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Spinal Cord Regeneration: From Gene to Transplants

Kwon, Brian K., MD, FRCS(C)*†; Tetzlaff, Wolfram, MD, PhD*

Basic Science
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The past 20 years has seen the emergence of many exciting and promising experimental therapeutic strategies to promote regeneration of the injured spinal cord in laboratory animals. A greater understanding of the pathophysiologic mechanisms that contribute to the initial and secondary cord injury may facilitate the development of neuroprotective strategies that preserve axonal function and prevent apoptotic cell death, thus optimizing neurologic function. Neurotrophic factors have been used to augment the poor intrinsic regenerative capacity of central nervous system neurons, and the need for sophisticated delivery of such trophic agents has stimulated the application of gene therapy in this context. In addition to augmenting the neuronal capacity to regenerate axons, many researchers are developing strategies to overcome the inhibitory environment into which these axons must grow. Characterizing the inhibitory elements of the glial scar at the site of injury and of myelin in the distal tracts is therefore a focus of intense scientific interest. To this effect, a number of strategies have also been developed to bridge the injury site and facilitate axonal growth across the lesion with a variety of cellular substrates. These include fetal tissue transplants, stem cells, Schwann cells, and olfactory ensheathing cells. With the collaboration of basic scientists and clinicians, it is hoped that these experimental strategies coupled with a greater understanding of the neurobiology of spinal cord injury will be translatable to the clinical setting in the near future.

From *Collaboration on Repair Discoveries and the

†Division of Spine Surgery, Department of Orthopaedics, University of British Columbia, and Vancouver General Hospital, Vancouver, British Columbia, Canada.

Device status category: 1.

Conflict of interest category: 12.

Address reprint requests to

Wolfram Tetzlaff, MD, PhD

Department of Zoology

University of British Columbia

6270 University Boulevard

Columbia, Vancouver V6T 1Z4

British Columbia

Canada

E-mail:tetzlaff@cord.ubc.ca

Clinical research focused on the initial resuscitation, surgical stabilization, medical treatment, and long-term rehabilitation of the patient has led to dramatic improvements in the overall care of the victims of spinal cord injury (SCI). However, with regards to the central issue of paraplegia, it is the exciting progress in basic science over the past two decades that has ignited the interest and hope of clinicians, scientists, and patients that a cure for paralysis is most likely achievable. The need for further neurobiologic understanding and for the eventual translation of knowledge from bench to bedside has made the field of spinal cord regeneration a common ground for rich collaboration between clinicians and scientists. The purpose of this review is to update clinicians on recent basic science advances in spinal cord regeneration that are conceivably translatable to the clinical setting. Within the context of spinal cord regeneration, most spine surgeons who treat patients with these injuries are likely to be interested in three things: what can be done acutely to minimize neurologic injury, what can be done during the acute surgical management to enhance neurologic function, and finally, what can be done for the chronically injured patient with longstanding paralysis and an essentially stable neurologic deficit? Before addressing these questions, we begin with a brief discussion about the animal models used in the studies of spinal cord regeneration that are discussed subsequently. Because it is impossible to comprehensively cover the entire field of spinal cord regeneration, we apologize in advance to our colleagues whose contributions are not referenced in this review. A summary of obstacles to regeneration and strategies to overcome these is illustrated in Figure 1.

Figure 1

Figure 1

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Animal Models in Spinal Cord Regeneration Research

Animal models are extremely important for studying SCI in the laboratory setting and provide an essential in vivo arena for the development of experimental therapeutic strategies. A wide variety of animals have been used in this regard, but at the present time the rat and mouse are most popular, the latter being of particular utility for its transgenic potential. The injury paradigms are similarly of a wide spectrum, with each possessing its own advantages and disadvantages.

Models in which the spinal cord is fully transected ensure the absolute completeness of the injury, making it somewhat easier to evaluate the effectiveness of interventions, with regards to both axonal regeneration and functional recovery. Partial transection models attempt to selectively injure tracts of the spinal cord and are widely used because of the relative ease of animal care after surgery. In addition, some partial transection models allow a comparison with the contralateral uninjured side. However, it can be difficult to determine the anatomic completeness of the lesion and to determine whether observed functional improvement is caused by regeneration of the injured tract or compensation from other spared systems. Axonal tracers are useful in interpreting whether axons that are observed distal to the injury site are those that have regenerated or those that have escaped injury in the first place. For example, a retrograde axonal tracer that is picked up by the axons and transported back to the cell body can be applied distal to the site of injury; the completeness of the injury can then be confirmed by demonstrating the absence of tracer proximally in the cell bodies.

These transection models have endured some criticism for their lack applicability to the vast majority of spinal cord injuries caused by nonpenetrating trauma, but their role in studying axonal regeneration should not be underestimated. Nonetheless, there has been growing interest in models that are representative of the more common “blunt” SCI. Beginning with a crude weight drop model by Allen in 1911, 6 many models have been developed to deliver a blunt contusion force to the spinal cord. There are a number of issues that arise from the use of such models, including variability in the amount of force being delivered and variations of the resultant pathologic and functional injury. The most commonly used contusion model has been the New York University impactor, in which a 10-g impactor is dropped from a variable height onto the exposed dorsal surface of the spinal cord. 53 This device has been reported to cause reproducible spinal cord lesions and functional deficits, and it was adopted for use in the multicenter animal spinal cord injury study. An electromechanical impactor has been developed at Ohio State University that, via a feedback mechanism, allows for a more consistent evaluation of the impact delivered to the dorsal spinal cord. 124 These models apply a single blow and therefore do not simulate ongoing compression, nor do they simulate the clinical scenario in which bone fragments are driven into the ventral surface of the cord. A “clip compression” model was introduced by Rivlin and Tator 109 in which a modified aneurysmal clip produces the SCI with circumferential compression. Although this may not reproduce the dynamic nature of the impact, it does appear to be consistent in its production of injuries and it does allow study of variable periods of compression. These models provide a good setting for the functional and histologic evaluation of experimental neuroprotective agents and of agents that enhance plasticity. However, because of the incomplete nature of injury and the anatomic complexity of the tracts, it is very difficult to verify anatomic axonal regeneration in these contusion models.

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Minimizing Neurologic Injury Acutely

The acutely traumatized spinal cord is subjected to significant neurotoxicity, with ionic fluxes, alterations in local blood flow, and inflammatory processes all contributing to the initial and evolving secondary neurologic injury. A comprehensive review of the relevant pathophysiology and of the debated merits of methylprednisolone in this context is included elsewhere within this volume and therefore will not be covered in detail here. Suffice it to say that although much excitement has been generated for regenerative strategies involving neurotrophic factors and cellular transplantation, the initial advances for patients may be borne out in neuroprotective strategies designed not to regenerate but rather to preserve and optimize native neurologic function by maintaining axonal function and preventing cell death. Innovative work 1–4,68–70,91,111,117,118,126,138,139 has shed much insight into the importance of ionic fluxes in white matter pathology and have raised the intriguing possibility that axonal function might be preserved by the local application of ion channel antagonists to the site of spinal cord damage. Also, in addition to the necrotic death of cells at the site of trauma, the apoptotic demise of neurons and oligodendrocytes has been demonstrated in animal models of contusive injury and in postmortem studies of human patients with spinal cord injuries. 11,29,38,75 Although necrosis and apoptosis represent morphologically distinct phenomena, cell death may indeed occur along a continuum between the two, 77 allowing various degrees of therapeutic intervention. Secondary tissue damage may be reduced by limiting excitotoxicity with glutamate receptor antagonists, 111,132 by nitric oxide synthase inhibition, 112 by targeting the molecular apoptotic pathway, 114 by providing neurotrophic factors to the injury site, 97 or by modulating the inflammatory response of the injured tissue. 116 The potential for these neuroprotective strategies to be refined and incorporated into an early spinal cord decompression and stabilization procedure has led to the initiation of clinical trials in this regard.

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Enhancing Axonal Regeneration After Acute Spinal Cord Injury

Whereas the neuroprotective strategies discussed above are aimed at minimizing secondary damage, another line of basic science research focuses on strategies to promote axonal regeneration. Axonal regeneration in the central nervous system (CNS) fails to occur conceptually for two reasons: 1) the injured neurons have a limited intrinsic ability to regenerate, and 2) the environment into which they must send axons is not permissive to regeneration.

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Characterizing the Intrinsic Regenerative Ability

The capacity of CNS neurons to regenerate their injured axons was first described in 1908 by Tello in the laboratory of the Spanish neuroscientist Ramon y Cajal and was subsequently largely forgotten. 102 Seminal experiments in the 1980s 30,107,108 reiterated and extended these findings by demonstrating that when presented with permissive conditions (i.e., a peripheral nerve graft), some CNS neurons are indeed able to regenerate their injured axons. We now know that this regenerative competence is unequal among different neuronal types 87,137 and also varies significantly with neuronal age 26 and distance from the site of injury. 43,107 A landmark study by Richardson and Issa in 1984 demonstrated that the response of the neuronal cell body to axonal injury plays a pivotal role in the regenerative capacity of the neuron. 106 In this study the central spinal projection of a dorsal root ganglion cell was shown to regenerate into a peripheral nerve transplant only after the peripheral projection had been previously transected. It was subsequently demonstrated that transection of the peripheral axon induces changes in gene expression in the parent neuron that are not seen after injury of the central process. 113 These observations suggested that these neurons did indeed possess the appropriate regenerative machinery but required the initial stimulus of the peripheral transection to activate it and thus become “regeneration-capable.” Significant interest has therefore been generated to delineate these genetic mechanisms that must be initiated at the cell body level, with hopes that such understanding will allow for strategies to persuade otherwise incompetent neurons to regenerate their injured axons.

A number of genes have been shown to be up-regulated or constitutively expressed in association with axonal growth, both within a developmental and regenerative context; these have collectively been termed “regeneration-associated genes” or RAGs (reviewed recently by Fernandes and Tetzlaff 44). The products of these genes include transcription factors such as c-jun, which mediates subsequent gene expression, 54,61 cytoskeletal proteins involved in axonal extension such as Tα1-tubulin, 43,82 cytoplasmic growth cone proteins involved in mediating signal transduction such as GAP-43 and CAP-23, 48,119 and cell adhesion molecules such as L1 and NCAM involved in growth cone guidance. 12,62,137 The importance of these genes in axonal regeneration has been extrapolated from the correlation of their up-regulation with axonal growth and the absence of their expression with regenerative failure. 8,12,43,113,128 Regeneration of the central (spinal) axons of the dorsal root ganglion recently has been demonstrated after transgenic overexpression of GAP-43 and CAP-23 in combination, but not when each of these genes was expressed alone. 16 These findings imply a prerequisite rather than associative role for the expression of these growth cone proteins for axonal elongation and also serve to highlight the fact that multiple gene products acting in combination are necessary for an effective regenerative response. In summary, although injured axons may intrinsically be capable of short disorganized terminal sprouting and in some cases even slow axonal elongation, 7,14 the sustained and distant growth of axons requires the participation of the cell body, manifested by the expression of a number of RAGs. 44,121

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Augmenting the Neurons’ Intrinsic Regenerative Ability

Having identified some of the components that are seemingly important for axonal regeneration in the CNS, the problem then becomes identifying methods to manipulate them in a favorable manner. Neurotrophic factors have emerged as leading candidates for enhancing the regenerative capacity of CNS neurons because of their stimulation of a large number of known and probably unknown RAGs. Our laboratory has previously demonstrated that after axotomy of the rubrospinal tract at the thoracic level, the infusion of brain-derived neurotrophic factor (BDNF) or neurotrophin (NT)-4/5 into the vicinity of the red nucleus resulted in the up-regulation of GAP-43 and Tα1-tubulin and the promotion of axonal regeneration into peripheral nerve transplants grafted into the injury site. 65 Without this neurotrophic application to the cell body, the rubrospinal neurons fail to express RAGs after thoracic axotomy and fail to regenerate into peripheral nerve grafts. 43,64 We have similarly found BDNF application to the thoracic axotomy site ineffective at promoting RAG expression. Others, however, have encountered some regenerative success with the direct application of neurotrophic factors to the SCI site. Using an adult rat model in which the dorsal columns were crushed, Bradbury et al 17 demonstrated sensory axonal regeneration after the intrathecal application of NT-3, but not with BDNF. With a similar intrathecal administration of NT-3, NGF, and glial-derived neurotrophic factor after complete crush injuries of cervical dorsal roots in a rat model, Ramer et al 98 demonstrated not only regrowth of damaged axons across the dorsal root entry zone but also functional reconnection within the spinal cord. As in the Bradbury et al experiments, 17 BDNF was not found to be effective. These studies highlight the differential responsiveness of neuronal types to the various neurotrophic factors, which may be caused by the variable expression of their respective receptors, not only between different populations of neurons and glial cells but also among different locations of the same neuron.

Despite these observed variations in effectiveness, neurotrophic factors are powerful tools that will undoubtedly remain a cornerstone in future regenerative strategies, in one form or another. The studies described above are examples of a simple infusion of the neurotrophic factor. The desire to develop more sophisticated and longer-lasting delivery systems has led to the introduction of gene transfer technology, in which cells genetically altered to produce trophic factors are inserted into the spinal cord 15,52,90,130 (ex vivo gene therapy) or the native tissue is transfected with a neurotrophic gene, usually via a viral vector 59,110,142 (in vivo gene therapy). Obviously, the clinical translation of such strategies will require the resolution of some of the potential safety hazards involved with viral vectors and the recognized side effects of neurotrophic factor infusion. In the animal setting neurotrophic delivery by ex vivo gene transfer was demonstrated by Liu et al 76 who transplanted genetically modified fibroblasts producing BDNF into a rat cervical hemisection injury model and showed some rubrospinal tract growth and functional recovery in a limb usage test. The precise neurologic basis of this function test is not entirely clear, however, making it difficult to establish the extent to which the improved performance was caused by regenerating axons versus enhanced local plasticity. The application of neurotrophic factors is evolving into new therapeutic strategies that combine them with cellular transplantation methods of overcoming the inhibitory environment experienced by CNS axons, a topic that will be addressed subsequently.

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Characterizing the Inhibitory Central Nervous System Environment: Myelin and the Glial Scar

The previously mentioned classic work by David and Aguayo, 30 which demonstrated that injured CNS axons incapable of regenerating within their native environment were able to grow into peripheral nerve grafts, pointed not only to an intrinsic regenerative ability possessed by the neurons but also to the inhibitory nature of the CNS environment for axonal growth. The enormous interest stimulated by these findings has resulted in the delineation of many of the obstacles for axonal regeneration within the CNS and the development of strategies to overcome them. The CNS inhibition of axonal regeneration can be broadly divided into inhibitory factors related to CNS myelin and to the nonpermissive nature of the cyst and glial scar that forms in response to injury. Pioneering work in the late 1980s by Martin Schwab and colleagues led to the immunologic identification of NI-35 and NI-250, and neurite inhibitory epitopes associated with CNS myelin, 25,115 which include what has subsequently been cloned as Nogo-A. 27 This has led to the exciting finding that neutralization of these inhibitory factors with the IN-1 antibody results in axonal regeneration within the normally nonpermissive CNS environment. 20,24 A partially humanized, recombinant fragment of this IN-1 antibody has been shown to promote regeneration and sprouting of injured corticospinal axons in a rat injury model. 20 The human nature of this antibody potentially represents an important step in the translation of this strategy to the clinical setting. The discovery of myelin associated glycoprotein by Mukhopadhyay et al 89 and McKerracher et al 79 in 1994 has yielded another important neurite inhibitor whose axonal inhibition has been shown to be surmountable by priming with BDNF and glial-derived neurotrophic factor. 23 The possibility that myelin may contain multiple molecular inhibitors, each requiring a specific blocking agent, has prompted promising investigation into the promotion of axonal regeneration by immunologically targeting the myelin sheath as a whole. 37,58

Axonal growth also is impeded at the site of injury by a gliotic scar 31 and cavity, which form as the complex result of cell death, inflammation, and tissue degradation. 45,46 The scar is composed of reactive astrocytes, microglial cells, some oligodendrocytes, and meningeal cells, and it appears that in addition to forming a complex three-dimensional physical barrier to axonal regeneration, these cells secrete a number of molecules that are inhibitory to growth. 47 These molecules include proteoglycans (such as phosphocan, neurocan, brevican, and NG2), 41 collagens, 123 semaphorins, 93,95 and ephrins. 83 Generally speaking, the inhibitory properties of these molecules have been documented in isolation under in vitro conditions; their significance in vivo as axonal inhibitors is difficult to assess. A further understanding is needed of the cellular and molecular aspects of glial scarring and their intimate association with the inflammatory processes initiated by the injury. Techniques that inhibit or remove the cellular components of the scar 85 or that promote the degradation of inhibitory proteoglycans 88 have been attempted experimentally and offer some promise that, with further refinement, they may become elements of a clinically relevant therapeutic strategy. Another line of interesting research that has emerged recently involves the targeting of the intracellular pathways that mediate axonal growth, such as the Rho signaling pathway. Rho is a small protein that binds guanine triphosphatase and is part of a second messenger signaling system on which many pathways that regulate the actin cytoskeleton of the axonal growth cone converge. The recent application of a Rho inhibitor (C3 enzyme) promoted the sprouting of axons in the crushed rat optic nerve. 67 Another intracellular pathway involved in growth cone advance is regulated by cyclic nucleotides such as cyclic AMP and cyclic GMP; it may be possible to reverse the repulsive nature of some of the factors within myelin into stimulators of axonal growth by targeting such pathways. 122

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Overcoming the Inhibitory Central Nervous System Environment With Cellular Transplantation

In an attempt to overcome or circumvent this gliotic barrier and promote functional regeneration across the injury site, a number of intraspinal cellular transplantation techniques have been developed that, by the mere nature of their administration, are of significant relevance for spine surgeons. Primary candidates for such transplantation strategies include neural stem cells, fetal cells, Schwann cells, and olfactory ensheathing cells (OECs). It is noteworthy that not all cell transplantation strategies are necessarily aimed at achieving the same thing. The undifferentiated nature of neural stem cells, for example, gives them the potential to mature into both neuronal and glial phenotypes and hence raises the possibility that, within the injury site, they might form neurons to relay synaptic information across the lesion or alternatively form glial cells to provide neurotrophic support, a permissive growth matrix, and myelination for spared and newly generated axons. 78 Fetal neuronal tissue transplants similarly offer the possibility of replacing neuronal and glial cells lost during the initial or secondary injury. 104 Schwann cells and OECs are considered to provide neurotrophic support, a suitable growth substrate, and remyelination for regenerating or spared axons. 60 The role of neurotrophic factors in cellular survival and differentiation and for the promotion of axonal elongation makes them important adjuncts to virtually all cellular transplantation strategies.

In animal models of SCI, fetal tissue transplants have shown robust survival, 5,50 enhancement of host neuronal survival, 13,18 and promotion of axonal elongation. 9,34 In addition, improved distal function after fetal tissue transplantation in transection, hemisection, and contusion models of injury has been demonstrated. 35,84,103,120,125,127 A case report of the safe use of human fetal spinal cord tissue for obliterating a post-traumatic syrinx was published by Falci et al 39 in 1997. These promising results have culminated in the initiation of a Phase 1 clinical trial for testing the safety and feasibility of this technique in chronically injured patients with post-traumatic syringomyelia. 104 The preliminary findings of this study suggesting overall safety were presented in October 2000, and we anxiously await the final conclusions of these authors. 136 The use of fetal tissue in the future holds some promise, but on its way to becoming a clinical strategy for spinal cord regeneration it will have to contend with the issues of limited availability and the complex ethical considerations surrounding its procurement.

Neural stem cells can also be acquired from embryonic tissue and thus would be subject to similar ethical and availability constraints as fetal tissue if not for the somewhat surprising discovery of their presence in the mature CNS, long considered to consist entirely of postmitotic cells. 105,134 In most applications neural stem cells are harvested and then cultured in vitro before being transplanted into the brain or spinal cord, 49,135 but recent reports by Fallon et al 40 and Kojima and Tator 66 have demonstrated the exciting possibility of stimulating the proliferation, migration, and differentiation of these cells in vivo as well. The pluripotency and therapeutic potential of neuronal stem cells in SCI have been demonstrated by McDonald et al 78 who transplanted progenitor cells into the spinal cord of adult rats after a contusion injury and observed improved hindlimb motor function in association with the differentiation of the progenitor cells into neurons, oligodendrocytes, and astrocytes. Other authors have recently demonstrated that embryonic stem cells grafted into a demyelinated spinal cord could differentiate into oligodendrocytes that subsequently remyelinate host axons. 21,74 Although much about these neural and embryonic stem cells has yet to be elucidated, an accelerated interest in their basic biology makes them good contenders for future regeneration strategies.

The long-recognized permissiveness of peripheral nerves to axonal growth is largely attributable to the Schwann cells, and the relative ease with which they can be acquired and expanded in culture has made them attractive candidates for promoting regeneration in patients with SCI. 22 A landmark study in 1996 by Cheng et al 28 demonstrated corticospinal tract regeneration and functional recovery after bridging a rat spinal cord transection with 18 tiny intercostal nerve grafts stabilized with a fibrin glue containing acidic fibroblast growth factor. Despite immense worldwide interest in the application of peripheral nerves and Schwann cells in this regard, these results have not been able to be reproduced, a problem that Dr. Cheng attributes to deviations from the original experimental protocol (personal communication). Attempts to apply a Schwann cell bridging approach in humans have been undertaken in Taiwan by Dr. Cheng, and we eagerly await the results of this study. Schwann cells have also served as a foundation for developing combination strategies with neurotrophic factors. In an adult rat transection model, Schwann cell transplants supplemented with BDNF and NT-3 infusions were found to increase axonal elongation compared with transplants alone. 141 Genetic manipulation of Schwann cells to increase their secretion of neurotrophic factors, such as BDNF 80 and NGF, 131,133 has also been demonstrated to improve their promotion of axonal growth.

Whereas Schwann cell transplants have demonstrated their ability to incorporate into the host lesion, bridge transected spinal cord stumps, attract axonal growth into the graft, and myelinate the entering axons, their utility has been hindered by their poor migration and the reluctance of the axons to leave the graft and re-enter the CNS environment. 94,140 The unwillingness of regenerating axons to grow back into the CNS is likely influenced by the local glial scar and inhibitory myelin components encountered at the graft–host interface. Because these are not necessarily insurmountable obstacles, continued work with Schwann cell bridging techniques is warranted. In the meantime, however, much attention has been directed to a cell that appears capable of accompanying axons regenerating across the graft–host interface: the olfactory ensheathing cell (OEC).

Olfactory ensheathing cells are distinct glial cells bearing resemblance to Schwann cells and astrocytes and are found in both the peripheral nervous system and CNS in association with the olfactory axons. 33,101 Olfactory epithelial neurons are unique in that throughout life they undergo a constant turnover and grow axons that are accompanied by OECs from the peripheral nervous system into the mature, normally inhibitory, CNS. 36 Their ability to escort axons across the interface from the peripheral nervous system to CNS has identified them as a potential solution for this problem seen with Schwann cell bridging. 33,92,100 This exploding line of research on OECs has recently culminated in the bridging of a complete spinal cord transection in the rat by a group led by Ramon-Cueto et al. 99 These investigators demonstrated long-distance regeneration of the corticospinal tract in addition to noradrenergic and serotonergic fibers in the distal cord stump, which was associated with significant functional recovery (climbing on a platform). The experience of other authors using OECs in partial SCI models 71,72 appears to corroborate the general concept that these cells provide a favorable substrate for growth across an injury site and possibly also a source of myelination, although the potential for Schwann cell contamination makes the origin of the observed myelin somewhat uncertain. Recent reports by Kato et al 63 and Barnett et al 10 have described the acquisition and purification of human OECs and have evidence to suggest that they are able to remyelinate adult rat CNS axons when transplanted into areas of demyelination within the spinal cord.

Like neural stem cells, recent studies have generated much excitement about the regenerative potential of OECs, but similarly, much still needs to be learned about their basic biology. Because the olfactory system is somewhat inaccessible, the possibility of acquiring the cells from the nasal mucosa would certainly enhance the applicability of this technique. Nevertheless, the exciting properties demonstrated to this date make it likely that OECs will play an important role in initial clinical regeneration strategies.

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Enhancing Neurologic Function in the Chronic Setting

Although most neurobiologic experiments concerning regeneration of the traumatized spinal cord are performed in the acute injury setting, the overwhelming majority of patients with spinal cord injuries have long-standing neurologic deficits that are considered chronic. An important question arises as to how applicable the strategies developed for the acutely injured will be in the chronic context. It is reasonable to anticipate an important role for the aforementioned neurotrophic factors, cellular transplantation, and methods for overcoming axonal inhibition by myelin and glial scarring, although the few studies that have been performed thus far have indicated that their effectiveness in a chronic setting falls short of their performance acutely. 55,129 A good example of this was provided by Houle and Ye 56 who showed a 50% decrease in the regenerative response of rat brain stem neurons when ciliary neurotrophic factor was applied 8 weeks after spinal cord hemisection as compared with administration at 4 weeks postinjury. In a recent report by Decherchi and Gauthier, 32 a peripheral nerve graft was implanted rostral to a cervical hemisection lesion acutely and chronically (3 weeks postinjury). These authors found 85% fewer brain stem and spinal cord neurons extending into the grafts implanted chronically as compared with those implanted acutely.

One of the important considerations that arises from chronic injury studies such as these is the survival response of the neuronal cell body to the axonal injury. A total of 25–50% of the neurons in the rubrospinal nucleus, for example, have been reported to die as early as 4 weeks after axotomy of its tract, 42,51,57,96 with the remaining cells becoming highly atrophic. The application of fetal cell transplants 19,86 and neurotrophic factors 57,65 has been shown to prevent some of this cell loss. We have evaluated the survival and regenerative capacity of rubrospinal neuron in rats that have undergone a cervical hemisection 1 year earlier. At the end of this year, an intracranial infusion of BDNF for 7 days to the vicinity of the rubrospinal nucleus resulted in equal numbers of cells in the injured red nucleus compared with uninjured, suggesting that these neurons are not dying but rather are atrophying so significantly as to become indistinguishable from the surrounding glial stroma. 81 In addition, we have observed this cell body neurotrophic application to be associated with an up-regulation in RAGs and the promotion of rubrospinal axon regeneration into peripheral nerve transplants, even as late as 1 year postinjury. 73 These results are encouraging for the development of therapeutic strategies for chronically injured patients, but more work is necessary to improve on the delivery of neurotrophic factor to the neuronal cell bodies and to integrate cellular transplantation techniques.

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Conclusion

Although much progress has been made in the field of spinal cord regeneration in the past 20 years, it would appear that a great deal of work lies ahead of us. The axonal regeneration witnessed in some of the most promising strategies to date often represents quantitatively few fibers and is frequently measured in terms of millimeters. This is not to suggest that such regeneration would be necessarily insignificant to a patient, whose ability to breathe without mechanical ventilation or to transfer independently from bed to chair might only require short lengths of axonal growth coupled with remyelination and an as yet undefined degree of native plasticity within the cord. Simply put, the goalposts must be set realistically, but with cautious optimism. Yet the inertia created by collaboration among basic scientists and clinicians with an appreciation of both the complexity of the biologic problem and the necessity for combination therapies will undoubtedly translate into clinically useful therapeutic strategies for this devastating injury in the near future.

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Key Points

  • Neuroprotective strategies to minimize secondary damage by maintaining axonal function and preventing cell death will undoubtedly play a role in future therapies, particularly as our understanding of the pathophysiology of acute SCI increases.
  • Neurons of the mature CNS in general have limited regenerative capacity. The activation or up-regulation of a battery of regeneration-associated genes appears to be necessary for axonal regeneration and can enhance this capacity.
  • Neurotrophic factors have widespread effects throughout the nervous system and will likely play a role in promoting neurologic recovery either alone or, more likely, in combination with other strategies.
  • The glial scar that forms at the site of injury and the myelin distal to it impose important impediments to axonal regeneration. The molecules responsible for these inhibitory effects and the molecular pathways within the growth cone that mediate this inhibition are potential targets for therapeutic intervention.
  • Cellular substrates that bridge the injury site aim to promote axonal growth and/or provide neuronal relays. Although facilitating axonal growth into the cellular graft seems readily achievable, olfactory ensheathing glial cells appear to possess the unique capacity of escorting the regenerating axons out of the graft and back into the otherwise inhibitory CNS.
  • The overwhelming majority of experimental therapies are being developed in acute injury models, raising questions about their applicability in the large population of chronically injured patients. Although the effectiveness of many of these strategies appears to decline as time elapses from injury to intervention, it may be possible to restore the regenerative capacity of chronically injured neurons by stimulating the re-expression of those genes responsible for promoting axonal growth.
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Keywords:

animal models; neuroprotection; axonal regeneration; growth cone guidance; myelin inhibition; cellular bridging]Spine 2001;26:S13–S22

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