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Journal of N eurological P hysical Therapy From Animal Models to Humans: Strategies for Promoting CNS Axon
Regeneration and Recovery of Limb Function after Spinal Cord Injury
Lawrence Moon,1 Mary Bartlett Bunge1,2 ABSTRACT
CNS axon tracts and recovery of limb function in animal There are currently no fully restorative therapies for human spinal cord injury (SCI). Here, we briefly review the Although beyond the scope of this review, there are different types of human SCI pathology as well as the most other extremely important efforts aimed at restoring nor- commonly used rodent and nonhuman primate models of mal function after SCI. For example, following the initial SCI that are used to simulate these pathologies and to test insult to the spinal cord, further structure and function is potential therapies. We then discuss various high profile lost through active secondary processes.2 Substantial effort (sometimes controversial) experimental strategies that have has been devoted to limiting this secondary damage reported CNS axon regeneration and functional recovery of through development of neuroprotective measures.3 limb movement using these animal models of SCI. We par- Substantial effort also has been dedicated to improving ticularly focus upon strategies that have been tested both in upon the small degree of endogenous repair that proceeds rodents and in nonhuman primates, and highlight those spontaneously in the spinal cord.4,5 For example, strategies which are currently transitioning to clinical tests or trials in for improving conduction through spared axons have been humans. Finally we discuss ways in which animal studies tested in animals and in humans.6 Strategies for treating might be improved and what the future may hold for physi- pain and sexual, bladder, or bowel dysfunction and auto- cal therapists involved in rehabilitation of humans with SCI.
nomic dysreflexia are also beyond the remit of this article.
Key Words: plasticity, SCI, regeneration, animal, human, ther- Readers are directed to excellent recent reviews of these fields in this volume and elsewhere.1,3,6,7 The present review will first outline different types of INTRODUCTION
human SCI and will discuss how contemporary animal Spinal cord injury (SCI) affects hundreds of thousands of models of SCI attempt to simulate these pathologies. Next, people worldwide, with massive associated health care and we will discuss cellular and molecular strategies for improv- ing CNS axon regeneration and recovery of limb function results most notoriously in flaccid paralysis and loss of nor- after SCI. Special consideration will be given to experi- mal sensation in the limbs below the level of the lesion.
mental strategies that have been evaluated in nonhuman Spinal cord injury may also lead to debilitating pain, spas- primates and/or are currently transitioning to clinical stud- ticity, impairments in breathing and coughing, bowel or ies or trials.7 We will highlight where strategies remain con- bladder problems, and reduced reproductive ability or sex- troversial, and where safety or efficacy barriers to translat- ual sensation.1 People with SCI may suffer from autonomic ing experimental strategies exist. The aim is to inform dysreflexia and may be at increased risk for stroke, decubi- readers of exciting laboratory advances, but to temper this tus ulcers, fractures, and depression. There are no fully with a realistic understanding of the challenges that remain restorative clinical therapies for SCI.
in developing effective and safe therapies for humans.
In attempts to develop therapeutic strategies for over- coming each of these sequelae, researchers often rely upon HUMAN SCI IS PATHOLOGICALLY HETEROGENEOUS
animal models of SCI. These enhance our understanding of The response of the human spinal cord to injury has the cellular and molecular response of the mammalian been studied using imaging techniques as well as by histo- spinal cord to injury, and they allow us to evaluate the safety logical staining and inspection of autopsy material.8 These and efficacy of potential therapies for improving outcome.
studies reveal 4 classes of lesion. In one study of 48 speci- Use of animal models has shown that dysfunction following mens,8 in 33% of cases, contusion injuries resulted in cavity SCI results from interruption of descending and ascending and cyst formation, typically with some sparing of white spinal axons and loss of both myelin and cells including matter (or glial tissue) but with the external glial limiting neurons, oligodendrocytes, and astrocytes.
membrane (the glia limitans) remaining intact. In 29% of regenerate following injury to the peripheral nervous sys- cases, massive compression or maceration occurred due to tem (PNS) but few, if any, axons regenerate long distances vertebral displacement whereas in 21% of cases, laceration following injury to the central nervous system (CNS).1 This occurred due to cord penetration by foreign bodies or bony review will discuss strategies for promoting regeneration of fragments. After compression, maceration or laceration, 1Post Doctoral Associate,The Miami Project to Cure Paralysis, Miami, FL ([email protected])2Professor, Departments of Cell Biology and Anatomy and Neurological Surgery, University of Miami Miller School of Medicine, Journal of N eurological P hysical Therapy substantial breaks in the glia limitans led to the epicenter phases, but here we prefer to specify precise numbers of being filled predominantly with connective tissue; cysts and hours, days, months, or years. The majority of animal stud- cavities were less prominent. In 17% of cases, solid core ies involve interventions within minutes, hours, or days of injuries manifested as either central cord syndrome (with injury. There are far fewer studies reporting improvements loss of myelin and axons, but with preservation of gray mat- in CNS axon regeneration or functional outcome after inter- ter), or as chronic cord compression (with loss of myelin vening more than one month postinjury23–28 and this imbal- and/or motor neurons in the epicenter, but with preserva- ance requires much redressing if CNS axon regeneration is tion of axonal integrity). The extent of demyelination, the to be promoted in individuals with long-standing injuries.
invasion of host cells and the deposition of growth- Additionally, the majority of studies only assess outcomes inhibitory molecules (see below) at sites of human SCI also over a period of weeks or months. Relatively few studies that report improvements in CNS axon regeneration and Various animal models of SCI have been developed in functional recovery go on to determine whether these order to model each of these different types of human changes remain stable beyond 2 or 3 months.
injury. The relative merits and limitations of these models Data from animal models show that only a small per- will be discussed next, since these directly affect the ability centage of CNS axons need to be spared to mediate rea- of the researcher to develop safe and effective strategies for sonable locomotion. Partial locomotion on a treadmill is improving CNS axon regeneration and motor function after possible following lesions that spare only 5% to 10% of axons in adult mammalian thoracic spinal cord.29,30 Forexample, following spinal cord compression in the adult ANIMAL MODELS OF SCI ARE DESIGNED TO
cat, remaining nerve fibers became myelinated by Schwann SIMULATE DIFFERENT TYPES OF HUMAN SCI
cells and were able to support spontaneous walking.31 The vast majority of mammalian SCI research is con- These data are encouraging as they suggest that only a small ducted using adult mice and rats, although some work has percentage of axons need to regenerate and form func- been done using cats and dogs, and increasing work is tional connections to restore useful function.
being done using nonhuman primates. Contusion injuries It is important to bear in mind that rodent models only in the human are typically modeled using devices that dis- approximate normal human locomotion and dysfunction place11-12 or impact13 the exposed spinal cord. Compression after SCI. Bipedal locomotion (humans and birds) differs injuries may be simulated by subdural insertion and infla- radically from quadrupedal locomotion (rodents and most tion of a balloon14 or by clip compression.15 Methods for mammals) or partially upright locomotion (nonhuman pri- inducing contusion via vertebral displacement have also mates) in terms of posture, kinematics, physiology, and recently been developed.16 Laceration injuries are usually anatomy. Further, whereas many studies show that rodent modeled by surgical incision, including complete transec- spinal cords respond to injury in ways similar to that of the tion,17,18 dorsal hemisection,19 or lateral hemisection.20 In all human,32,33 it is clear that there are differences in responses cases, the aim is to produce reproducible graded (or com- to SCI between species and even between strains of a given species.34 For example, whereas many human spinal cord The animal model selected by the researcher largely injuries become cystic or cavitated, very little cavitation depends upon the hypothesis to be tested. Researchers occurs following contusion injury to many strains of adult testing neuroprotective strategies often employ contusion mice.32 The histopathological response of the primate spinal or compression injuries. Those testing pro-regenerative cord to injury has been studied only relatively little, includ- strategies typically prefer models that involve transection of ing after injection of autologous fibroblasts into otherwise all tracts (complete) or some tracts (eg, dorsal column for uninjured rhesus monkeys35 and after contusion injury in sensory axons ascending in the fasciculi gracilis and cunea- rhesus monkeys.36–38 This situation is likely to improve since tus) as these allow one to distinguish regenerating axons a graded model of contusion injury in marmosets has from axons that were spared or that sprouted a collateral.21 recently been developed39 and a number of groups are now Cervical injuries are typically chosen to test therapies for testing potential therapeutics in nonhuman primates (see improvement of forelimb movement while thoracic injuries below). Given the differences between rodent and primate are usually selected to evaluate therapies for improvement nervous systems, it seems reasonable to test pro-regenera- tive therapies in nonhuman primates for safety and efficacy.
In the USA alone, a large number of people live with The paucity of safety and efficacy studies using nonhuman existing spinal cord injuries and a substantial number primates makes it somewhat remarkable therefore that so receive injuries each year (prevalence ~250,000; incidence many clinical studies and trials are in progress.7 ~11,00022). Different temporal opportunities to intervene In summary, researchers who test therapeutics designed therapeutically therefore exist. Thus the time between the to improve function after SCI select animal models that sim- injury and the time of treatment and of evaluation in an ani- ulate particular aspects of the human pathology. Animal mal model is of critical importance. ‘Acute,’ ‘subchronic,’ models indicate that axon regeneration by a small percent- and ‘chronic’ have all been used to describe postinjury age of injured CNS neurons may be sufficient to restore par- Journal of N eurological P hysical Therapy tial function. As will be reviewed next, factors intrinsic and grafts 6 months after implantation abolished electrophysio- extrinsic to neurons limit CNS axon regeneration.40,41 logical and behavioral recovery, interpreted to mean that Modulation of these intrinsic and extrinsic factors can pro- functional improvements were dependent on long tract mote CNS axon regeneration and recovery of function in regeneration. In another study, axons from the cortex and animal models of SCI. As will be described, these successes brainstem nuclei (including vestibular, red and raphe nuclei, are leading to clinical tests and trials.
and reticular formation) were also reported as regeneratingbeyond transplants into the distal spinal cord, accompanied EXTRINSIC FACTORS
by improvements in hindlimb function including instances Transplants of peripheral nerve promote CNS axon
regeneration and functional repair after SCI
Using a similar strategy, peripheral nerves have been Axon regeneration and functional recovery occurs spon- grafted autologously into nonhuman primates (cynomolo- taneously (if incompletely) after injury to the adult mam- gous monkeys) after lateral spinal hemisection20 with evi- malian PNS. Early experiments therefore attempted to pro- dence for regeneration of at least some spinal axons at 4 mote CNS axon regeneration by transplantation of PNS seg- months postinjury. No functional differences were detected ments into the mammalian CNS. Advances in retrograde trac- between control and experimental monkeys in this study.
ing techniques allowed the unambiguous demonstration that This approach also has been used to treat one human neurons in many (but not all) different regions of the CNS case of clinically complete SCI, with a report of limited can regenerate axons into PNS grafts.42 Transplants of periph- functional recovery.54 This study did not include control eral nerves as a strategy for promoting CNS axon regenera- subjects and therefore additional work remains to be done tion and functional repair are in part attractive because autol- to determine whether therapies involving peripheral nerve ogous grafts reduce the risk of immune rejection and obviate bridge grafting safely and effectively improve outcome after the consequent need for immunosuppression.
There are limits, however, on the ability of PNS grafts to promote axon regeneration and functional repair after SCI.
Transplants of Schwann cells promote CNS axon
In rat models of SCI, supraspinal axons (eg, from the red regeneration and functional repair after SCI
nucleus and from cortex) do not extend axons into periph- Axon regeneration through peripheral nerves is largely eral nerve grafts placed into thoracic sites of SCI.43–45 Further, due to the presence of growth-promoting Schwann cells CNS axons that do extend into peripheral nerve grafts rarely (SCs).55 While grafts of peripheral nerve are suited to bridg- exit grafts distally to form synapses or to restore function ing regular gaps in the injured human spinal cord, many of unless routed directly to appropriate targets.46 the cysts and cavities that often form after SCI are irregularly As a result of these limitations, combination strategies shaped and below the cord surface, and therefore less have been tested and found to enhance long distance CNS amenable to bridging with segments of peripheral nerve.
nerve regeneration and motor recovery following SCI and Purified SCs have therefore attracted attention as candidates implantation of peripheral nerve. For example, in adult rats, for promoting CNS axon regeneration and functional repair following creation of a gap in the dorsal columns, trans- after SCI because they can be transplanted as suspensions.
plants of peripheral nerve support regeneration of the cen- Additionally, use of mitogens allows rat or human (but, to tral branch of sensory axons from lumbar dorsal root gan- date, not mouse) SCs from small biopsies of adult peripheral glia (DRG)47 and some of these axons grow out of grafts nerve to be dramatically expanded in culture56 to produce when growth factors are infused rostrally.48,49 sufficient numbers to bridge large gaps in the spinal cord.57 In several studies, multiple segments of intercostal Purified populations of rat SCs from peripheral nerve peripheral nerve have been transplanted autologously fol- have been transplanted into rat models of SCI, injected as lowing complete thoracic transection and creation of a 5 suspensions 1 week after contusion injury,58 or implanted mm gap. Nerves were routed from white matter to distal within channels containing extracellular matrix (Matrigel or gray matter and secured using fibrin glue containing acidic fibrin) immediately following lateral hemisection59 or com- fibroblast growth factor. In studies using adult rats, corti- plete transection.18 After transection and implantation of cospinal (CST) axons regenerated through and beyond the SCs, sensory and spinal axons with cell bodies near the nerve bridges50 and hindlimb functional recovery was grafts extend axons into these bridge grafts, are myeli- reported.51 Importantly, this work recently has been repro- nated,18 and are electrophysiologically active.60 After contu- sion and implantation of SCs, less cavitation is observed and somatosensory and motor cortex-evoked potentials demon- sensory and spinal axons extend axons into grafts, and are strated recovery of electrophysiological connectivity from myelinated.58 In the transection model, regenerating axons motor cortex to hindlimb muscles and from sciatic nerves do not leave the grafts distally to reinnervate the host: com- to sensorimotor cortex.52 Extent of recovery in hindlimb bination therapies including SCs have therefore been the function in the open field correlated positively with motor evoked potential amplitudes in hindlimbs. In some cases, Various combinations of therapies have been reported weight supported stepping was reported. Transection of to enhance long distance CNS nerve regeneration and Journal of N eurological P hysical Therapy recovery of hindlimb function following SCI and implanta- their bodies up a wire grid inclined at 45º and onto a hori- tion of Schwann cells. Following transection of adult rat thoracic spinal cord, SCs have been transplanted with deliv- Primate72 and human OEG73 derived from olfactory bulb ery of growth factors including brain derived neurotrophic or lamina propria, are now being tested in rodent and non- factor (BDNF)61 or BDNF with neurotrophin-3 (NT-3).17 The human primate models of SCI and demyelination.74 Over SCs have also been transplanted in combination with anti- 500 transplants of cells from human fetal olfactory bulbs inflammatory steroids (methylprednisolone),62 and with have been performed in humans in China with reports of other regeneration-promoting cells including olfactory modest rapid improvement in function.75 Because in many ensheathing glia (OEG).63 Increased regeneration of CNS cases mild recovery of function is reported to occur within axons into and beyond bridges was reported in several of 1 or 2 days, it undoubtedly does not depend upon long dis- these studies. Following thoracic contusion in adult rats, tance regeneration of CNS axons but perhaps on secretion SCs compared to OEG were found to be less effective than of trophic factors or induction of local changes in circuitry.
To date, comprehensive follow-up studies have not been Purified populations of human SCs from peripheral performed, so it is difficult to gauge the long-term safety nerve have also been transplanted into the injured rodent spinal cord (within channels containing Matrigel).64 avoid rejection of grafted cells, nude rats (that have attenu- Do transplants of immune/inflammatory cells pro-
ated immune systems and were treated with methylpred- mote or impair CNS axon regeneration and func-
tional repair after SCI?
regeneration of brainstem-spinal axons into Schwann cell Spontaneous axon regeneration in the PNS also has been grafts was observed and propriospinal neurons regenerated partly attributed to the abundance of inflammatory cells up to 2.6 mm distal to grafts. Mild functional improvements including macrophages. Macrophages in the CNS derive were also reported, including extensive movements of predominantly from resident microglia but also come from ankles, knees, and hips (sweeping without weight bearing) blood-borne monocytes.76 Monocytes enter the CNS more and occasional weight bearing in stance. Weight-supported slowly and in fewer number than in the PNS and they differ stepping was only observed in a single rat.
phenotypically from monocytes that have entered the To date, SCs have not been transplanted into humans fol- PNS.77,78 For these reasons, it has been hypothesized that lowing SCI. A number of hurdles remain. Since transplanting transplants of monocytes preactivated by exposure to PNS SCs alone affords only a small level of recovery of function, it tissue may boost CNS axon regeneration and functional remains important to find the most effective and repro- ducible combination therapy involving SCs. It will also be In one study, homologous monocytes, activated by prein- important to ensure that transplantation of SCs is safe.
cubation with PNS tissue, were implanted immediately fol- Although mitogen stimulated human SCs do not form tumors lowing transection of adult rat thoracic spinal cord, result- after implantation into mouse PNS, it will be important to ing in improved CNS axon regeneration.79 Partial recovery check this after implantation into the injured spinal cord.65 of hindlimb movements was observed during open field One important step towards human clinical trials would be locomotor testing. Hindlimb muscle responses were also to test the safety and efficacy of transplanting autologous SCs evoked by cortical stimulation. Finally, loss of function after into nonhuman primates following contusive SCI.39 re-transection was taken to indicate a dependence of recov-ery upon axon regeneration. In a more recent paper, mono- Transplants of OEG promote CNS axon regeneration
cytes were activated by preincubation with skin, and func- and functional repair after SCI
tional recovery was reported following implantation into a Central nervous system axon regeneration and func- tional recovery also has been reported when OEG are trans- In stark contrast to these findings, several studies indi- planted immediately after SCI66–69 or after a delay of 426 or 8 cate that inflammatory cells aggravate outcome after SCI.
weeks.27 In one study using adult rats, following lateral Subpopulations of macrophages within the injured spinal hemisection of the cervical spinal cord, injection of OEG cord are found in regions that subsequently cavitate81 and improved respiratory function and enhanced performance intraspinal injection of a macrophage activator (zymosan) on a climbing task.69 In other studies using adult rats, fol- worsens cavitation.82 Finally, pharmacological depletion of lowing thoracic transection and implantation of OEG into macrophages at 1, 3, and 6 days after injury (using clo- spinal cord stumps, regeneration of long descending tracts dronate) actually improves functional recovery after contu- important for locomotion has been reported,26,68 even up to sion injury in adult rats.83 These data argue that transplan- 2.5 cm distally, for serotonergic axons.70 Recovery has also tation of inflammatory cells risks worsening the original been reported in adult rats after transection and implanta- injury and may increase disability after SCI.
tion of OEG when testing hindlimb function using an Transplantation of inflammatory cells for treatment of inclined plane.70 Between 3 and 7 months post-transplanta- SCI is therefore extremely controversial. Replication and tion, many transplanted rats were capable of propelling within-experiment comparison of these rodent studies by Journal of N eurological P hysical Therapy independent laboratories would be extremely valuable.
Fetal cell grafts slightly reduce neurological dysfunction Testing of autologous (not allografted), activated monocytes following human SCI.89 In one study, 41 patients with clini- would also be informative. To date, we know of no studies cally complete thoracic spinal cord lesions and progressive investigating the safety or efficacy of transplanting immune syringomyelia received multiple subpial grafts of human neo- or inflammatory cells into injured nonhuman primate cortex from 8 to 9 week old fetuses usually within 6 to 12 spinal cord. Despite this, clinical trials involving transplan- months of injury (cited in reference 89). A trend towards tation of autologous, activated monocytes are currently in improved neurological outcome over a few dermatomal lev- progress, supported by Proneuron Biotechnologies.7 els was observed relative to patients who had laminectomy,durotomy, and lysis of adhesions. There were no reports of Transplants of embryonic/fetal spinal cord promote
morbidity, mortality, or increased pain. However, it should be CNS axon regeneration and motor recovery after SCI
noted that as yet, there is no available evidence for graft sur- Fetal spinal cord transplants have been shown to pro- vival or CNS axon regeneration in humans. Fetal cell grafts mote regeneration of CNS axons following resection lesions have also been used in an attempt to prevent additional loss of adult rat spinal cord and in contused adult rat and cat of function in cases of progressive syringomyelia and to date spinal cord. A variety of host axons from distances up to 6 there appear to have been few negative outcomes associated mm away grow short (<500µm) distances into, and inner- with transplantation.93,94 Therapeutic strategies involving tis- vate, the grafts including serotonergic and coerulospinal sue derived from human embryos or fetuses remain ethically axons.84 Innervation of the host tissue by transplanted neu- controversial and therefore clinical trials are additionally rons is also observed. Despite the attenuation in scar for- mation induced by transplantation of immature tissue, thenumber of axons to cross the host/graft interfaces in either Transplants of progenitor cells may improve motor
direction is usually relatively small. Modest but significant recovery following SCI
functional recovery is observed following transplantation of Multipotent progenitor cells (cells that have the capacity immature tissue in rats84 and in cats 1 to 3 months after con- to differentiate into many different cell types) and stem tusion SCI.85 For example, although there was no improve- cells (cells having the capacity to self-renew indefinitely ment on inclined plane or grid walking up to 3 months and differentiate into any cell type) appear to exist in adults postimplantation, 2 indices of gait analysis (base of support as well as in embryos and these may have the potential to and stride length) showed statistically significant improve- replace cells of different types that die after SCI. Since it is ment.86,87 Improvements in locomotion were also observed in practice difficult (if not impossible) to establish that a following delayed transplantation of immature tissue into precursor cell is capable of indefinite division and of differ- contusion injured adult cat spinal cord and electrophysio- entiation into all possible phenotypes, here we prefer to use logical evidence suggested ascending host axons made the term progenitor. Transplants of purified progenitor cells synapses with graft neurons.88 However, recovery does not from embryos and adult tissues are now being tested in ani- appear to depend on long distance growth into, through mal models of SCI.95 The following cell types are among and beyond grafts and the authors of those studies specu- those that have been transplanted in animal models of SCI: lated that transplants behave as relays, affording transmis-sion of signals via the transplanted neurons which are human umbilical cord progenitors,96 bone marrow stromal innervated by proximal host neurons and project in turn to cells,97 murine cell-line cells,98 and populations of multipo- distal host neurons. Grafts may also provide growth factors tent cells derived from human fetal brain99 and human or may improve conduction in spared axons, perhaps by To date, however, where multipotent cells promote Transplants of embryonic tissue may also enhance func- recovery of function in animal models of CNS injury, axons tion of spared circuitry caudal to the lesion thereby improv- do not appear to regenerate through and beyond grafts.96,98 ing local somatic and visceral responses. One study demon- One study has shown that immortalized neural progenitors strated that serotonergic neurons from the embryonic raphe promote PNS axon growth following SCI.101 Alternative nucleus grafted caudal to a complete cord transection in the explanations for recovery of function include cell replace- adult rat influenced penile erectile function90 and enhanced ment, creation of new neuronal relays, myelination, neuro- weight support and treadmill locomotion in spinal rats.91 Since grafts of fetal/embryonic tissue promote recovery Researchers also have recently begun investigating trans- but without concomitant long distance axon regeneration plants of multipotent cells with restricted fates. Grafts of beyond the graft/host interface, co-treatment strategies glial-restricted precursors promote mild ingrowth of CST have been evaluated. In one study, fetal tissue transplanted Grafted neuronal-restricted precursors extend 2 to 4 weeks after injury, when combined with neu- axons de novo after grafting into the intact spinal cord105 rotrophin delivery, promoted regrowth of axons caudal to although differentiation into mature neurons appears to be the transection site, resulting in plantar foot placement and inhibited after grafting into the contusion-injured spinal weight-supported stepping in adult rats.92 Journal of N eurological P hysical Therapy Recently, neural progenitors derived from 8 week old the transplant site.112,114 Forelimb use was also partially human fetuses have been transplanted into immunosup- restored, and more so when transplants were placed early pressed nonhuman primates 9 days following cervical con- after injury rather than when delayed 6 weeks.28 tusion injury.107 There was a statistically significant differ- Although many neurons die after axotomy, severely atro- ence in spontaneous locomotion within the cage and in phied neurons can be rescued. In one study, severe atrophy forelimb grip power between marmosets transplanted with of rubrospinal neurons could be reversed by applying cells relative to those transplanted with culture medium BDNF to the cell bodies up to one year after injury.25 only. Eight weeks following transplantation, histopathology Further, this treatment promoted the regeneration of these revealed reduced cavitation but no evidence of tumor for- rubrospinal axons into peripheral nerve transplants mation. Some surviving grafted cells expressed markers characteristic of neurons or glia and there was evidence for Growth factors have also been delivered to the nonhu- man primate spinal cord using autologous fibroblasts genet- Autologous transplants of progenitor cells remain to be tested in rodent or primate models of SCI. Future experi- implantation into rhesus monkeys, sensory and putative ments using animals may also show whether it is possible coerulospinal axons were found to regenerate into NGF- to induce regeneration and repair by stimulating the secreting grafts. Since these monkeys had not been given response of endogenous adult progenitors. Finally, several deliberate spinal cord injuries, further work is required to clinical studies are attempting to evaluate whether trans- show whether delivery of growth factors promote CNS plantation of progenitor cells improves outcome after axon regeneration and functional recovery in injured non- Disappointingly, clinical trials using systemic delivery of Delivery of growth factors promotes CNS axon
high doses of neurotrophins for various nervous system dis- regeneration and motor recovery after SCI
orders have run into a number of difficulties, including Growth factors potently promote axon regeneration induction of side effects including severe muscular pain, during nervous system development and after PNS injury.
fever, depression, and hallucinations.116 Targeted delivery of Following contusion or transection injury to the adult rat physiological doses of growth factors (eg, by using viral spinal cord, however, message levels for many growth fac- vectors) may prove safer and more efficacious.
tors remain almost undetectable (including neurotrophins3 and 4), or only briefly and slightly upregulated (including Scar tissue modifiers enhance CNS axon regeneration
nerve growth factor (NGF) and glial cell line-derived neu- and functional recovery after SCI
Extrinsic factors that limit CNS axon regeneration also rotrophin receptors on neurons remain absent (trkA), unal- include factors associated with myelin and the injury site.
tered (trkB, trkC), or only slightly upregulated (p75).108 Brain Spinal cord injury sites, particularly those resulting from derived neurotrophic factor may not be available for signal- penetrating injury, are also typically filled with multiple lay- ing through its high affinity receptor (trkB) since there is a ers of fibroblasts. This scar tissue represents a formidable large, fast, and sustained upregulation of competitor bindingsites (truncated trkB receptors) that may dominate as non- physical barrier to CNS axon regeneration,117 the cells often functional sinks for the active neurotrophin.109 In summary, being oriented perpendicular to the neuraxis (appearing after SCI, CNS neurons may lack the ability to respond to geometrically impenetrable) and containing molecular generally low levels of most growth factors.
inhibitors of growth including chondroitin sulfate gly- Consequently, to promote CNS axon regeneration and functional recovery after SCI, supplementary growth fac- In adult rats, degradation of CS GAGs by delivery of the tors have been delivered to neuronal cell bodies (eg, in bacterial enzyme, chondroitinase ABC, promotes regenera- brainstem or cortex) or SCI sites by direct injection,110 by tion of injured CNS axons119 and recovery of function after osmotic minipump,17 by application within fibrin glue,111 or dorsal hemisection.120 Following spinal cord hemisection in by gene transfer techniques.61,112-114 Delivery of growth fac- adult rats, delivery of chondroitinase ABC also promotes tors can enhance long distance anatomical, electrophysio- regrowth of axons from spinal cord neurons into grafts of logical, and behavioral recovery of CNS axons in vivo. For peripheral nerve121 and regrowth of CNS axons into spinal example, following dorsal hemisection lesions of adult rat cord beyond hemichannel bridges containing SCs and spinal cord and immediate implantation of NT-3-secreting Matrigel.122 Following complete transection and implanta- fibroblasts, CST axons grew up to 8 mm distal to the injury tion of channels containing SCs and Matrigel, delivery of site with reduced locomotor deficits on a gridwalk task 1 chondroitinase ABC and immunoglobulin promotes regen- and 3 months postinjury.113 Following cervical lateral hemi- eration of serotonergic axons beyond grafts.123 Repeated section and implantation of BDNF-expressing fibroblasts intrathecal delivery of chondroitinase ABC also promotes (together with methylprednisolone and immunosuppres- hindlimb recovery following severe (but not moderate) tho- sion), up to 7% of rubrospinal neurons regenerated beyond Journal of N eurological P hysical Therapy A number of safety and delivery issues remain to be tack- those involving transection.111,136 It will also be important to led. Delivery of bacterial proteins to humans carries a risk evaluate whether anti-Nogo strategies have any adverse out- of nonspecific immune responses. Further, repeated dosing comes in animal models of SCI. One study indicates no (as is typically performed in animal models of SCI using increase in tail flick (interpreted as no increase in nocicep- chondroitinase ABC) may not be possible if the immune sys- tion) 5 weeks following dorsal overhemisection and deliv- tem develops responses to neutralize these foreign pro- ery of IN-1 in adult rats.137 Clinical trials using antibodies teins. Thus efforts are underway to develop smaller, less against Nogo-A are currently being planned in association immunogenic yet enzymatically active variants of chon- A peptide inhibitor (NEP1-40) targeting a receptor for matrix metalloproteinases) that cleave CS GAGs might also Nogo-A (NgR) has also been developed138 in order to pre- avoid these side effects. Encouragingly, a preparation of vent neurons from responding to this inhibitor.
chondroitinase ABC is currently being tested in Phase 2 clin- Remarkably, subcutaneous treatment with NEP1-40 one ical trials for herniated lumbar discs125 and FDA approval for week after thoracic dorsal hemisection promotes growth of this therapy might accelerate clinical trials for SCI. In due CST axons and serotonergic fibers and a degree of locomo- course, chondroitinase ABC should be tested for safety and tor recovery.139 Intrathecal delivery of NEP1-40 also pro- efficacy in nonhuman primate models of SCI.
motes regrowth of rubrospinal axons and a degree of func-tional recovery after rat SCI.140 Biogen Idec Inc. holds a Therapies that target myelin inhibitors promote CNS
license for NgR-related therapeutics and clinical trials may growth and functional recovery
Intact and injured CNS myelin contains a number of dif- ferent growth inhibitory molecules.126 These include Nogo- INTRINSIC FACTORS
A, myelin-associated glycoprotein (MAG) and oligodendro- Cyclic adenosine monophosphate
Various strategies exist to boost CNS axon regeneration have been developed to target and overcome these by targeting molecules intrinsic to neurons. For example, inhibitors of axon growth, most notably the antibody IN-1, levels of cyclic adenosine monophosphate (cAMP) are which binds to Nogo-A and neutralizes its inhibitory higher in young, growing neurons than in older neurons.
Levels of cAMP are low in neurons in a state of growth arrest, The CNS axon growth and recovery of limb function such as those cultured on growth-inhibitory MAG, and deliv- have been reported in various animal models of SCI follow- ery of cAMP analogs relieves this growth-arrest in vitro.141 ing delivery of anti-Nogo therapeutics.
To test the hypothesis that elevation of cAMP boosts experiments, small numbers of adult rat CST axons were CNS axon regeneration in adult rats, cAMP analogs have observed approximately 1 cm beyond sites of thoracic dor- been injected into adult rat DRG one week prior to thoracic sal hemisection following implantation of hybridoma cells dorsal column hemisection. In 2 studies, centrally project- secreting IN-1.19,127 Further, between 7 and 12 weeks postle- ing sensory axons regrew better within injury sites.142,143 sion, low threshold stimulation of hindlimbs enhanced the Combining strategies, preinjury administration of cAMP incidence of paw placing responses in 8 of 10 animals with postinjury delivery of NT-3, promotes regeneration of treated with IN-1 antibodies but in no control animals, and sensory axons within and beyond transplants of bone mar- this effect was abolished by bilateral sensorimotor cortex row stromal cells.144 In these studies, however, it should be ablation.128 Administration of IN-1 antibodies also potenti- noted that administration of cAMP or NT-3 alone did not ated CST axon growth after dorsal hemisection in adult rats promote regeneration of axons beyond grafts.
and implantation of embryonic tissue127 or following injec- Since prophylactic treatments for SCI are inappropriate, tion of NT-3 either immediately after SCI129 or after a delay postinjury delivery of drugs that boost cAMP levels are of 2 or 8 weeks.24 IN-1 also induced CNS axons to sprout being tested. Postinjury delivery of a cAMP analog pro- collaterals, for example, following unilateral injury of the motes CNS axon regeneration after SCI in fish.145 CST at the level of the brainstem.130,131 A fall in the levels of endogenous cAMP after SCI in adult Recently, and promisingly, IN-1 has been shown to pro- rats can be prevented by delivery of the phosphodiesterase mote growth of CST axons following unilateral lesioning in inhibitor, rolipram, which prevents the hydrolysis of 4 out of 5 marmoset monkeys tested.132 Recombinant, cAMP.146 After cervical hemisection in adult rats, delivery of humanized fragments of IN-1 and immunoglobulin G class rolipram promotes growth of serotonergic axons into fetal antibodies against Nogo-A have so far been tested in 2 rat tissue transplants and enhances placing of the impaired stroke models133 and in a dorsal hemisection model of SCI.134 forelimb during rearing.147 After spinal cord contusion, adult To date, however, no reports exist of testing anti-Nogo rats injected intraspinally with SCs and a cyclic AMP analog therapies in contusion or compression models of SCI. This together with subcutaneous delivery of rolipram exhibited is important because anti-Nogo antibodies apparently do white matter sparing, increased numbers of spinal and not promote long distance CNS axon regeneration in all supraspinal axons beyond the injury/transplant site and models tested,130,131,135 particularly in larger injuries such as Journal of N eurological P hysical Therapy Prior to human clinical tests or trials, solo or combina- ated with mild expression of a limited set of regeneration- tion methods for elevating cAMP levels after injury need to associated genes,158 particularly when injury occurs close to be evaluated for safety and efficacy, at least in rodent mod- els of SCI and preferably also in nonhuman primates.
transplants of peripheral nerve express various regenera- Therapeutic windows of delivery of cAMP analogs need to be defined and doses and methods of delivery established.
In general, however, after injury to the CNS, neurons fail to re-express the majority of these genes, and do not regen- Modulation of small enzymes (GTPases) boosts CNS
axon regeneration and recovery of function after
transection, DRG neurons express GAP-43 in their regener- dorsal hemisection
ating peripheral projections but not in their nonregenerat- Many different extrinsic factors that impact upon axon ing central projections following central axotomy in dorsal regeneration have common final pathways, many signaling columns.162 It is therefore encouraging that overexpression intracellularly via small enzymes that interact with guano- of regeneration-associated genes causes injured CNS neu- sine triphosphate (GTP; a chemical compound that is used rons to regenerate axons. For example, simultaneous over- as a source of energy during synthesis of proteins). This has expression of GAP43 and CAP23 in transgenic mice causes been shown for CS GAGs as well as MAG, OMgp, and Nogo- enhanced neurite growth from explanted DRG in vitro and A.148,149 These GTPases include Rho and Rac family mem- boosts regeneration of centrally projecting axons from lum- bers. Injured spinal cords of adult rats contain increased bar DRG into peripheral nerve grafts implanted in thoracic levels of message for at least 5 different small GTPases including RhoA, RhoB, and Rac1.150 Rho and Rac GTPases Experiments overexpressing regeneration-associated generally act antagonistically; while Rho family members genes in CNS neurons in nonhuman primates have not yet retard growth, Rac family members promote neurite growth been performed and to our knowledge, therapies specifi- in vitro.151 Consequently, modulators of small GTPases have cally designed to boost expression of pro-regenerative been evaluated as methods for promoting CNS axon regen- genes in neurons after SCI are not yet being considered for eration and functional recovery in animal models of SCI.
Rho GTPase protein is activated after SCI and inhibition A number of hurdles remain. In the combination strat- of Rho (using C3 botulinum toxin) promotes CNS axon egy described above,163 relatively few neurons regenerated regeneration and a degree of functional recovery following and there was no report of testing for recovery of function.
dorsal hemisection injury in adult rats.152 Rho kinase acts as Future experiments may identify additional, more potent a downstream effector of Rho, and pharmacologic inhibi- regeneration-promoting genes. For example, novel regener- tion of Rho kinase promotes CNS axon regeneration fol- ation-associated genes have been identified in neurons dur- lowing dorsal hemisection in adult rats.153 Improved cell-permeable inhibitors of Rho are currently recently identified a novel set of regeneration-associated being tested. For example, BioAxone Therapeutic Inc. is genes that are expressed by injured spinal neurons that developing technologies to target Rho signaling. Strategies regenerate into grafts of SCs placed in transected adult rat to stimulate Rac in vivo remain to be developed. To date, spinal cord.165 Future experiments are underway to identify activators of Rac and inhibitors of Rho and Rho kinase have genes that potently promote neurite growth in cell culture neither been tested in rodent models of contusion injury systems. Targeted upregulation of these growth-promoting nor in any nonhuman primate models of SCI.
genes (eg, using viral vectors) may prove effective in pro- Other inhibitors of CNS axon regeneration intrinsic to moting CNS axon regeneration and functional recovery in neurons likely exist, and with the advent of molecular knockdown methods including RNA interference, attemptsto promote CNS axon regeneration through targeting of Physical therapy enhances recovery of function after
regeneration-inhibiting genes and proteins may become Although we know of no evidence yet that physical ther- apy or rehabilitation directly improves CNS axon regenera- Overexpressing regeneration-promoting molecules
tion after SCI, environmental enrichment, physical therapy, in neurons promotes CNS axon regeneration
and motor training have been shown to improve limb func- As axons elongate during development, PNS and CNS tion in animal models of SCI (including after transection166 neurons express a range of genes154 including growth asso- and contusion167–169) and in humans with SCI.170–173 ciated protein 43 kDa (GAP-43) and cortical cytoskeleton- To date, very few studies have combined rehabilita- associated protein 23 kDa (CAP-23). On maturation, most tion/physical therapy with strategies for promoting CNS neurons downregulate expression of these genes. After axon regeneration and recovery of limb function. In one injury to the PNS, neurons that re-extend axons re-express recent study from our laboratory, following transection of many of these “regeneration-associated” genes.155–157 Injured adult rat thoracic spinal cord, OEG were transplanted with CNS neurons do sprout processes locally, and this is associ- Journal of N eurological P hysical Therapy through provision of motor enrichment housing (MEH): a multiple types of progenitor cells. In many cases these large, multilevel cage filled with ramps, textured surfaces, claims have been made relative to controls that were not and hanging food rewards. The return of hindlimb joint transplanted with cells, but relative to injection of movement was assessed weekly for 22 weeks starting 1 medium.63,107 Very few studies have directly compared dif- week postinjury and was compared between animals ferent cell types within a single experiment,58 so it is very housed in MEH and those maintained in basic housing (BH).
difficult to know which cell type affords particular benefits All rats recovered a small amount of hindlimb movement, over others. In the future, additional control or comparison but recovery was not accelerated or enhanced by MEH.
groups should be included such as transplants of other cell Recovered hindlimb movements were, however, sustained up to 22 weeks postinjury in most rats in MEH, whereas Worryingly, some pro-regenerative therapies have been most rats kept in BH progressively lost recovered hindlimb shown to promote growth of sensory axons within the PNS movements after 9 weeks. Furthermore, MEH decreased or CNS. This includes progenitor cells,175 neurotrophin ther- mortality, and improved health. We conclude that motor apies,176 anti-inhibitor therapies,110 and regeneration-gene rehabilitation can be of substantial benefit when using overexpression therapies.163 In light of this, rigorous testing transplantation strategies to treat SCI.
for changes in nociception should take place in animal In the future it seems very likely that rehabilitation/phys- models of SCI before moving to the human patient. This is ical therapy will play a key role in many strategies designed especially important as many humans with SCI rate neuro- to promote CNS axon regeneration and recovery of limb logic pain as one of the worst sequelae of SCI.177 Great care function. These combination strategies will likely first be should therefore be taken to ensure that any clinical ther- tested in rodent, then primate models of SCI, and then, if apy does not induce neurological pain as a side effect.
safe and efficacious, in human clinical trials.
TOWARDS THE FUTURE
Increasing numbers of experimental studies using ani- In cases of anatomically complete injury in animal mod- mal models of SCI are resulting in reports of improvements els, the degree of recovery of hindlimb movement obtained in CNS axon regeneration and recovery of limb function.
by any single transplant therapy remains functionally slight.
Combinations of these strategies in animal models and in In general, following transection and intervention of any humans may lead to additive improvements in outcome kind in adult rats, plantar placement and weight support on after SCI. Many of these combination strategies may involve hindlimbs during stepping is not consistently observed. This rehabilitation measures and, as clinical tests and trials pro- includes: (1) transplantation of glia obtained from nasal ceed, it seems likely that the demand for physical therapists mucosa or olfactory bulb;26,68 (2) transplantation of activated with a good working knowledge of pro-repair strategies for (3) implantation of peripheral nerve grafts secured across transection gaps with fibrin glue containingacidic fibroblast growth factor;51 and (4) transplantation of ACKNOWLEDGEMENTS
OEG, with or without SC bridges;71 and (5) implantation of Supported by Christopher Reeve Paralysis Foundation SC bridges and OEG with delivery of chondroitinase ABC.123 Research Consortium grants, NINDS 09923 and the Miami To our knowledge there exists only one study that reports Project to Cure Paralysis (to M.B.B). With thanks to Dr.
frequent plantar placement and weight-supported stepping Caitlin Hill for suggestions for improving the manuscript in adult rats with transection injuries.92 This study utilized and Diana Masella for assistance with manuscript prepara- delayed transplantation of fetal tissue in combination with In cases of anatomically incomplete injury, the degree of REFERENCES
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