated axon 1993; 11: 257–267. growth on astrocytic scars. Exp Neurol 1995; 136: 32–43. 108 Eclancher F, Kehrli P, Labourdette G, Sensenbrenner M. 91 Zuo J, Neubauer D, Dyess K, Ferguson TA, Muir D. Basic fibroblast growth factor (bFGF) injection activates Degradation ofchondroitin sulfateproteoglycan en- the glial reaction in the injured adult rat brain. Brain Res hances the neurite-promoting potential ofspinal cord 1996; 737: 201–214. tissue. Exp Neurol 1998; 154: 654–662. 109 Bansal R, Kumar M, Murray K, Pfeiffer SE. Develop- 92 Bradbury EJ et al. Chondroitinase ABC promotes mental and FGF-2-mediated regulation ofsyndecans functional recovery after spinal cord injury. Nature (1–4) and glypican in oligodendrocytes. Mol Cell Neurosci 2002; 416: 636–640. 1996; 7: 276–288. 93 Chau CH et al. Chondroitinase ABC enhances axonal 110 Karthikeyan L, Flad M, Engel M, Meyer-Puttlitz B, regrowth through Schwann cell-seeded guidance channels Margolis RU, Margolis RK. Immunocytochemical and in after spinal cord injury. FASEB J 2004; 18: 194–196. situ hybridization studies ofthe heparan sulfateproteo-

Spinal Cord Orchestrating spinal cord repair LM Ramer et al 153

glycan, glypican, in nervous tissue. J Cell Sci 1994; 129 Pindon A, Berry M, Hantai D. Thrombomodulin as a 107(Part 11): 3213–3222. new marker oflesion-induced astrogliosis: involvement of 111 Litwack ED, Stipp CS, Kumbasar A, Lander AD. thrombin through the G-protein-coupled protease-acti- Neuronal expression ofglypican, a cell-surfaceglycosyl- vated receptor-1. J Neurosci 2000; 20: 2543–2550. phosphatidylinositol-anchored heparan sulfate proteogly- 130 Jaworski DM. Differential regulation oftissue inhibitor of can, in the adult rat nervous system. J Neurosci 1994; 14: metalloproteinase mRNA expression in response to 3713–3724. intracranial injury. Glia 2000; 30: 199–208. 112 Funderburgh JL et al. Sequence, molecular properties, 131 Muir EM et al. Matrix metalloproteases and their and chromosomal mapping ofmouse lumican. Invest inhibitors are produced by overlapping populations of Ophthalmol Vis Sci 1995; 36: 2296–2303. activated astrocytes. Brain Res Mol Brain Res 2002; 100: 113 Funderburgh JL. Keratan sulfate: structure, biosynthesis, 103–117. and function. Glycobiology 2000; 10: 951–958. 132 Mori T, Iijima N, Kitabatake K, Kohsaka S. Alpha 2- 114 Bareyre FM, Haudenschild B, Schwab ME. Long-lasting macroglobulin is an astroglia-derived neurite-promoting sprouting and gene expression changes induced by the factor for cultured neurons from rat central nervous monoclonal antibody IN-1 in the adult spinal cord. system. Brain Res 1990; 527: 55–61. J Neurosci 2002; 22: 7097–7110. 133 Logan A, Baird A, Berry M. Decorin attenuates gliotic 115 Seo H, Geisert Jr EE. A keratan sulfate proteoglycan scar formation in the rat cerebral hemisphere. Exp Neurol marks the boundaries in the cortical barrel fields ofthe 1999; 159: 504–510. adult rat. Neurosci Lett 1995; 197: 13–16. 134 Mendis DB, Ivy GO, Brown IR. SC1, a brain extra- 116 Cole GJ, McCabe CF. Identification ofa developmentally cellular matrix glycoprotein related to SPARC and regulated keratan sulfate proteoglycan that inhibits follistatin, is expressed by rat cerebellar astrocytes cell adhesion and neurite outgrowth. Neuron 1991; 7: following injury and during development. Brain Res 1007–1018. 1996; 730: 95–106. 117 McCabe CF, Cole GJ. Expression ofthe barrier- 135 Mendis DB, Ivy GO, Brown IR. Induction ofSC1 mRNA associated proteins EAP-300 and claustrin in the devel- encoding a brain extracellular matrix glycoprotein related oping central nervous system. Brain Res Dev Brain Res to SPARC following lesioning of the adult rat forebrain. 1992; 70: 9–24. Neurochem Res 2000; 25: 1637–1644. 118 Geisert Jr EE, Bidanset DJ. A central nervous system 136 Richardson PM, Issa VM. Peripheral injury enhances keratan sulfate proteoglycan: localization to boundaries central regeneration ofprimary sensory neurones. Nature in the neonatal rat brain. Brain Res Dev Brain Res 1993; 1984; 309: 791–793. 75: 163–173. 137 Chong MS, WoolfCJ, Haque NS, Anderson PN. Axonal 119 Robson JA, Geisert Jr EE. Expression ofa keratin sulfate regeneration from injured dorsal roots into the spinal cord proteoglycan during development ofthe dorsal lateral ofadult rats. J Comp Neurol 1999; 410: 42–54. geniculate nucleus in the ferret. J Comp Neurol 1994; 340: 138 Neumann S, WoolfCJ. Regeneration ofdorsal column 349–360. fibers into and beyond the lesion site following adult 120 Jones LL, Tuszynski MH. Spinal cord injury elicits spinal cord injury. Neuron 1999; 23: 83–91. expression ofkeratan sulfateproteoglycans by macrophages, reactive microglia, and oligodendrocyte progeni- 139 Bulsara KR, Iskandar BJ, Villavicencio AT, Skene JH. tors. J Neurosci 2002; 22: 4611–4624. A new millenium for spinal cord regeneration: growth- associated genes. Spine 2002; 27: 1946–1949. 121 Miller B, Sheppard AM, Pearlman AL. Developmental expression ofkeratan sulfate-likeimmunoreactivity dis- 140 Qiu J, Cai D, Filbin MT. A role for cAMP in regeneration tinguishes thalamic nuclei and cortical domains. J Comp during development and after injury. Prog Brain Res Neurol 1997; 380: 533–552. 2002; 137: 381–387. 122 Gilad GM, Kagan HM, Gilad VH. Lysyl oxidase, the 141 Chaisuksunt V et al. Axonal regeneration from CNS extracellular matrix-forming enzyme, in rat brain injury neurons in the cerebellum and brainstem ofadult rats: sites. Neurosci Lett 2001; 310: 45–48. correlation with the patterns ofexpression and distribu- 123 Kagan HM, Trackman PC. Properties and function tion ofmessenger RNAs forL1, CHL1, c-jun and growth- oflysyl oxidase. Am J Respir Cell Mol Biol 1991; 5: associated protein-43. Neuroscience 2000; 100: 87–108. 206–210. 142 Chong MS et al. GAP-43 expression in primary sensory 124 Kagan HM, Li W. Lysyl oxidase: properties, specificity, neurons following central axotomy. J Neurosci 1994; 14: and biological roles inside and outside ofthe cell. J Cell 4375–4384. Biochem 2003; 88: 660–672. 143 Ramer MS, Priestley JV, McMahon SB. Functional 125 Ellison JA et al. Osteopontin and its integrin receptor regeneration ofsensory axons into the adult spinal cord. alpha(v)beta3 are upregulated during formation of the Nature 2000; 403: 312–316. glial scar after focal stroke. Stroke 1998; 29: 1698–1706. 144 Ramer MS et al. Neurotrophin-3-mediated regeneration 126 Wang X et al. Delayed expression ofosteopontin after and recovery ofproprioception followingdorsal rhizo- focal stroke in the rat. J Neurosci 1998; 18: 2075–2083. tomy. Mol Cell Neurosci 2002; 19: 239–249. 127 Fu Y, Hashimoto M, Ino H, Murakami M, Yamazaki M, 145 Qiu J et al. Spinal axon regeneration induced by elevation Moriya H. Spinal root avulsion-induced upregulation of ofcyclic AMP. Neuron 2002; 34: 895–903. osteopontin expression in the adult rat spinal cord. Acta 146 Cai D, Qiu J, Cao Z, McAtee M, Bregman BS, Filbin Neuropathol (Berl) 2004; 107: 8–16. MT. Neuronal cyclic AMP controls the developmental 128 Ellison JA, Barone FC, Feuerstein GZ. Matrix remodel- loss in ability ofaxons to regenerate. J Neurosci 2001; 21: ing after stroke. De novo expression ofmatrix proteins 4731–4739. and integrin receptors. Ann NY AcadSci 1999; 890: 147 Cai D, Deng K, Mellado W, Lee J, Ratan RR, Filbin MT. 204–222. Arginase I and polyamines act downstream from cyclic

Spinal Cord Orchestrating spinal cord repair LM Ramer et al 154

AMP in overcoming inhibition ofaxonal growth MAG growth factor in the adult spinal cord. J Neurosci 2000; and myelin in vitro. Neuron 2002; 35: 711–719. 20: 4435–4445. 148 Hebenstreit GF et al. Rolipram in major depressive 164 Romero MI, Rangappa N, Garry MG, Smith GM. disorder: results ofa double-blind comparative study with Functional regeneration ofchronically injured sensory imipramine. Pharmacopsychiatry 1989; 22: 156–160. afferents into adult spinal cord after neurotrophin gene 149 Nikulina E, Tidwell JL, Dai HN, Bregman BS, Filbin therapy. J Neurosci 2001; 21: 8408–8416. MT. The phosphodiesterase inhibitor rolipram delivered 165 Ramer MS, Bradbury EJ, McMahon SB. Nerve growth after a spinal cord lesion promotes axonal regeneration factor induces P2X(3) expression in sensory neurons. and functional recovery. Proc Natl AcadSci USA 2004; J Neurochem 2001; 77: 864–875. 101: 8786–8790. 166 Tang XQ, Tanelian DL, Smith GM. Semaphorin3A 150 Pearse DD et al. cAMP and Schwann cells promote inhibits nerve growth factor-induced sprouting of noci- axonal growth and functional recovery after spinal cord ceptive afferents in adult rat spinal cord. J Neurosci 2004; injury. Nat Med 2004; 10: 610–616. 24: 819–827. 151 Houle JD, Johnson JE. Nerve growth factor (NGF)- 167 Krenz NR, Meakin SO, Krassioukov AV, Weaver LC. treated nitrocellulose enhances and directs the regenera- Neutralizing intraspinal nerve growth factor blocks tion ofadult rat dorsal root axons through intraspinal autonomic dysreflexia caused by spinal cord injury. neural tissue transplants. Neurosci Lett 1989; 103: 17–23. J Neurosci 1999; 19: 7405–7414. 152 Chuah MI, West AK. Cellular and molecular biology 168 Marsh DR, Wong ST, Meakin SO, MacDonald JI, ofensheathing cells. Microsc Res Tech 2002; 58: Hamilton EF, Weaver LC. Neutralizing intraspinal nerve 216–227. growth factor with a trkA-IgG fusion protein blocks the 153 Oudega M, Hagg T. Neurotrophins promote regeneration development ofautonomic dysreflexia in a clip-compres- ofsensory axons in the adult rat spinal cord. Brain Res sion model ofspinal cord injury. J Neurotrauma 2002; 19: 1999; 818: 431–438. 1531–1541. 154 Xu XM, Guenard V, Kleitman N, Aebischer P, Bunge 169 Hempstead BL. The many faces of p75NTR. Curr Opin MB. A combination ofBDNF and NT-3 promotes Neurobiol 2002; 12: 260–267. supraspinal axonal regeneration into Schwann cell grafts 170 MacPhee IJ, Barker PA. Brain-derived neurotrophic in adult rat thoracic spinal cord. Exp Neurol 1995; 134: factor binding to the p75 neurotrophin receptor reduces 261–272. TrkA signaling while increasing serine phosphorylation in 155 Bregman BS, McAtee M, Dai HN, Kuhn PL. Neuro- the TrkA intracellular domain. J Biol Chem 1997; 272: trophic factors increase axonal growth after spinal cord 23547–23551. injury and transplantation in the adult rat. Exp Neurol 171 Kimpinski K, Jelinski S, Mearow K. The anti-p75 1997; 148: 475–494. antibody, MC192, and brain-derived neurotrophic factor 156 Lu P, Yang H, Jones LL, Filbin MT, Tuszynski MH. inhibit nerve growth factor-dependent neurite growth Combinatorial therapy with neurotrophins and cAMP from adult sensory neurons. Neuroscience 1999; 93: promotes axonal regeneration beyond sites ofspinal cord 253–263. injury. J Neurosci 2004; 24: 6402–6409. 172 Kohn J, Aloyz RS, Toma JG, Haak-Frendscho M, Miller 157 Bradbury EJ, Khemani S, Von R, King, Priestley JV, FD. Functionally antagonistic interactions between the McMahon SB. NT-3 promotes growth oflesioned adult TrkA and p75 neurotrophin receptors regulate sympa- rat sensory axons ascending in the dorsal columns ofthe thetic neuron growth and target innervation. J Neurosci spinal cord. Eur J Neurosci 1999; 11: 3873–3883. 1999; 19: 5393–5408. 158 Hendriks WT, Ruitenberg MJ, Blits B, Boer GJ, 173 Yeo TT et al. Absence ofp75NTR causes increased basal Verhaagen J. Viral vector-mediated gene transfer of forebrain cholinergic neuron size, choline acetyltransfer- neurotrophins to promote regeneration ofthe injured ase activity, and target innervation. J Neurosci 1997; 17: spinal cord. Prog Brain Res 2004; 146: 451–476. 7594–7605. 159 Ramer MS, McMahon SB, Priestley JV. Axon regenera- 174 Ferri CC, Moore FA, Bisby MA. Effects offacialnerve tion across the dorsal root entry zone. Prog Brain Res injury on mouse motoneurons lacking the p75 low-affinity 2001; 132: 621–639. neurotrophin receptor. J Neurobiol 1998; 34: 1–9. 160 Zhang Y, Dijkhuizen PA, Anderson PN, Lieberman AR, 175 Boyd JG, Gordon T. The neurotrophin receptors, trkB Verhaagen J. NT-3 delivered by an adenoviral vector and p75, differentially regulate motor axonal regenera- induces injured dorsal root axons to regenerate into tion. J Neurobiol 2001; 49: 314–325. the spinal cord ofadult rats. J Neurosci Res 1998; 54: 176 Zhou XF et al. Satellite-cell-derived nerve growth factor 554–562. and neurotrophin-3 are involved in noradrenergic sprout- 161 Bennett DL et al. A distinct subgroup ofsmall DRG cells ing in the dorsal root ganglia following peripheral nerve express GDNF receptor components and GDNF is injury in the rat. Eur J Neurosci 1999; 11: 1711–1722. protective for these neurons after nerve injury. J Neurosci 177 Ramer MS, Thompson SW, McMahon SB. Causes and 1998; 18: 3059–3072. consequences ofsympathetic basket formationin dorsal 162 Iwakawa M, Mizoi K, Tessler A, Itoh Y. Intraspinal root ganglia. Pain 1999; (Suppl 6): S111–S120. implants offibrin glue containing glial cell line-derived 178 Walsh GS, Krol KM, Kawaja MD. Absence ofthe p75 neurotrophic factor promote dorsal root regeneration neurotrophin receptor alters the pattern ofsympathosen- into spinal cord. Neurorehabil Neural Repair 2001; 15: sory sprouting in the trigeminal ganglia ofmice 173–182. overexpressing nerve growth factor. J Neurosci 1999; 19: 163 Romero MI, Rangappa N, Li L, Lightfoot E, Garry MG, 258–273. Smith GM. Extensive sprouting ofsensory afferents and 179 Scott ALM, Borisoff JF, Ramer MS. Deafferentation and hyperalgesia induced by conditional expression ofnerve neurotrophin-mediated intraspinal sprouting: a central

Spinal Cord Orchestrating spinal cord repair LM Ramer et al 155

role for the p75 neurotrophin receptor. Eur J Neurosci 199 Richardson PM, McGuinness UM, Aguayo AJ. Axons 2004, in press. from CNS neurons regenerate into PNS grafts. Nature 180 Oudega M, Rosano C, Sadi D, Wood PM, Schwab ME, 1980; 284: 264–265. Hagg T. Neutralizing antibodies against neurite growth 200 Cheng H, Cao Y, Olson L. Spinal cord repair in adult inhibitor NI-35/250 do not promote regeneration of paraplegic rats: partial restoration ofhind limb function. sensory axons in the adult rat spinal cord. Neuroscience Science 1996; 273: 510–513. 2000; 100: 873–883. 201 Lee YS, Hsiao I, Lin VW. Peripheral nerve grafts and 181 Goldberger ME. Locomotor recovery after unilateral aFGF restore partial hindlimb function in adult para- hindlimb deafferentation in cats. Brain Res 1977; 123: plegic rats. J Neurotrauma 2002; 19: 1203–1216. 59–74. 202 Fraidakis MJ, Spenger C, Olson L. Partial recovery after 182 Ballermann M, McKenna J, Whishaw IQ. A grasp-related treatment ofchronic paraplegia in rat. Exp Neurol 2004; deficit in tactile discrimination following dorsal column 188: 33–42. lesion in the rat. Brain Res Bull 2001; 54: 237–242. 203 Cheng H, Liao KK, Liao SF, Chuang TY, Shih YH. 183 Liu CN, Chambers WW. Intraspinal sprouting of Spinal cord repair with acidic fibroblast growth factor as a dorsal root axons. Archs Neurol Psychiatr 1998; 79: treatment for a patient with chronic paraplegia. Spine 46–61. 2004; 29: E284–E288. 184 Jeffery ND, Fitzgerald M. Effects ofred nucleus ablation 204 Steeves J, Fawcett J, Tuszynski M. Report ofinterna- and exogenous neurotrophin-3 on corticospinal axon tional clinical trials workshop on spinal cord injury terminal distribution in the adult rat. Neuroscience 2001; february 20–21, 2004, vancouver, Canada. Spinal Cord 104: 513–521. 2004; 42: 591–597. 185 Illis LS. Experimental model ofregeneration in the central 205 Tadie M et al. Partial return ofmotor functionin nervous system. II. The reaction ofglia in the synaptic paralyzed legs after surgical bypass of the lesion site by zone. Brain 1973; 96: 61–68. nerve autografts three years after spinal cord injury. 186 Illis L. Spinal cord synapses in the cat: the reaction ofthe J Neurotrauma 2002; 19: 909–916. boutons terminaux at the motoneurone surface to 206 Hall SM. The effect ofinhibiting Schwann cell mitosis on experimental denervation. Brain 1964; 87: 555–572. the re-innervation ofacellular autograftsin the peripheral 187 Illis L. Changes in spinal cord synapses and a possible nervous system ofthe mouse. Neuropathol Appl Neurobiol explanation for spinal shock. Exp Neurol 1963; 8: 1986; 12: 401–414. 328–335. 207 Hall SM. Regeneration in cellular and acellular autografts 188 Illis LS. Experimental model ofregeneration in the central in the peripheral nervous system. Neuropathol Appl nervous system. I. Synaptic changes. Brain 1973; 96: Neurobiol 1986; 12: 27–46. 47–60. 208 Fawcett JW, Keynes RJ. Peripheral nerve regeneration. 189 Illis L. Spinal cord synapses in the cat: the normal Annu Rev Neurosci 1990; 13: 43–60. appearances by light microscope. Brain 1964; 87: 543–554. 190 Muir GD, Steeves JD. Sensorimotor stimulation to 209 Mirsky R et al. Schwann cells as regulators ofnerve improve locomotor recovery after spinal cord injury. development. J Physiol Paris 2002; 96: 17–24. Trends Neurosci 1997; 20: 72–77. 210 Casella GT, Bunge RP, Wood PM. Improved method for 191 Dunlop SA, Steeves JD. Neural activity and facilitated harvesting human Schwann cells from mature peripheral recovery by training after CNS injury: implications for nerve and expansion in vitro. Glia 1996; 17: 327–338. rehabilitation. Top Spinal CordInj Rehabil 2003; 8: 211 Guest JD, Hesse D, Schnell L, Schwab ME, Bunge MB, 92–103. Bunge RP. Influence ofIN-1 antibody and acidic FGF- 192 Llewellyn-Smith IJ, Weaver LC. Changes in synaptic fibrin glue on the response ofinjured corticospinal tract inputs to sympathetic preganglionic neurons after spinal axons to human Schwann cell grafts. J Neurosci Res 1997; cord injury. J Comp Neurol 2001; 435: 226–240. 50: 888–905. 193 Belyantseva IA, Lewin GR. Stability and plasticity of 212 Guest JD, Rao A, Olson L, Bunge MB, Bunge RP. The primary afferent projections following nerve regeneration ability ofhuman Schwann cell graftsto promote and central degeneration. Eur J Neurosci 1999; 11: regeneration in the transected nude rat spinal cord. Exp 457–468. Neurol 1997; 148: 502–522. 194 Wang SD, Goldberger ME, Murray M. Plasticity of 213 Bunge MB. Bridging the transected or contused adult rat spinal systems after unilateral lumbosacral dorsal rhizo- spinal cord with Schwann cell and olfactory ensheathing tomy in the adult rat. J Comp Neurol 1991; 304: 555–568. glia transplants. Prog Brain Res 2002; 137: 275–282. 195 Carlstedt T. Dorsal root innervation ofspinal cord 214 Xu XM, Guenard V, Kleitman N, Bunge MB. Axonal neurons after dorsal root implantation into the spinal regeneration into Schwann cell-seeded guidance channels cord ofadult rats. Neurosci Lett 1985; 55: 343–348. grafted into transected adult rat spinal cord. J Comp 196 Smith KJ, Kodama RT, Liuzzi FJ. Is CNS trauma a Neurol 1995; 351: 145–160. prerequisite for the elongation of CNS axons into 215 Xu XM, Chen A, Guenard V, Kleitman N, Bunge MB. denervated peripheral nerve? Brain Res 1992; 575: 79–85. Bridging Schwann cell transplants promote axonal 197 Schnell L, Schneider R, Kolbeck R, Barde YA, Schwab regeneration from both the rostral and caudal stumps of ME. Neurotrophin-3 enhances sprouting ofcorticospinal transected adult rat spinal cord. J Neurocytol 1997; 26: tract during development and after adult spinal cord 1–16. lesion. Nature 1994; 367: 170–173. 216 Xu XM, Zhang SX, Li H, Aebischer P, Bunge MB. 198 Thallmair M, Metz GA, Z’Graggen WJ, Raineteau O, Regrowth ofaxons into the distal spinal cord through a Kartje GL, Schwab ME. Neurite growth inhibitors Schwann-cell-seeded mini-channel implanted into hemi- restrict plasticity and functional recovery following sected adult rat spinal cord. Eur J Neurosci 1999; 11: corticospinal tract lesions. Nat Neurosci 1998; 1: 124–131. 1723–1740.

Spinal Cord Orchestrating spinal cord repair LM Ramer et al 156

217 Bunge MB. Transplantation ofpurified populations of 233 Ramon-Cueto A, Plant GW, Avila J, Bunge MB. Long- Schwann cells into lesioned adult rat spinal cord. J Neurol distance axonal regeneration in the transected adult rat 1994; 242: S36–S39. spinal cord is promoted by olfactory ensheathing glia 218 Chen A, Xu XM, Kleitman N, Bunge MB. Methylpred- transplants. J Neurosci 1998; 18: 3803–3815. nisolone administration improves axonal regeneration 234 Li Y, Field PM, Raisman G. Repair ofadult rat into Schwann cell grafts in transected adult rat thoracic corticospinal tract by transplants ofolfactoryensheathing spinal cord. Exp Neurol 1996; 138: 261–276. cells. Science 1997; 277: 2000–2002. 219 Pinzon A, Calancie B, Oudega M, Noga BR. Conduction 235 Li Y, Field PM, Raisman G. Regeneration ofadult rat ofimpulses by axons regenerated in a Schwann cell graft corticospinal axons induced by transplanted olfactory in the transected adult rat thoracic spinal cord. J Neurosci ensheathing cells. J Neurosci 1998; 18: 10514–10524. Res 2001; 64: 533–541. 236 Nash HH, Borke RC, Anders JJ. Ensheathing cells and 220 Plant GW, Bates ML, Bunge MB. Inhibitory proteogly- methylprednisolone promote axonal regeneration and can immunoreactivity is higher at the caudal than the functional recovery in the lesioned adult rat spinal cord. rostral Schwann cell graft-transected spinal cord interface. J Neurosci 2002; 22: 7111–7120. Mol Cell Neurosci 2001; 17: 471–487. 237 Ramon-Cueto A, Cordero MI, Santos-Benito FF, Avila 221 Graziadei PP, Monti Graziadei GA. Neurogenesis and J. Functional recovery ofparaplegic rats and motor axon neuron regeneration in the olfactory system of mammals. regeneration in their spinal cords by olfactory ensheathing III. Deafferentation and reinnervation ofthe olfactory glia. Neuron 2000; 25: 425–435. bulb following section of the fila olfactoria in rat. 238 Lu J, Feron F, Ho SM, Mackay-Sim A, Waite PM. J Neurocytol 1980; 9: 145–162. Transplantation ofnasal olfactorytissue promotes partial 222 Doucette JR, Kiernan JA, Flumerfelt BA. The re- recovery in paraplegic adult rats. Brain Res 2001; 889: innervation of olfactory glomeruli following transection 344–357. ofprimary olfactoryaxons in the central or peripheral 239 Huang H et al. Influence ofpatients’ age on functional nervous system. J Anat 1983; 137(Part 1): 1–19. recovery after transplantation of olfactory ensheathing 223 Deckner ML, Risling M, Frisen J. Apoptotic death of cells into injured spinal cord injury. Chin MedJ (Engl) olfactory sensory neurons in the adult rat. Exp Neurol 2003; 116: 1488–1491. 1997; 143: 132–140. 240 Bianco JI, Perry C, Harkin DG, Mackay-Sim A, Feron F. 224 Harding J, Graziadei PP, Monti Graziadei GA, Margolis Neurotrophin 3 promotes purification and proliferation of olfactory ensheathing cells from human nose. Glia FL. Denervation in the primary olfactory pathway of 2004; 45: 111–123. mice. IV. Biochemical and morphological evidence for neuronal replacement following nerve section. Brain Res 241 Verdu E, Garcia-Alias G, Fores J, Lopez-Vales R, 1977; 132: 11–28. Navarro X. Olfactory ensheathing cells transplanted in lesioned spinal cord prevent loss ofspinal cord parench- 225 Kern RC, Quinn B, Rosseau G, Farbman AI. Post- yma and promote functional recovery. Glia 2003; 42: traumatic olfactory dysfunction. Laryngoscope 2000; 110: 275–286. 2106–2109. 242 Verdu E et al. Effects ofensheathing cells transplanted 226 Ramon-Cueto A, Nieto-Sampedro M. Regeneration into into photochemically damaged spinal cord. Neuroreport the spinal cord oftransected dorsal root axons is 2001; 12: 2303–2309. promoted by ensheathing glia transplants. Exp Neurol 243 Takami T, Oudega M, Bates ML, Wood PM, Kleitman 1994; 127: 232–244. N, Bunge MB. Schwann cell but not olfactory ensheath- 227 Navarro X et al. Ensheathing glia transplants promote ing glia transplants improve hindlimb locomotor perfor- dorsal root regeneration and spinal reflex restitution after mance in the moderately contused adult rat thoracic multiple lumbar rhizotomy. Ann Neurol 1999; 45: spinal cord. J Neurosci 2002; 22: 6670–6681. 207–215. 244 Plant GW, Christensen CL, Oudega M, Bunge MB. 228 Taylor JS, Muneton-Gomez VC, Eguia-Recuero R, Delayed transplantation ofolfactoryensheathing glia Nieto-Sampedro M. Transplants ofolfactorybulb en- promotes sparing/regeneration ofsupraspinal axons in the sheathing cells promote functional repair of multiple contused adult rat spinal cord. J Neurotrauma 2003; 20: dorsal rhizotomy. Prog Brain Res 2001; 132: 641–654. 1–16. 229 Pascual JI, Gudino-Cabrera G, Insausti R, Nieto- 245 Ruitenberg MJ et al. Ex vivo adenoviral vector-mediated Sampedro M. Spinal implants ofolfactoryensheathing neurotrophin gene transfer to olfactory ensheathing cells promote axon regeneration and bladder activity after glia: effects on rubrospinal tract regeneration, lesion bilateral lumbosacral dorsal rhizotomy in the adult rat. size, and functional recovery after implantation in J Urol 2002; 167: 1522–1526. the injured rat spinal cord. J Neurosci 2003; 23: 230 Gomez VM et al. Transplantation ofolfactoryensheath- 7045–7058. ing cells fails to promote significant axonal regeneration 246 Ramer LM, Au E, Richter MW, Liu J, Tetzlaff W, from dorsal roots into the rat cervical cord. J Neurocytol Roskams AJ. Peripheral olfactory ensheathing cells 2003; 32: 53–70. reduce scar and cavity formation and promote regenera- 231 Riddell JS, Enriquez-Denton M, Toft A, Fairless R, tion after spinal cord injury. J Comp Neurol 2004; 473: Barnett SC. Olfactory ensheathing cell grafts have 1–15. minimal influence on regeneration at the dorsal root 247 Chuah MI et al. Olfactory ensheathing cells promote entry zone following rhizotomy. Glia 2004; 47: 150–167. collateral axonal branching in the injured adult rat spinal 232 Ramer LM, Richter MW, Roskams AJ, Tetzlaff W, cord. Exp Neurol 2004; 185: 15–25. Ramer MS. Peripherally-derived olfactory ensheathing 248 Barnett SC, Chang L. Olfactory ensheathing cells and cells do not promote primary afferent regeneration CNS repair: going solo or in need ofa friend? Trends following dorsal root injury. Glia 2004; 47: 189–206. Neurosci 2004; 27: 54–60.

Spinal Cord Orchestrating spinal cord repair LM Ramer et al 157

249 Li Y, Decherchi P, Raisman G. Transplantation of transection in the chick embryo. J Neurobiol 1990; 21: olfactory ensheathing cells into spinal cord lesions 918–937. restores breathing and climbing. J Neurosci 2003; 23: 266 Hasan SJ, Keirstead HS, Muir GD, Steeves JD. Axonal 727–731. regeneration contributes to repair ofinjured brainstem– 250 Lu J, Feron F, Mackay-Sim A, Waite PM. Olfactory spinal neurons in embryonic chick. J Neurosci 1993; 13: ensheathing cells promote locomotor recovery after 492–507. delayed transplantation into transected spinal cord. Brain 267 Varga ZM, Schwab ME, Nicholls JG. Myelin-associated 2002; 125: 14–21. neurite growth-inhibitory proteins and suppression of 251 Keyvan-Fouladi N, Raisman G, Li Y. Functional repair regeneration ofimmature mammalian spinal cord in ofthe corticospinal tract by delayed transplantation of culture. Proc Natl AcadSci USA 1995; 92: 10959–10963. olfactory ensheathing cells in adult rats. J Neurosci 2003; 268 Steeves JD. Cues from developmental models of spinal 23: 9428–9434. cord regeneration for the repair of the injured adult CNS. 252 Wewetzer K, Verdu E, Angelov DN, Navarro X. In: Ingoglia NA, Murray M (eds). Axonal Regeneration in Olfactory ensheathing glia and Schwann cells: two of a the Central Nervous System. Marcel Dekker: Basel 2000 kind? Cell Tissue Res 2002; 309: 337–345. pp 129–159. 253 Williams SK, Franklin RJ, Barnett SC. Response of 269 Bregman BS, Kunkel-Bagden E, Reier PJ, Dai HN, olfactory ensheathing cells to the degeneration and McAtee M, Gao D. Recovery of function after spinal regeneration ofthe peripheral olfactorysystem and the cord injury: mechanisms underlying transplant-mediated involvement ofthe neuregulins. J Comp Neurol 2004; 470: recovery of function differ after spinal cord injury in 50–62. newborn and adult rats. Exp Neurol 1993; 123: 3–16. 254 Menei P, Montero-Menei C, Whittemore SR, Bunge RP, 270 Houle JD, Reier PJ. Transplantation offetalspinal cord Bunge MB. Schwann cells genetically modified to secrete tissue into the chronically injured adult rat spinal cord. human BDNF promote enhanced axonal regrowth across J Comp Neurol 1988; 269: 535–547. transected adult rat spinal cord. Eur J Neurosci 1998; 10: 271 Zurita M, Vaquero J, Oya S, Montilla J. Functional 607–621. recovery in chronic paraplegic rats after co-grafts of fetal 255 Cao L et al. Olfactory ensheathing cells genetically brain and adult peripheral nerve tissue. Surg Neurol 2001; modified to secrete GDNF to promote spinal cord repair. 55: 249–254. Brain 2004; 127: 535–549. 272 Anderson DK, Howland DR, Reier PJ. Fetal neural 256 Lakatos A, Franklin RJ, Barnett SC. Olfactory ensheath- grafts and repair of the injured spinal cord. Brain Pathol ing cells and Schwann cells differ in their in vitro 1995; 5: 451–457. interactions with astrocytes. Glia 2000; 32: 214–225. 273 Lakatos A, Franklin RJ. Transplant mediated repair of 257 Lakatos A, Barnett SC, Franklin RJ. Olfactory ensheath- the central nervous system: an imminent solution? Curr ing cells induce less host astrocyte response and chon- Opin Neurol 2002; 15: 701–705. droitin sulphate proteoglycan expression than Schwann cells following transplantation into adult CNS white 274 Jakeman LB, Reier PJ. Axonal projections between fetal matter. Exp Neurol 2003; 184: 237–246. spinal cord transplants and the adult rat spinal cord: 258 Woodhall E, West AK, Vickers JC, Chuah MI. Olfactory a neuroanatomical tracing study oflocal interactions. ensheathing cell phenotype following implantation in J Comp Neurol 1991; 307: 311–334. the lesioned spinal cord. Cell Mol Life Sci 2003; 60: 275 Duchossoy Y, Kassar-Duchossoy L, Orsal D, Stettler O, 2241–2253. Horvat JC. Reinnervation ofthe biceps brachii muscle 259 Cui Q, Pollett MA, Symons NA, Plant GW, Harvey AR. following cotransplantation of fetal spinal cord and A new approach to CNS repair using chimeric peripheral autologous peripheral nerve into the injured cervical nerve grafts. J Neurotrauma 2003; 20: 17–31. spinal cord ofthe adult rat. Exp Neurol 2001; 167: 260 Pearse DD, Marcillo AE, Oudega M, Lynch MP, Wood 329–340. PM, Bunge MB. Transplantation ofSchwann cells and 276 Coumans JV et al. Axonal regeneration and functional olfactory ensheathing glia after spinal cord injury: recovery after complete spinal cord transection in rats by does pretreatment with methylprednisolone and interleu- delayed treatment with transplants and neurotrophins. kin-10 enhance recovery? J Neurotrauma 2004; 21: J Neurosci 2001; 21: 9334–9344. 1223–1239. 277 Wirth III ED et al. Feasibility and safety of neural tissue 261 Boyd JG, Lee J, Skihar V, Doucette R, Kawaja MD. transplantation in patients with syringomyelia. J Neuro- LacZ-expressing olfactory ensheathing cells do not trauma 2001; 18: 911–929. associate with myelinated axons after implantation into 278 Thompson FJ et al. Neurophysiological assessment ofthe the compressed spinal cord. Proc Natl AcadSci USA feasibility and safety of neural tissue transplantation in 2004; 101: 2162–2166. patients with syringomyelia. J Neurotrauma 2001; 18: 262 Sims TJ, Gilmore SA. Regrowth ofdorsal root axons into 931–945. a radiation-induced glial-deficient environment in the 279 Chow SY et al. Characterization and intraspinal grafting spinal cord. Brain Res 1994; 634: 113–126. ofEGF/bFGF-dependent neurospheres derived from 263 Sims TJ, Gilmore SA. Regeneration ofdorsal root axons embryonic rat spinal cord. Brain Res 2000; 874: 87–106. into experimentally altered glial environments in the rat 280 Lu P, Jones LL, Snyder EY, Tuszynski MH. Neural stem spinal cord. Exp Brain Res 1994; 99: 25–33. cells constitutively secrete neurotrophic factors and 264 Ramer MS. Spontaneous functional viscerosensory re- promote extensive host axonal growth after spinal cord generation into the adult brainstem. J Neurosci 2003; 23: injury. Exp Neurol 2003; 181: 115–129. 9770–9775. 281 Roy NS et al. Telomerase immortalization ofneuronally 265 Shimizu I, Oppenheim RW, O’Brien M, Shneiderman A. restricted progenitor cells derived from the human fetal Anatomical and functional recovery following spinal cord spinal cord. Nat Biotechnol 2004; 22: 297–305.

Spinal Cord Orchestrating spinal cord repair LM Ramer et al 158

282 Ogawa Y et al. Transplantation of in vitro-expanded fetal 301 Felts PA, Smith KJ, Tilt E. Blood–brain barrier function neural progenitor cells results in neurogenesis and in central demyelinating lesions repaired by Schwann cell functional recovery after spinal cord contusion injury in remyelination. Ann NY AcadSci 1991; 633: 615–616. adult rats. J Neurosci Res 2002; 69: 925–933. 302 Franklin RJ. Remyelination by transplanted olfactory 283 Cao Q, Benton RL, Whittemore SR. Stem cell repair of ensheathing cells. Anat Rec 2003; 271B: 71–76. central nervous system injury. J Neurosci Res 2002; 68: 303 Franklin RJ. Why does remyelination fail in multiple 501–510. sclerosis? Nat Rev Neurosci 2002; 3: 705–714. 284 Cao QL, Zhang YP, Howard RM, Walters WM, Tsoulfas 304 Kocsis JD, Akiyama Y, Lankford KL, Radtke C. Cell P, Whittemore SR. Pluripotent stem cells engrafted into transplantation ofperipheral-myelin-formingcells to the normal or lesioned adult rat spinal cord are restricted repair the injured spinal cord. J Rehabil Res Dev 2002; to a glial lineage. Exp Neurol 2001; 167: 48–58. 39: 287–298. 285 Teng YD et al. Functional recovery following traumatic 305 Devon R, Doucette R. Olfactory ensheathing cells spinal cord injury mediated by a unique polymer scaffold myelinate dorsal root ganglion neurites. Brain Res 1992; seeded with neural stem cells. Proc Natl AcadSci USA 589: 175–179. 2002; 99: 3024–3029. 306 Ramon-Cueto A, Perez J, Nieto-Sampedro M. In vitro 286 Rabinovich SS et al. Transplantation treatment ofspinal enfolding of olfactory neurites by p75 NGF receptor cord injury patients. BiomedPharmacother 2003; 57: positive ensheathing cells from adult rat olfactory bulb. 428–433. Eur J Neurosci 1993; 5: 1172–1180. 287 Calza L et al. Stem cells and nervous tissue repair: from 307 Plant GW et al. Purified adult ensheathing glia fail to in vitro to in vivo. Prog Brain Res 2004; 146: 75–91. myelinate axons under culture conditions that enable 288 Rothstein JD, Snyder EY. Reality and immortality – Schwann cells to form myelin. J Neurosci 2002; 22: neural stem cells for therapies. Nat Biotechnol 2004; 22: 6083–6091. 283–285. 308 Blakemore WF, Patterson RC. Suppression ofremyelina- 289 Jimenez Hamann MC, Tsai EC, Tator CH, Shoichet MS. tion in the CNS by X-irradiation. Acta Neuropathol Novel intrathecal delivery system for treatment of spinal (Berl) 1978; 42: 105–113. cord injury. Exp Neurol 2003; 182: 300–309. 309 Franklin RJ, Gilson JM, Franceschini IA, Barnett SC. 290 Cao X, Shoichet MS. Investigating the synergistic effect of Schwann cell-like myelination following transplantation combined neurotrophic factor concentration gradients to ofan olfactorybulb-ensheathing cell line into areas of guide axonal growth. Neuroscience 2003; 122: 381–389. demyelination in the adult CNS. Glia 1996; 17: 217–224. 291 Kapur TA, Shoichet MS. Immobilized concentration 310 Imaizumi T, Lankford KL, Waxman SG, Greer CA, gradients ofnerve growth factorguide neurite outgrowth. Kocsis JD. Transplanted olfactory ensheathing cells J BiomedMater Res 2004; 68A: 235–243. remyelinate and enhance axonal conduction in the 292 Patist CM, Mulder MB, Gautier SE, Maquet V, Jerome demyelinated dorsal columns ofthe rat spinal cord. R, Oudega M. Freeze-dried poly(D,L-lactic acid) macro- J Neurosci 1998; 18: 6176–6185. porous guidance scaffolds impregnated with brain-derived 311 Kato T, Honmou O, Uede T, Hashi K, Kocsis JD. neurotrophic factor in the transected adult rat thoracic Transplantation ofhuman olfactoryensheathing cells spinal cord. Biomaterials 2004; 25: 1569–1582. elicits remyelination ofdemyelinated rat spinal cord. Glia 293 Tsai EC, Dalton PD, Shoichet MS, Tator CH. Synthetic 2000; 30: 209–218. hydrogel guidance channels facilitate regeneration of 312 Smith PM, Sim FJ, Barnett SC, Franklin RJ. SCIP/Oct-6, adult rat brainstem motor axons after complete spinal Krox-20, and desert hedgehog mRNA expression during cord transection. J Neurotrauma 2004; 21: 789–804. CNS remyelination by transplanted olfactory ensheathing 294 Geller HM, Fawcett JW. Building a bridge: engineering cells. Glia 2001; 36: 342–353. spinal cord repair. Exp Neurol 2002; 174: 125–136. 313 Smith PM, Lakatos A, Barnett SC, Jeffery ND, Franklin 295 Friedman JA, Windebank AJ, Moore MJ, Spinner RJ, RJ. Cryopreserved cells isolated from the adult canine Currier BL, Yaszemski MJ. Biodegradable polymer grafts olfactory bulb are capable of extensive remyelination for surgical repair of the injured spinal cord. Neurosurgery following transplantation into the adult rat CNS. Exp 2002; 51: 742–751. Neurol 2002; 176: 402–406. 296 Novikova LN, Novikov LN, Kellerth JO. Biopolymers 314 Lakatos A, Smith PM, Barnett SC, Franklin RJ. and biodegradable smart implants for tissue regeneration Meningeal cells enhance limited CNS remyelination by after spinal cord injury. Curr Opin Neurol 2003; 16: transplanted olfactory ensheathing cells. Brain 2003; 126: 711–715. 598–609. 297 Smith EJ, Blakemore WF, McDonald WI. Central 315 Radtke C et al. Remyelination ofthe nonhuman primate remyelination restores secure conduction. Nature 1979; spinal cord by transplantation ofH-transferasetransgenic 280: 395–396. adult pig olfactory ensheathing cells. FASEB J 2004; 18: 298 Jeffery ND, Blakemore WF. Locomotor deficits induced 335–337. by experimental spinal cord demyelination are abolished 316 Blakemore WF, Crang AJ. The use ofcultured auto- by spontaneous remyelination. Brain 1997; 120(Part 1): logous Schwann cells to remyelinate areas ofpersistent 27–37. demyelination in the central nervous system. J Neurol Sci 299 Bunge RP. Expanding roles for the Schwann cell: 1985; 70: 207–223. ensheathment, myelination, trophism and regeneration. 317 Franklin RJ, Crang AJ, Blakemore WF. Type 1 astro- Curr Opin Neurobiol 1993; 3: 805–809. cytes fail to inhibit Schwann cell remyelination of CNS 300 Blakemore WF, Patterson RC. Observations on the axons in the absence ofcells ofthe O-2A lineage. Dev interactions ofSchwann cells and astrocytes following Neurosci 1992; 14: 85–92. X-irradiation ofneonatal rat spinal cord. J Neurocytol 318 Blesch A, Tuszynski MH. Cellular GDNF delivery 1975; 4: 573–585. promotes growth ofmotor and dorsal column sensory

Spinal Cord Orchestrating spinal cord repair LM Ramer et al 159

axons after partial and complete spinal cord transections previously non-responsive region. Brain Res 1976; 116: and induces remyelination. J Comp Neurol 2003; 467: 181–204. 403–417. 338 Darian-Smith C, Gilbert CD. Axonal sprouting accom- 319 Jasmin L, Janni G, Moallem TM, Lappi DA, Ohara PT. panies functional reorganization in adult cat striate Schwann cells are removed from the spinal cord after cortex. Nature 1994; 368: 737–740. effecting recovery from paraplegia. J Neurosci 2000; 20: 339 Bareyre FM, Kerschensteiner M, Raineteau O, Metten- 9215–9223. leiter TC, Weinmann O, Schwab ME. The injured spinal 320 Keirstead HS, Levine JM, Blakemore WF. Response of cord spontaneously forms a new intraspinal circuit in the oligodendrocyte progenitor cell population (defined adult rats. Nat Neurosci 2004; 7: 269–277. by NG2 labelling) to demyelination ofthe adult spinal 340 van Meeteren NL, Brakkee JH, Hamers FP, Helders PJ, cord. Glia 1998; 22: 161–170. Gispen WH. Exercise training improves functional 321 McTigue DM, Wei P, Stokes BT. Proliferation of NG2- recovery and motor nerve conduction velocity after sciatic positive cells and altered oligodendrocyte numbers in the nerve crush lesion in the rat. Arch Phys MedRehabil 1997; contused rat spinal cord. J Neurosci 2001; 21: 3392–3400. 78: 70–77. 322 Watanabe M, Toyama Y, Nishiyama A. Differentiation 341 Bizzi E, Tresch MC, Saltiel P, d’Avella A. New ofproliferatedNG2-positive glial progenitor cells in a perspectives on spinal motor systems. Nat Rev Neurosci remyelinating lesion. J Neurosci Res 2002; 69: 826–836. 2000; 1: 101–108. 323 McDonald JW et al. Transplanted embryonic stem cells 342 Eidelberg E, Walden JG, Nguyen LH. Locomotor control survive, differentiate and promote recovery in injured rat in macaque monkeys. Brain 1981; 104: 647–663. spinal cord. Nat Med 1999; 5: 1410–1412. 343 Eidelberg E, Story JL, Walden JG, Meyer BL. Anatomi- 324 Sasaki M, Honmou O, Akiyama Y, Uede T, Hashi K, cal correlates ofreturn oflocomotor functionafterpartial Kocsis JD. Transplantation ofan acutely isolated bone spinal cord lesions in cats. Exp Brain Res 1981; 42: 81–88. marrow fraction repairs demyelinated adult rat spinal 344 Steeves JD, Tetzlaff W. Engines, accelerators, and brakes cord axons. Glia 2001; 35: 26–34. on functional spinal cord repair. Ann NY AcadSci 1998; 325 Akiyama Y, Radtke C, Kocsis JD. Remyelination ofthe 860: 412–424. rat spinal cord by transplantation ofidentified bone 345 Barbeau H, Rossignol S. Recovery oflocomotion after marrow stromal cells. J Neurosci 2002; 22: 6623–6630. chronic spinalization in the adult cat. Brain Res 1987; 412: 326 Inoue M, Honmou O, Oka S, Houkin K, Hashi K, Kocsis 84–95. JD. Comparative analysis ofremyelinating potential of 346 Gazula VR, Roberts M, Luzzio C, Jawad AF, Kalb RG. focal and intravenous administration of autologous bone Effects oflimb exercise afterspinal cord injury on motor marrow cells into the rat demyelinated spinal cord. Glia neuron dendrite structure. J Comp Neurol 2004; 476: 2003; 44: 111–118. 130–145. 327 Sherratt RM, Bostock H, Sears TA. Effects of4- 347 de Leon RD, Hodgson JA, Roy RR, Edgerton VR. aminopyridine on normal and demyelinated mammalian Locomotor capacity attributable to step training versus nerve fibres. Nature 1980; 283: 570–572. spontaneous recovery after spinalization in adult cats. 328 Targ EF, Kocsis JD. 4-Aminopyridine leads to restora- J Neurophysiol 1998; 79: 1329–1340. tion ofconduction in demyelinated rat sciatic nerve. Brain 348 de Leon RD, Hodgson JA, Roy RR, Edgerton VR. Res 1985; 328: 358–361. Retention ofhindlimb stepping ability in adult spinal cats 329 Blight AR. Effect of4-aminopyridine on axonal conduc- after the cessation of step training. J Neurophysiol 1999; tion-block in chronic spinal cord injury. Brain Res Bull 81: 85–94. 1989; 22: 47–52. 349 Steeves JD, Jordan LM. Localization ofa descending 330 Shi R, Blight AR. Compression injury ofmammalian pathway in the spinal cord which is necessary for spinal cord in vitro and the dynamics ofaction potential controlled treadmill locomotion. Neurosci Lett 1980; 20: conduction failure. J Neurophysiol 1996; 76: 1572–1580. 283–288. 331 Shi R, Kelly TM, Blight AR. Conduction block in acute 350 Loy DN et al. Functional redundancy ofventral and chronic spinal cord injury: different dose–response spinal locomotor pathways. J Neurosci 2002; 22: characteristics for reversal by 4-aminopyridine. Exp 315–323. Neurol 1997; 148: 495–501. 351 Basso DM. Neuroanatomical substrates offunctional 332 Potter PJ et al. Randomized double-blind crossover trial recovery after experimental spinal cord injury: implica- offampridine-SR(sustained release 4-aminopyridine) in tions ofbasic science research forhuman spinal cord patients with incomplete spinal cord injury. J Neuro- injury. Phys Ther 2000; 80: 808–817. trauma 1998; 15: 837–849. 352 Wernig A, Muller S, Nanassy A, Cagol E. Laufband 333 Jensen JM, Shi R. Effects of4-aminopyridine on stretched therapy based on ‘rules ofspinal locomotion’ is effective mammalian spinal cord: the role ofpotassium channels in in spinal cord injured persons. Eur J Neurosci 1995; 7: axonal conduction. J Neurophysiol 2003; 90: 2334–2340. 823–829. 334 Calford MB. Dynamic representational plasticity in 353 Wernig A, Nanassy A, Muller S. Maintenance of sensory cortex. Neuroscience 2002; 111: 709–738. locomotor abilities following Laufband (treadmill) ther- 335 Calford MB. Mechanisms for acute changes in sensory apy in para- and tetraplegic persons: follow-up studies. maps. Adv Exp Med Biol 2002; 508: 451–460. Spinal Cord 1998; 36: 744–749. 336 Raineteau O, Schwab ME. Plasticity ofmotor systems 354 Wirz M, Colombo G, Dietz V. Long term effects of after incomplete spinal cord injury. Nat Rev Neurosci locomotor training in spinal humans. J Neurol Neurosurg 2001; 2: 263–273. Psychiatr 2001; 71: 93–96. 337 Basbaum AI, Wall PD. Chronic changes in the response 355 Bavetta S, Hamlyn PJ, Burnstock G, Lieberman AR, ofcells in adult cat dorsal horn followingpartial Anderson PN. The effects ofFK506 on dorsal column deafferentation: the appearance ofresponding cells in a axons following spinal cord injury in adult rats: neuro-

Spinal Cord Orchestrating spinal cord repair LM Ramer et al 160

protection and local regeneration. Exp Neurol 1999; 158: inhibits glutamate release after spinal injury in rats. 382–393. Neuroreport 1998; 9: 1655–1659. 356 Constantini S, Young W. The effects ofmethylpredniso- 373 Yick LW, So KF, Cheung PT, Wu WT. Lithium chloride lone and the ganglioside GM1 on acute spinal cord injury reinforces the regeneration-promoting effect of chondroi- in rats. J Neurosurg 1994; 80: 97–111. tinase ABC on rubrospinal neurons after spinal cord 357 De Ley G, Leybaert L. Effect offlunarizine and injury. J Neurotrauma 2004; 21: 932–943. methylprednisolone on functional recovery after experi- 374 Wang MS, Gold BG. FK506 increases the regeneration of mental spinal injury. J Neurotrauma 1993; 10: 25–35. spinal cord axons in a predegenerated peripheral nerve 358 Diaz-Ruiz A et al. Lipid peroxidation inhibition in spinal autograft. J Spinal CordMed 1999; 22: 287–296. cord injury: cyclosporin-A vs methylprednisolone. Neuro- 375 von Meyenburg J, Brosamle C, Metz GA, Schwab ME. report 2000; 11: 1765–1767. Regeneration and sprouting ofchronically injured corti- 359 Haghighi SS et al. Effects ofmethylprednisolone and MK- cospinal tract fibers in adult rats promoted by NT-3 and 801 on functional recovery after experimental chronic the mAb IN-1, which neutralizes myelin-associated spinal cord injury. Spinal Cord 2000; 38: 733–740. neurite growth inhibitors. Exp Neurol 1998; 154: 583–594. 360 Kim DH, Jahng TA. Continuous brain-derived neuro- 376 Azanchi R, Bernal G, Gupta R, Keirstead HS. Combined trophic factor (BDNF) infusion after methylprednisolone demyelination plus Schwann cell transplantation therapy treatment in severe spinal cord injury. J Korean MedSci increases spread ofcells and axonal regeneration follow- 2004; 19: 113–122. ing contusion injury. J Neurotrauma 2004; 21: 775–788. 361 Lankhorst AJ, ter Laak MP, Hamers FP, Gispen WH. 377 Keirstead HS, Morgan SV, Wilby MJ, Fawcett JW. Combined treatment with alphaMSH and methylpredni- Enhanced axonal regeneration following combined de- solone fails to improve functional recovery after spinal myelination plus schwann cell transplantation therapy injury in the rat. Brain Res 2000; 859: 334–340. in the injured adult spinal cord. Exp Neurol 1999; 159: 362 Mu X, Azbill RD, Springer JE. Riluzole and methyl- 225–236. prednisolone combined treatment improves functional 378 Yick LW, Wu W, So KF, Yip HK, Shum DK. recovery in traumatic spinal cord injury. J Neurotrauma Chondroitinase ABC promotes axonal regeneration of 2000; 17: 773–780. Clarke’s neurons after spinal cord injury. Neuroreport 363 Sheng H, Spasojevic I, Warner DS, Batinic-Haberle I. 2000; 11: 1063–1067. Mouse spinal cord compression injury is ameliorated by 379 King VR, Phillips JB, Brown RA, Priestley JV. The effects intrathecal cationic manganese(III) porphyrin catalytic oftreatment with antibodies to transforminggrowth antioxidant therapy. Neurosci Lett 2004; 366: 220–225. factor beta1 and beta2 following spinal cord damage in 364 Takami T, Oudega M, Bethea JR, Wood PM, Kleitman the adult rat. Neuroscience 2004; 126: 173–183. N, Bunge MB. Methylprednisolone and interleukin-10 380 Oudega M, Hagg T. Nerve growth factor promotes reduce gray matter damage in the contused Fischer rat regeneration ofsensory axons into adult rat spinal cord. thoracic spinal cord but do not improve functional Exp Neurol 1996; 140: 218–229. outcome. J Neurotrauma 2002; 19: 653–666. 381 Richardson PM, Verge VM. The induction ofa regen- 365 Ibarra A, Hauben E, Butovsky O, Schwartz M. The erative propensity in sensory neurons following peripheral therapeutic window after spinal cord injury can accom- axonal injury. J Neurocytol 1986; 15: 585–594. modate T cell-based vaccination and methylprednisolone 382 Chong MS, WoolfCJ, Turmaine M, Emson PC, in rats. Eur J Neurosci 2004; 19: 2984–2990. Anderson PN. Intrinsic versus extrinsic factors in 366 Baffour R et al. Synergistic effect ofbasic fibroblast determining the regeneration ofthe central processes of growth factor and methylprednisolone on neurological rat dorsal root ganglion neurons: the influence ofa function after experimental spinal cord injury. J Neuro- peripheral nerve graft. J Comp Neurol 1996; 370: 97–104. surg 1995; 83: 105–110. 383 Oudega M, Varon S, Hagg T. Regeneration ofadult rat 367 Li L, Xu Q, Wu Y, Hu W, Gu P, Fu Z. Combined therapy sensory axons into intraspinal nerve grafts: promoting ofmethylprednisolone and brain-derived neurotrophic effects ofconditioning lesion and graftpredegeneration. factor promotes axonal regeneration and functional Exp Neurol 1994; 129: 194–206. recovery after spinal cord injury in rats. Chin MedJ 384 Blits B, Dijkhuizen PA, Boer GJ, Verhaagen J. Intercostal (Engl) 2003; 116: 414–418. nerve implants transduced with an adenoviral vector 368 Haghighi SS, Clapper A, Johnson GC, Stevens A, encoding neurotrophin-3 promote regrowth ofinjured rat Prapaisilp A. Effect of4-aminopyridine and single-dose corticospinal tract fibers and improve hindlimb function. methylprednisolone on functional recovery after a chronic Exp Neurol 2000; 164: 25–37. spinal cord injury. Spinal Cord 1998; 36: 6–12. 385 Fernandez E, Pallini R, Mercanti D. Effects oftopically 369 Guth L, Zhang Z, Roberts E. Key role for pregnenolone administered nerve growth factor on axonal regeneration in combination therapy that promotes recovery after in peripheral nerve autografts implanted in the spinal cord spinal cord injury. Proc Natl AcadSci USA 1994; 91: ofrats. Neurosurgery 1990; 26: 37–42. 12308–12312. 386 Hiebert GW, Khodarahmi K, McGraw J, Steeves JD, 370 Kuchner EF, Hansebout RR. Combined steroid and Tetzlaff W. Brain-derived neurotrophic factor applied to hypothermia treatment ofexperimental spinal cord injury. the motor cortex promotes sprouting ofcorticospinal Surg Neurol 1976; 6: 371–376. fibers but not regeneration into a peripheral nerve 371 Kuchner EF, Hansebout RR, Pappius HM. Effects of transplant. J Neurosci Res 2002; 69: 160–168. dexamethasone and oflocal hypothermia on early and 387 Kwon BK, Liu J, Oschipok L, Teh J, Liu ZW, Tetzlaff W. late tissue electrolyte changes in experimental spinal cord Rubrospinal neurons fail to respond to brain-derived injury. J Spinal Disord 2000; 13: 391–398. neurotrophic factor applied to the spinal cord injury site 372 Yamamoto K, Ishikawa T, Sakabe T, Taguchi T, Kawai 2 months after cervical axotomy. Exp Neurol 2004; 189: S, Marsala M. The hydroxyl radical scavenger Nicaraven 45–57.

Spinal Cord Orchestrating spinal cord repair LM Ramer et al 161

388 Levi AD, Dancausse H, Li X, Duncan S, Horkey L, ofsensory axons into the injured spinal cord. Exp Neurol Oliviera M. Peripheral nerve grafts promoting central 1997; 148: 433–443. nervous system regeneration after spinal cord injury in the 403 Tobias CA, Dhoot NO, Wheatley MA, Tessler A, Murray primate. J Neurosurg Spine 2002; 96: 197–205. M, Fischer I. Grafting of encapsulated BDNF-producing 389 Novikova LN, Novikov LN, Kellerth JO. Differential fibroblasts into the injured spinal cord without immune effects ofneurotrophins on neuronal survival and axonal suppression in adult rats. JNeurotrauma2001; 18: 287–301. regeneration after spinal cord injury in adult rats. J Comp 404 Bregman BS, Broude E, McAtee M, Kelley MS. Neurol 2002; 452: 255–263. Transplants and neurotrophic factors prevent atrophy of 390 Storer PD, Houle JD, Oblinger M, Jones KJ. Combina- mature CNS neurons after spinal cord injury. Exp Neurol tion ofgonadal steroid treatment and peripheral nerve 1998; 149: 13–27. grafting results in a peripheral motoneuron-like pattern of 405 Broude E, McAtee M, Kelley MS, Bregman BS. Fetal beta II-tubulin mRNA expression in axotomized hamster spinal cord transplants and exogenous neurotrophic rubrospinal motoneurons. J Comp Neurol 2002; 449: support enhance c-Jun expression in mature axotomized 364–373. neurons after spinal cord injury. Exp Neurol 1999; 155: 391 Ye JH, Houle JD. Treatment ofthe chronically injured 65–78. spinal cord with neurotrophic factors can promote axonal 406 Castellanos DA, Tsoulfas P, Frydel BR, Gajavelli S, Bes regeneration from supraspinal neurons. Exp Neurol 1997; JC, Sagen J. TrkC overexpression enhances survival and 143: 70–81. migration ofneural stem cell transplants in the rat spinal 392 Bamber NI, Li H, Lu X, Oudega M, Aebischer P, Xu cord. Cell Transplant 2002; 11: 297–307. XM. Neurotrophins BDNF and NT-3 promote axonal re- 407 Han PJ, Shukla S, Subramanian PS, Hoffman PN. Cyclic entry into the distal host spinal cord through Schwann AMP elevates tubulin expression without increasing cell-seeded mini-channels. Eur J Neurosci 2001; 13: intrinsic axon growth capacity. Exp Neurol 2004; 189: 257–268. 293–302. 393 Blits B, Oudega M, Boer GJ, Bartlett BM, Verhaagen J. 408 Gautier SE et al. Poly(alpha-hydroxyacids) for applica- Adeno-associated viral vector-mediated neurotrophin tion in the spinal cord: resorbability and biocompatibility gene transfer in the injured adult rat spinal cord improves with adult rat Schwann cells and spinal cord. J Biomed hind-limb function. Neuroscience 2003; 118: 271–281. Mater Res 1998; 42: 642–654. 394 Iannotti C, Li H, Yan P, Lu X, Wirthlin L, Xu XM. Glial 409 Guenard V, Kleitman N, Morrissey TK, Bunge RP, cell line-derived neurotrophic factor-enriched bridging Aebischer P. Syngeneic Schwann cells derived from adult transplants promote propriospinal axonal regeneration nerves seeded in semipermeable guidance channels en- and enhance myelination after spinal cord injury. Exp hance peripheral nerve regeneration. J Neurosci 1992; 12: Neurol 2003; 183: 379–393. 3310–3320. 410 Novikov LN, Novikova LN, Mosahebi A, Wiberg M, 395 Oudega M, Xu XM, Guenard V, Kleitman N, Bunge MB. Terenghi G, Kellerth JO. A novel biodegradable implant A combination ofinsulin-like growth factor-Iand for neuronal rescue and regeneration after spinal cord platelet-derived growth factor enhances myelination but injury. Biomaterials 2002; 23: 3369–3376. diminishes axonal regeneration into Schwann cell grafts in 411 Oudega M et al. Axonal regeneration into Schwann cell the adult rat spinal cord. Glia 1997; 19: 247–258. grafts within resorbable poly(alpha-hydroxyacid) gui- 396 Tuszynski MH, Weidner N, McCormack M, Miller I, dance channels in the adult rat spinal cord. Biomaterials Powell H, Conner J. Grafts of genetically modified 2001; 22: 1125–1136. Schwann cells to the spinal cord: survival, axon growth, 412 Paino CL, Bunge MB. Induction ofaxon growth into and myelination. Cell Transplant 1998; 7: 187–196. Schwann cell implants grafted into lesioned adult rat 397 Weidner N, Blesch A, Grill RJ, Tuszynski MH. Nerve spinal cord. Exp Neurol 1991; 114: 254–257. growth factor-hypersecreting Schwann cell grafts augment 413 Paino CL, Fernandez-Valle C, Bates ML, Bunge MB. and guide spinal cord axonal growth and remyelinate Regrowth ofaxons in lesioned adult rat spinal cord: central nervous system axons in a phenotypically appro- promotion by implants ofcultured Schwann cells. priate manner that correlates with expression ofL1. J Neurocytol 1994; 23: 433–452. J Comp Neurol 1999; 413: 495–506. 414 Schlosshauer B, Muller E, Schroder B, Planck H, Muller 398 Bakshi A, Fisher O, Dagci T, Himes BT, Fischer I, HW. Rat Schwann cells in bioresorbable nerve guides to Lowman A. Mechanically engineered hydrogel scaffolds promote and accelerate axonal regeneration. Brain Res for axonal growth and angiogenesis after transplantation 2003; 963: 321–326. in spinal cord injury. J Neurosurg Spine 2004; 1: 322–329. 415 Chauhan NB, Figlewicz HM, Khan T. Carbon filaments 399 Bloch J, Fine EG, Bouche N, Zurn AD, Aebischer P. direct the growth ofpostlesional plastic axons after Nerve growth factor- and neurotrophin-3-releasing gui- spinal cord injury. Int J Dev Neurosci 1999; 17: dance channels promote regeneration ofthe transected rat 255–264. dorsal root. Exp Neurol 2001; 172: 425–432. 416 Dupont-Versteegden EE, Murphy RJ, Houle JD, Gurley 400 Houle JD. Regeneration ofdorsal root axons is related to CM, Peterson CA. Mechanisms leading to restoration of specific non-neuronal cells lining NGF-treated intraspinal muscle size with exercise and transplantation after nitrocellulose implants. Exp Neurol 1992; 118: 133–142. spinal cord injury. Am J Physiol Cell Physiol 2000; 279: 401 Houle JD, Ziegler MK. Bridging a complete transection C1677–C1684. lesion ofadult rat spinal cord with growth factor-treated 417 Peterson CA, Murphy RJ, Dupont-Versteegden EE, nitrocellulose implants. J Neural Transplant Plast 1994; 5: Houle JD. Cycling exercise and fetal spinal cord 115–124. transplantation act synergistically on atrophied muscle 402 Prewitt CM, Niesman IR, Kane CJ, Houle JD. Activated following chronic spinal cord injury in rats. Neurorehabil macrophage/microglial cells can promote the regeneration Neural Repair 2000; 14: 85–91.

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