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Focus Paper

Strategies to Promote Neural Repair and Regeneration After Spinal Cord Injury

Kwon, Brian K. MD, PhD, FRCSC; Fisher, Charles G. MD, MPH, FRCSC; Dvorak, Marcel F. MD, FRCSC; Tetzlaff, Wolfram MD, PhD

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doi: 10.1097/01.brs.0000175186.17923.87
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The permanence and irreversibility of the paralysis associated with spinal cord injury have been recognized and accepted for thousands of years,1 with hope emerging in only the latter part of the 20th century that advances in our scientific understanding of the neurobiology of spinal cord injury might produce therapies for this devastating condition.2 The sequelae of spinal cord paralysis are most profoundly manifested in the loss of voluntary motor, sensory, urologic, and sexual function, but significant abnormalities of the respiratory, cardiovascular, gastrointestinal, and musculoskeletal systems are common.3 In adult patients, mechanical failure of the spinal column in association with nonpenetrating spinal cord injury can lead to progressive kyphotic, and less commonly, scoliotic deformity.4 In skeletally immature patients, spinal cord injuries are associated with a high incidence of progressive deformity, with chronic spinal column instability, asymmetric growth arrest, and neuromuscular imbalance contributing to the paralytic collapse of the spine. The role of neuromuscular imbalance is demonstrated by the near 100% incidence of scoliosis in children paralyzed before the age of 10 years or before the adolescent growth spurt,5,6 a statistic that in itself represents the relevance of emerging therapies for spinal cord injury in the discussion of spinal deformity and its management.

Current Areas of Focus in Spinal Cord Research

The mechanical forces imparted to the spinal cord during fractures and dislocations of the spine disrupt the cord’s local neuroglial architecture (the “primary” damage) and initiate a complex pattern of acute pathophysiologic processes that are thought to exacerbate the derangement at and around the epicenter of injury (the “secondary” damage) (reviewed by Kwon et al7). It is well recognized that such nonpenetrating injuries to the spinal column rarely result in complete transection of the spinal cord. Even in individuals who are deemed to have functionally “complete” spinal cord injuries graded as “A” by the American Spinal Injury Association (ASIA), the chronically injured spinal cord remains anatomically in-continuity and is often characterized by a peripheral rim of intact tissue encircling a cystic cavity,8,9 a gross morphology that can be reasonably reproduced in animal models of a dorsal contusion spinal cord injury (Figure 1).

Figure 1
Figure 1:
Gross histology of the spinal cord after contusion injury. This figure depicts the damage to a rat spinal cord (Sprague-Dawley) 12 weeks after a dorsal contusion injury (Ohio State University Impactor, 1.2 mm injury) at the injury epicenter (C), 3 mm away (B), and 6 mm away (A) (myelin-stained with Luxol Fast Blue, counterstained with hematoxylin and eosin). Note how the injury epicenter (C) is characterized by a peripheral rim tissue encircling a cystic cavity, and that 3 mm away from this (B) the cord still remain grossly disrupted, with little gray-white matter distinction, before assuming a more normal appearance (A). Neuroprotective interventions are aimed at minimizing the extent of damage seen in B and C. The appearance of the injury epicenter at C represents the difficult environment that must be traversed by growing axons in order for functional reconnections to occur across the injury site.

The implications of this observation of the cord histomorphology are numerous. One is that if the entire spinal cord at the site of impact does not completely succumb to the initial mechanical injury and subsequent secondary pathophysiologic processes, then an intervention that attenuates these processes and maximizes the extent of this spared tissue could potentially result in improved neurologic function. Cautious optimism for such a strategy is derived from the observation in both humans9,10 and animals11–13 that some distal cord function can in some cases be mediated by surprisingly small amounts of spared spinal cord at the injury site, documented at less than 10% in some of these studies (Figure 2). Such is the rationale for neuroprotective strategies after acute injury, of which methylprednisolone is the most widely recognized, administered, and contentiously debated pharmacologic agent. Such pharmacologic agents will be further discussed.

Figure 2
Figure 2:
The MRI evolution of cord injury and sparing at injury site. This 48-year-old man was struck by a motor vehicle while riding his bicycle, suffering a C3–C4 fracture-dislocation and severe spinal cord injury requiring immediate intubation for respiratory failure. Note the severe edema and hemorrhage within his spinal cord on the T2-weighted MRI sagittal (A) and axial (B) views taken 18 hours after injury. Six weeks after posterior decompression and fusion, his imaging was repeated (C and D), having surprisingly regained Grade 4 out of 5 strength in his left quadriceps, ankle dorsiflexors, and plantarflexors. Note the peripheral sparing of spinal cord tissue at the injury site along the left side of the cord (arrow) and the cystic cavity that has already encompassed the remainder of his cord. The fact that this extent of spinal cord tissue can mediate near-normal strength in the lower extremity provides a compelling rationale for neuroprotective therapies that maximize this spared tissue.

The second implication is that, while some tissue may remain spared at the injury site, the spinal cord environment that is established over time, to even the untrained eye (Figure 1), appears extremely nonpermissive to axonal growth. This environment embodies a major challenge for neural regeneration, and as such is the focus of much neuroscientific study that aims to characterize the molecular obstacles and inhibitors to axonal regeneration and develop means of circumventing them. For example, the strategy of transplanting cellular substrates such as stem cells into the injured spinal cord aims to provide a more permissive and encouraging environment for axonal growth. Research into the challenges of axonal regeneration has identified not only external obstacles to axonal growth but also the low “intrinsic” regenerative competence of central nervous system (CNS) neurons that contributes to the failure of neural repair after spinal cord injury. These extrinsic and intrinsic impediments to axonal regeneration will be discussed further as well.

Finally, if some cord tissue is typically spared, even after the secondary damage induced by the pathophysiologic processes initiated acutely after injury, then a substrate exists for strategies that might enhance this remaining tissue’s ability to mediate distal function. For example, axons that traverse the injury site but have been stripped of their myelin sheath are the target of potassium channel blocking agents such as fampridine (Acorda Therapeutics, Hawthorne, NY) which, by altering the voltage potential across the axonal membrane, facilitates impulse conduction. Transplantation of cells that could remyelinate axons also has the potential to improve signal transmission across the injury site. Furthermore, if these precious few axons that survive and traverse the injury site could be induced to extend collateral sprouts that might reinnervate distal or even local neural circuitry, some functional recovery could be achieved. Such “plasticity” is postulated to be one of the principal effects of physical rehabilitation. It may indeed also be chiefly responsible for the functional recovery that is observed in animal models with experimental axonal regeneration therapies (although it is, of course, hoped that such therapies would produce long-distance axonal regeneration from axons that are interrupted at the injury site).

Within this framework of neural repair challenges, it is important that realistic expectations are maintained in the research and clinical communities vis-à-vis the achievability of a “cure” for paralysis. Clearly, we are at an exciting time for repair strategies, as a number of international clinical trials have been initiated; however, even the most promising axonal regeneration and neuroprotective strategies alone or in combination are almost certainly not going to restore a complete quadriplegic individual to normal physical function. Nevertheless, the process must begin somewhere, and even short distances of axonal regrowth or small victories in neuroprotection have the potential to make an enormous impact on the quality of life for patients with spinal cord injury. With this in mind, we first discuss the acute pathophysiologic responses to spinal cord injury and neuroprotective strategies, past, present, and future, that act to attenuate them. We then discuss the obstacles to axonal regeneration after spinal cord injury and how these may be overcome with therapies such as neurotrophic factors and cellular transplantation.

Pathophysiologic Processes That Contribute to Secondary Injury

Laboratory research in spinal cord injury uses a large number of animal models in which the spinal cord is subjected to either a blunt or sharp injury. Examples of the former include contusion injuries induced by a dorsally applied force (weight drop14–16 or impactor17,18), clip compression,19,20 and more recently, lateral spinal dislocation.21 Because of their conceptual resemblance to human nonpenetrating injuries, these models are most useful for studying pathophysiologic processes and neuroprotective strategies after spinal cord injury.22

These models have depicted an increasingly complex array of secondary pathophysiologic processes that are rapidly initiated after the mechanical injury to the spinal cord. Intraparenchymal hemorrhage, disruption of the blood–brain barrier, thrombosis, vasospasm, and the loss of pressure autoregulation are vascular aberrations that are intimately linked to the development of local ischemia, a fundamental component of secondary damage in the injury penumbra.23 Concomitantly, an influx of inflammatory cells, including neutrophils and macrophages, the activation of microglia, and the expression of cytokines such as IL-1β, TNFα, IL-6, LIF, and a myriad of matrix metalloproteinases contribute to the secondary injury process.24–28 Free radical generation and lipid peroxidation can propagate the local destruction of cellular membranes, and the oxidative stress can fatally disrupt intracellular molecular and biochemical homeostasis of neurons and glial cells.29,30 Such cells are also subjected to excitotoxicity caused by the extracellular release and accumulation of glutamate and the activation of N-methyl-D-aspartate (NMDA) and non-NMDA receptors.31 These components of the secondary injury cascade, i.e., microvascular ischemia, inflammation, oxidative stress, and excitotoxicity, are not mutually independent but rather can synergistically act to promote further destruction through the necrotic and apoptotic demise of neurons and glial cells. They are mentioned only briefly here, as all have been recognized not only as important components of the secondary injury response, but also potential targets for intervention.

Neuroprotective Interventions

The concept of neuroprotection is based on the premise that attenuating these aforementioned pathophysiologic processes will improve the ultimate neurologic outcome. A substantial amount of data from animal studies supports this concept, but effective neuroprotection in human spinal cord injury has been exceedingly difficult to demonstrate. Improvements over the past 40 years in protocols for trauma stabilization, immobilization, and resuscitation are thought to be neuroprotective insofar as the proportion of complete spinal cord injuries appears to have decreased with a concomitant increase in the proportion of incomplete injuries over that period of time.32 Early surgical decompression has for many years been postulated to improve neurologic outcome after traumatic spinal cord injury, although human data to this point have been contradictory and a large-scale prospective multicenter trial to demonstrate the effect of early decompression has only recently begun. Data from this trial, entitled the “Surgical Treatment of Acute Spinal Cord Injury Trial” (STASCIS), are as yet unavailable but will hopefully provide insight on this important question.

Methylprednisolone and Other Previously Tested Pharmacologic Agents

The publishing of the second National Acute Spinal Cord Injury Study (NASCIS 2)33 in 1990 was heralded as the first convincing demonstration of a pharmacologic neuroprotection in humans, but more recent critiques34–36 of the execution and interpretation of this and the subsequent NASCIS 337 have led many prominent orthopedic, neurosurgical, and emergency medicine societies to speak out against methylprednisolone as a standard of care for individuals with acute spinal cord injuries.38,39 Additional pharmacologic agents that have been trialed in human spinal cord injury but have not demonstrated significant clinical efficacy include GM-1 ganglioside (Sygen),40 naloxone,33 thyrotropin releasing hormone,41 nimodipine,42 tirilazad mesylate,37 and gacyclidine (as yet unpublished negative results of a 280-patient prospective randomized trial43). The failure to demonstrate convincing efficacy in these human spinal cord injury trials stands in resounding contrast to the very promising results reported for each of these agents in preclinical animal studies (and for GM-1 ganglioside, even a small clinical study44). This fact serves as a sobering reminder of the important differences that exist between the human and animal condition, and should temper the enthusiasm for making strong predictions about the efficacy of neuroprotective agents based on animal results alone.

The debate over the merits of methylprednisolone has certainly pointed out the need for the development of more effective neurotherapeutic agents. While many compounds are evaluated in the laboratory setting, of particular interest to clinicians are those agents that are currently in widespread use for other human conditions that, almost surreptitiously, have been found to have neuroprotective properties. Human experience with such agents would therefore overcome many important safety and tolerance issues that would be understandably enormous for a trial on, for example, viral-mediated gene therapy. Leading the list of such agents are the antibiotic minocycline and the hormone erythropoietin.

Minocycline

Minocycline is a tetracycline derivative that is currently in common clinical use for the treatment of acne and chronic periodontitis. In addition to its broad spectrum of antibacterial activity, minocycline has been found to have a number of neuroprotective properties,45 including the inhibition of matrix metalloproteinases,46 the inhibition of microglial activation47–49 (both considered to be important aspects of neuroinflammation), and the prevention of programmed cell death.50–53 These neuroprotective properties have been demonstrated in promising results from animal models of ischemic stroke,54,55 Parkinson’s disease,56,57 Huntington disease,58,59 and amyotrophic lateral sclerosis.60–62 Such promising results have prompted the initiation of minocycline clinical trials for patients with Parkinson’s disease,63 Huntington disease,64 and amyotrophic lateral sclerosis.65 One should be aware, however, that not all studies of minocycline in this regard have been positive, and that negative or even deleterious effects have also been reported in animal studies of these neurologic disorders,66–69 illustrating the need for caution in the development of minocycline as a neuroprotective agent.70

Despite these conflicting reports, a number of laboratories have independently reported beneficial effects of minocycline in acute spinal cord injury. The first demonstration of this came from Wells et al in 2003, who observed that minocycline administration 1 hour after a spinal cord compression injury resulted in increased axonal sparing at the injury,71 results similar to those found by Lee et al after a contusion spinal cord injury.72 Stirling et al reported that minocycline treatment after a dorsal column transection reduced the apoptotic demise of oligodendrocytes and the activation of microglia/macrophages, and diminished axonal dieback from the injury site.53 The reduction in oligodendrocyte apoptosis was also reported by Teng et al, who demonstrated that minocycline inhibits cytochrome c release at the injury site.73 Importantly, all four of these studies reported that minocycline administration was associated with improved behavioral outcomes on locomotor and other functional testing. It is worth pointing out that the reproducibility of the minocycline effect by independent laboratories using different animal models of spinal cord injury, different dosing regimens, and different outcome measures is rather unique in the neuroscientific study of therapeutic agents, and lends credence to the potential efficacy of minocycline in this context. A pilot study to evaluate minocycline in patients with acute spinal cord injuries has been initiated in Calgary, Alberta, Canada.

Erythropoietin

Erythropoietin, a hormone produced mainly by the kidney in response to hypoxia, has long been recognized for its role in regulating erythropoiesis and is currently in widespread clinical use for the treatment of anemia related to such scenarios as chemotherapy, chronic renal failure, and autologous blood donation. It has also been subsequently discovered to have tissue-protective properties in animal models of stroke,74,75 multiple sclerosis,76 Parkinson’s disease,77 myocardial infarction,78,79 and spinal cord injury. In an ischemia model of spinal cord injury (induced by aortic occlusion), erythropoietin was found to prevent motoneuron apoptosis and to promote functional recovery.80 Neuroprotection after traumatic spinal cord injury was first demonstrated by Gorio et al, who found that after either a clip compression or a contusion injury to the rat spinal cord, the administration of erythropoietin immediately after injury promoted improved locomotor scores and tissue sparing at the injury site.81 Kaptanoglu et al subsequently demonstrated a dose-dependent reduction in lipid peroxidation and improved histologic appearance of the injury site with erythropoietin after contusive spinal cord injury.82 It is of interest that, while the inhibition of lipid peroxidation has been proposed as the major mechanism of action for methylprednisolone after spinal cord injury, in this study, the reduction of lipid peroxidation was significantly better in animals treated with high-dose erythropoietin than with the NASCIS 2 bolus dose of methylprednisolone (30 mg/kg).

A potential problem with the use of erythropoietin as a neuroprotective agent is the possibility that the stimulation of erythropoiesis (particularly in a nonanemic patient) could lead to an undesirable erythrocytosis and a prothrombotic state. In practical terms, this may not be a substantial problem for a short-term application of erythropoietin (such as acutely after spinal cord injury). Within this context, however, it has been recently demonstrated that erythropoietin may be modified in such a manner as to relinquish its erythropoietic properties but yet maintain its tissue-protective characteristics.83 Further study has demonstrated that the tissue-protective properties of erythropoietin may be mediated by different receptor interactions and intracellular signaling pathways than the erythropoietic properties.84 Most promisingly, a small prospective randomized double-blinded study of human patients with acute strokes revealed that a once daily injection of erythropoietin for 3 days was well tolerated and led to improved neurologic function and infarct size at an early time point.85 These animal and human results would strongly suggest that a clinical trial for spinal cord injuries is warranted.

Axonal Regeneration Therapies

The poor axonal regeneration and functional recovery after blunt spinal cord injury stand in stark contrast to the fairly robust axonal regeneration and functional recovery observed in even transected peripheral nerves after primary repair. The biologic factors that account for these differences are the subject of much research. In very simple conceptual terms, the success of neurons within the peripheral nervous system to regenerate is influenced by their ability to upregulate genetic programs necessary for axonal growth, and by the permissive growth environment provided by the Schwann cells within the injured peripheral nerve. Conversely, neurons within the CNS often fail to upregulate or sustain the expression of such genes as a reflection of their “intrinsic” growth incompetence, and must extend new axons into the inhibitory CNS environment.86 Therapies to promote axonal regeneration within the CNS therefore focus on either augmenting the neuron’s intrinsic ability to regenerate, or attenuating the inhibitory CNS environment into which they must regrow.

Augmenting Intrinsic Growth Propensity With Neurotrophic Factors

A commonly used approach for stimulating axonal growth within the injured CNS is the administration of neurotrophic factors. Neurotrophic factors are proteins that exert considerable influence on a wide spectrum of processes within the developing and mature nervous system, including neuronal survival, axonal growth, synaptic plasticity, and neurotransmission (reviewed by Tuszynski87) Since the identification of the first neurotrophic factor, nerve growth factor (NGF) by Levi-Montalcini and Hamburger over 5 decades ago,88,89 dozens of such factors have been uncovered. It is important to recognize that the extreme diversity of neurotrophic factors and their functions, in addition to the complex cytoarchitecture of the spinal cord, make it unrealistic to expect that the administration of a single trophic factor will, by itself, elicit the comprehensive regenerative response in all relevant neuronal and glial cell populations necessary for full recovery after spinal cord injury. While the induction of ectopic bone formation to facilitate spinal fusion appears to be achievable with the exogenous administration of single growth factors of the bone morphogenetic protein family (e.g., the commercially available forms of BMP-2 or BMP-7), the situation within the injured spinal cord is clearly more complex.

Like bone morphogenetic proteins, however, the biologic activity of neurotrophic factors depends on the target tissue possessing the appropriate receptors, and the presence of such receptors is an important consideration for the therapeutic application of exogenous growth factors. This has been well demonstrated in the rat rubrospinal system (a motor control system) after acute and chronic spinal cord injury. Following a cervical spinal cord injury in which the rubrospinal tract is cut, the immediate administration of brain derived neurotrophic factor (BDNF) into the spinal cord injury site promotes significant rubrospinal axonal regeneration and prevents axotomy-induced atrophy and/or death of rubrospinal neurons.90,91 This indicates that the acutely injured rubrospinal axons at the spinal cord injury site are responsive to BDNF and thereby conduct the appropriate intracellular signaling pathways to augment the intrinsic growth propensity of these CNS neurons. However, such a regenerative response is not elicited in the rubrospinal neurons if the administration of BDNF into the injury site is delayed by 6 to 8 weeks after injury.92,93 This loss of effectiveness in the “chronic” setting can be explained by the observation that the injured rubrospinal axons at the spinal cord injury site lose, over time, the full-length trkB receptors necessary to mediate BDNF activity (such receptors are present on uninjured rubrospinal axons and are thus presumed to mediate the effect of BDNF applied immediately after injury).94 Conversely, BDNF applied directly to the rubrospinal cell bodies within the brainstem either acutely95 or even 1 year after injury96 elicits a reversal of axotomy-induced neuronal atrophy, an upregulation of genes important for regeneration, and the promotion of rubrospinal axonal growth. Consistent with this, full-length trkB receptors are maintained on the cell bodies of rubrospinal neurons even 1 year after cervical injury.96 While this is but one example of one neurotrophic factor (BDNF) and its receptor (trkB) within one neuronal system (rubrospinal system), it illustrates the principle that the development of effective therapeutic strategies that use the administration of neurotrophic factors will require an understanding of the biologic responsiveness of the target tissue, responsiveness that may in fact change with time and thus differ between the acute and chronic injury settings.

Augmenting Intrinsic Growth Propensity by Elevating Intracellular Cyclic AMP

With the demonstrated ability of neurotrophic factors to augment the growth potential of CNS neurons after injury, it is obviously of great interest to elucidate the intracellular mechanisms that mediate this effect. One mechanism that has stimulated substantial interest over the last 5 years is the elevation of intracellular cyclic adenosine monophosphate (cAMP). Neurite outgrowth from cerebellar neurons is typically inhibited when cultured in vitro on nonpermissive substrates such as CNS myelin or myelin-associated glycoprotein (MAG). Filbin et al demonstrated, however, that this inhibition could be overcome and neurite growth could be facilitated by “priming” such neurons with an overnight exposure to the neurotrophic factors BDNF or glial-derived neurotrophic factor before plating them on the nonpermissive substrates.97 This promotion of the neuron’s ability to overcome the nonpermissive environment was mediated by a rise in the intracellular cAMP levels and could be reproduced by the exogenous application of dibutyryl cAMP (db cAMP), a cAMP analogue.

Subsequent in vivo experiments revealed that the injection of db cAMP into the dorsal root ganglions promoted the regeneration of the centrally projecting axons within the spinal cord after a dorsal column spinal cord injury, again demonstrating that the elevated cAMP levels within the neurons allowed them to grow axons over even long distances within the inhibitory CNS environment.98,99 Three recent in vivo studies in which the administration of cAMP (or alternatively, the inhibition of its hydrolytic breakdown) was combined with the of Schwann cells,100 bone marrow stromal cells,101 or embryonic spinal tissue102 into the injured spinal cord have demonstrated that the elevation of cAMP is an important determinant of the neuronal growth propensity and may facilitate both axonal regeneration and possibly even functional recovery after spinal cord injury. These studies demonstrate, in principle, the potential efficacy of using a “combinatorial” therapeutic strategy of integrating different treatment paradigms. An example of this would be elevating intracellular cAMP to augment the growth ability of the neuron, plus cell transplantation to provide a better growth environment for regenerating axons. It is widely thought that effective therapies for spinal cord injury in the future will require a combinatorial approach such as this.

Nonpermissive Environment of the Injured Spinal Cord: Myelin and the Glial Scar

As alluded to previously, the other conceptual obstacle to axonal regeneration after spinal cord injury is the inhibitory environment into which new axons must grow. A tremendous amount of research over the past two decades has gone into identifying the inhibitory CNS molecules that make the spinal cord less permissive to axonal regeneration than peripheral nerves. The two major impediments to axonal regeneration in the injured spinal cord are CNS myelin and the glial scar that is established at the injury site.

The full complement of inhibitory elements that reside within CNS myelin and obstruct axonal regeneration is currently unknown. The three best characterized inhibitory constituents include the protein called Nogo,101,103–105 MAG,106 and oligodendrocyte-myelin glycoprotein (OMgp)107 (reviewed by Grados-Munro and Fournier108). The discovery of the receptor for Nogo in 2001109 was followed by the somewhat surprising finding that this receptor also interacts with MAG and OMgp to mediate some of their inhibitory properties as well.110 This has prompted much interest in the Nogo receptor and its subsequent intracellular signaling as a potential target for therapeutic interventions, interventions that, by altering this single pathway, might attenuate the axonal growth-repulsive properties of multiple inhibitory molecules (i.e., Nogo, MAG, and OMgp).111 As proof of this concept, Strittmatter et al demonstrated that the intrathecal administration of a portion of the Nogo receptor that competitively binds to and neutralizes Nogo, MAG, and OMgp promoted axonal sprouting and functional recovery after a partial spinal cord injury in rats.112 It is worth noting, however, that despite increasing interest in Nogo and mounting enthusiasm for developing human therapies that target this molecule, studies on Nogo-deficient transgenic mice performed by three independent laboratories have come to contradictory conclusions regarding the in vivo importance of Nogo as an inhibitor of axonal growth.113–115 Furthermore, a very recent study of transgenic mice lacking the Nogo receptor demonstrated that neurite outgrowth of certain populations of cells was not enhanced, as one would expect in the absence of this receptor.116 Clearly, these contradictory observations emphasize the complexity of this biologic system and provide a compelling rationale for further investigation to better delineate the role of Nogo in spinal cord injuries before human translation.

The glial scar and cyst that become established at the injury site over time are thought to be the other major impediments to axonal regeneration after spinal cord injury. Some evidence exists to suggest that in the absence of glial scarring within the CNS, axonal regeneration can occur within CNS myelin,117 a finding that stirs some controversy as to whether the glial scar or CNS myelin is more responsible for inhibiting axonal regeneration after spinal cord injury (and is therefore the most attractive target for intervention). The predominant cellular constituent of the glial scar are astrocytes, which, in addition to ultimately forming a physical barrier to growing axons, also express a number of chondroitin sulfate proteoglycans (CSPGs), which are inhibitory to axonal growth (reviewed by Silver and Miller118).

A potentially promising therapeutic intervention to address the CSPG inhibition to axonal growth is to enzymatically degrade the proteoglycans, and in this regard, the enzyme chondroitinase ABC has received increasing attention. This enzyme removes a large proportion of the glycosaminoglycan chains from CSPGs, and the remaining protein core does not possess the same inhibitory properties as the intact macromolecule. The intrathecal infusion of chondroitinase ABC after a partial spinal cord lesion was reported to promote growth of corticospinal axons and result in functional recovery.119 The application of this strategy with cell transplantation therapies in order to attenuate the glial scar establishment at the host-transplant interface and thus facilitate the passage of axons through the graft and back into the spinal cord has also been successfully demonstrated.120

Cellular Transplantation

On observing the cystic cavity that often typifies the chronic spinal cord injury site, it is only intuitive to postulate that axonal regeneration could be facilitated by surgically filling that cavity with some form of growth-permissive cellular substrate. In this regard, a wide variety of cells have been investigated as therapeutic candidates for spinal cord injury, some of which have more recently been trialed in human patients. Primary candidates for such transplantation strategies include stem cells, bone marrow stromal cells, fetal tissue, Schwann cells, and olfactory ensheathing cells. It is worth noting that not all cell transplantation strategies are necessarily aimed at achieving the same thing. The undifferentiated nature of stem cells, for example, makes it possible for them to mature down neuronal or glial lineages, raising the potential for them to either form neurons to relay synaptic information across the injury, or to form glial cells that might, for example, remyelinate growing axons. Alternatively, the transplant may serve mainly to provide a favorable growth substrate for new axons and to subsequently remyelinate them to restore efficient conduction of action potentials (e.g., Schwann cells and olfactory ensheathing cells). In addition to their native properties, cells may be genetically modified ex vivo in order to confer specific properties that might be beneficial when transplanted, such as the augmented expression of neurotrophic factors.121 Because of recent developments and human experience with cell transplantation for spinal cord injury, this review will focus on only two of the many cellular substrates: Schwann cells and olfactory ensheathing cells.

Schwann Cells and the PNS-CNS Barrier

Schwann cells have long been recognized as the key cellular constituent of peripheral nerves that facilitates axonal regeneration. Indeed, seminal studies in the field of spinal cord research used this permissive growth environment, either as free-ending peripheral nerve transplants122 or as intercalary grafts bridging a completely transected spinal cord injury,123 to demonstrate axonal regeneration of CNS neurons after spinal cord injury. Further studies have shown that, in principle, this facilitation of axonal growth can be augmented by administering neurotrophic factors124 or by genetically manipulating Schwann cells to increase their neurotrophic factor production,125–127 again reflecting the potential efficacy of combinatorial therapeutic strategies.

A case report of the utilization of peripheral nerve transplantation for human chronic spinal cord injury was recently published by Cheng et al.128 The lead author, well known for his work with peripheral nerve transplants in acute123 and chronic129 rat spinal cord injury, used a similar approach by taking grafts from intercostal nerves and transplanting them into the spinal cord injury site of a 24-year-old man who, at the age of 20 years, had had a left-sided spinal cord hemisection in a stabbing incident. The authors reported an improvement in ASIA motor scores and motor-evoked potentials 2 years post-transplantation in this individual, although it is difficult to know how much of the recovery was attributable to the transplant versus the detethering and cyst decompression of his cord. A similar study in which autologous sural nerves segments have been used to bridge the spinal cord injury of 8 patients with complete spinal cord injuries from gunshot wounds has been performed by Dr. Tarcisio Barros at the University of Sao Paulo. Five-year follow-up results have suggested no improvement in neurologic function or electrophysiologic parameters.130

While many potential explanations exist for the limited success with peripheral nerve transplantation in these human reports, much animal literature has demonstrated that CNS axons that regenerate into such grafts are typically reluctant to leave the permissive PNS conditions and reenter the host, inhibitory CNS environment.131 This “PNS-CNS barrier” is likely related to the establishment of the glial scar and the presence of inhibitory myelin elements (discussed above) at the graft-host interface. Consistent with this, a recent study that combined Schwann cell transplants with chondroitinase ABC to attenuate this gliotic scar demonstrated that axons growing through the graft could indeed regenerate farther into the host CNS environment.120 This notwithstanding, the PNS-CNS barrier that inherently encumbers peripheral nerve and Schwann cell transplants has directed a great deal of attention toward a cell that appears capable of accompanying regenerating axons across this obstacle: the olfactory ensheathing cell.

Olfactory Ensheathing Cells

Sensory neurons within the olfactory epithelium (considered to be in the peripheral nervous system) are unique in that throughout adult life, they undergo a constant turnover and thus regenerate new axons that must extend through the cribriform plate and reconnect with second-order neurons in the olfactory bulb (considered to be in the inhibitory CNS). Olfactory ensheathing cells (OECs) are distinct glial cells that escort these growing axons across this PNS-CNS interface, and this property was identified as a potential solution for the problem encountered with Schwann cell and peripheral nerve transplants. The possibility that such cells could be harvested from an individual with a spinal cord injury, cultured in large quantities, and then transplanted into the same individual without risk of immunologic rejection gives them similar conceptual appeal as Schwann cells.

An important paper by Ramon-Cueto et al in 2000 accelerated international interest in what was already an expanding line of research into the therapeutic properties of OECs for CNS repair.132 These investigators reported that the transplantation of OECs into a complete spinal cord transection facilitated long-distance regeneration of corticospinal, noradrenergic, and serotonergic fibers across the lesion and into the distal spinal cord, observations that were associated with significant functional recovery. A large body of literature now exists on the use of OECs in spinal cord injury (reviewed by Barnett and Riddell133). While much of it is promising, the work from different laboratories using different techniques of cell acquisition, expansion, purification, and implantation into different animal models at different time points after injury has left many important questions to be answered.

Nonetheless, the human application of putative OECs has already begun in a number of centers around the world. These include Beijing, China, Lisbon, Portugal, and Brisbane, Australia, under the direction of Drs. Huang, Lima, and Mackay-Sim, respectively. For a more complete discussion of these and other clinical trials for spinal cord injuries, please see Steeves et al.130 It is important to note that only the project of Dr. Mackay-Sim in Australia has been designed as an epidemiologically sound Phase I clinical trial to evaluate the safety of purified OEC transplantation with four OEC and four placebo-treated patients and regularly scheduled assessments with blinded evaluators. Dr. Huang’s approach employs olfactory cells obtained from human fetuses aborted at 12 to 16 weeks’ gestation and, given the lack of strict inclusion criteria, placebo controls, nonblinded assessments, and standardized follow-up evaluations is more akin to an experimental treatment than a clinical trial. By February 2004, he had performed this transplantation on more than 300 patients with spinal cord injuries, and throughout the year has received increasing worldwide attention. Follow-up of these patients, and in particular, a reporting of safety and adverse events, has not been systematically and comprehensively documented. Dr. Lima’s trial in Portugal evaluated autologous transplants of olfactory mucosa in motor complete patients (ASIA A or B) and again lacks placebo controls and blinded assessment. Formal peer-reviewed reports of these human trials are anxiously awaited.

Conclusion

An accelerating pace of scientific discovery over the past three decades that has generated much hope for patients, scientists, and clinicians that a “cure for paralysis” will someday become a reality. While laboratory research in animal models of spinal cord injury has generated a number of promising experimental therapies, it has also revealed a daunting complexity of the neurobiologic challenges that impede neural repair after injury. A greater understanding of these obstacles will be necessary for the further development of therapeutic strategies. This review describes many encouraging reports of novel neuroprotective agents such as minocycline and erythropoietin, and a number of promising strategies for promoting axonal regeneration. These serve as the foundation for the cautious optimism that exists for the future, that in our generation we will see the emergence of truly effective therapies for patients with spinal cord injuries. The initiation of human trials for experimental therapies that have been extensively studied in the laboratory setting represents the forward movement of this knowledge but also speaks loudly for the need for these technologies to now be subject to epidemiologically sound and rigorous clinical evaluation. As the list of therapies available for human testing grows, equipoise will be necessary in this field of “spinal cord regeneration” that has traditionally been viewed as the realm of laboratory researchers but now demands leadership from clinicians with interest and expertise in clinical research methodology.

Key Points

  • An increased understanding of the acute pathophysiologic processes initiated after spinal cord injury is essential to the development of neuroprotective therapies.
  • While methylprednisolone remains a treatment option for acute spinal cord injury, a number of promising neuroprotective agents are being developed, many of which are already in clinical use for other indications.
  • Axonal regeneration of neurons within the central nervous system is limited by both their limited intrinsic capacity to regenerate and the inhibitory environment within the injured spinal cord.
  • Many experimental therapies are emerging from the laboratory as potential candidates for pilot clinical trials; spinal physicians will have an increasingly important leadership role in the design and execution of such trials.

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Keywords:

spinal cord injury; neuroprotection; axonal regeneration; minocycline; erythropoietin; clinical trials; neurotrophic factors; olfactory ensheathing cells

© 2005 Lippincott Williams & Wilkins, Inc.