Putting the Spinal Cord Together Again
Authors: Oded Behar, Keiko Mizuno, Simona Neumann, Clifford J. Woolf
Journal: Neuron (2000)

Humpty Dumpty sat on a wall, Humpty Dumpty had a great fall, all the King's horses and all the King's men couldn't put Humpty together again. In view of the anatomical arrangement of its ascending and descending fiber tracts, even local limited injury to the spinal cord typically has devastating consequences. Communication between that part of the body below the level of injury and the brain is disturbed, resulting in permanent para- or quadriplegia and an equivalent loss of sensation. These motor and sensory deficits are usually accompanied by positive symptoms, including pain and exaggerated motor and autonomic reflexes. Treatment for traumatic spinal cord injury has until now only had two aims, both conservative. The first at the time of injury is to prevent further damage by high-dose steroids and, where appropriate, stabilizing the vertebral column and removing compressive tumors and any source of infection. The second is rehabilitative, teaching the patient and their family to live with this disability by optimizing intact function and reducing problems like poor ventilation, bedsores, bladder infections, joint fixation, and muscle wastage. What are the realistic prospects for a much more ambitious treatment objective aimed at promoting regeneration and restoration of lost function? Can we put the injured spinal cord together again? That we can even begin to contemplate the feasibility of promoting regeneration in the spinal cord is a measure of the almost explosive success that has been achieved recently in elucidating how neuronal growth occurs in development and why it fails in the adult CNS. This success can be attributed to a multiplicity of approaches in diverse fields including genetics, biochemistry, molecular and cellular biology, and physiology. Genetic approaches in Drosophila and C. elegans together with biochemical and cellular approaches in mammals have, for example, revealed a large number of molecules that potentially have a role in axon guidance in development and in aborting growth in the adult. Other approaches that have an increasing bearing on our growing understanding of CNS regeneration include embryonic and adult stem cell biology, the analysis of intracellular signal transduction cascades, and the biology of cell death. The progress achieved, although very promising, is really just the beginning of a real understanding of regeneration, and much remains to be learned. We are not, unfortunately, even at the beginning of applying this knowledge to the clinic. This remains an enormously daunting problem, one whose complexity cannot be minimized, and which needs to be confronted with a realistic sense not only of what is needed but also what is possible. Clinical intervention clearly will be contingent on our understanding the neurobiology of regeneration. Successful regeneration depends upon the ability of injured axons to survive, regrow, and reconnect with their original targets, processes integral to normal development. Why then is the regenerative response abortive in the adult mammalian CNS? There are three explanations: death of the injured neurons, an inability of differentiated adult neurons to initiate or maintain axonal growth, and a lack of an environment permissive for such growth (Figure 1). The inability of the CNS environment to support growth appears to be due to both the presence of inhibitory and a lack of growth-promoting signals. There are three sources of such signals; on cells, in the extracellular matrix, and diffusible molecules. One major source of inhibition is myelin. Schwab and colleagues demonstrated some years ago that a monoclonal antibody generated against central myelin, IN-1, improved neuronal regeneration (reviewed by 19Tatagiba M. Brosamle C. Schwab M.E Neurosurgery. 1997; 40: 541-547PubMed Google Scholar). Since then, myelin-associated glycoprotein (MAG) has been shown to be a myelin-associated growth-inhibitory molecule (reviewed by 6Fawcett J.W. Asher R.A Brain Res. Bull. 1999; 49: 377-391Crossref PubMed Scopus (1473) Google Scholar). Recently, a major achievement has been the cloning and expression of a novel protein, Nogo-A, that is the specific target for IN-1's action (3Chen M.S. Huber A.B. van der Haar M.E. Frank M. Schnell L. Spillmann A.A. Christ F. Schwab M.E Nature. 2000; 403: 434-439Crossref PubMed Scopus (330) Google Scholar, 7GrandPre T. Nakamura F. Vartanian T. Strittmatter S.M Nature. 2000; 403: 439-444Crossref PubMed Scopus (1015) Google Scholar, 14Prinjha R. Moore S.E. Vinson M. Blake S. Morrow R. Christie G. Michalovich D. Simmons D.L. Walsh F.S Nature. 2000; 403: 383-384Crossref PubMed Scopus (546) Google Scholar). Only a small portion of Nogo-A is expressed on the surface (7GrandPre T. Nakamura F. Vartanian T. Strittmatter S.M Nature. 2000; 403: 439-444Crossref PubMed Scopus (1015) Google Scholar), and the cells may need to be damaged for significant quantities of Nogo-A to be released. Understanding the mechanism of action of Nogo-A and MAG requires the elucidation of their receptors, and this is eagerly awaited. In addition to Nogo-A and MAG, it is likely that other myelin-related molecules inhibit regeneration. When the animals' own immune system is engaged to produce antibodies against all myelin-associated molecules by means of autoimmunization to myelin, a regenerative response apparently greater than that produced using specific antibodies occurs (8Huang D.W. McKerracher L. Braun P.E. David S Neuron. 1999; 24: 639-647Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar). However, even this approach only leads to limited regeneration, suggesting that other factors are involved in preventing axonal regeneration. One potential growth obstacle is the glial scar tissue that forms at the site of injury. Using a microtransplantation technique avoiding glial scarring and damage to myelin, Davies and colleagues (4Davies S.J.A. Fitch M.T. Memberg S.P. Hall A.K. Raisman G. Silver J Nature. 1997; 390: 680-683Crossref PubMed Scopus (670) Google Scholar) demonstrated that dissociated adult dorsal root ganglion (DRG) neurons injected into the CNS grow axons for long distances on the outer surface of the myelin leaflet. Intact myelin is then apparently permissive for growth, and only damaged or exposed myelin appears to inhibit growth. Even when these axons encounter tracts undergoing Wallerian degeneration (5Davies S.J.A. Goucher D.R. Doller C. Silver J J. Neurosci. 1999; 19: 5810-5822Crossref PubMed Google Scholar), they continue to grow robustly; one explanation may be that they do not directly contact the myelin debris, while another may be that they do not express the receptors for myelin inhibitors. However, when the DRG axons encounter an established scar, their growth halts. The molecules responsible are unknown. Candidates include developmental growth cone inhibitory guidance cues. Two such molecules, semaphorin 3A (13Pasterkamp R.J. Giger R.J. Ruitenberg M.J. Holtmaat A.J.G.D. De Wit J. De Winter F. Verhaagen J Mol. Cell. Neurosci. 1999; 13: 143-166Crossref PubMed Scopus (277) Google Scholar) and EphB3 (11Miranda J.D. White L.A. Marcillo A.E. Willson C.A. Jagid J. Whittemore S.R Exp. Neurol. 1999; 156: 218-222Crossref PubMed Scopus (130) Google Scholar), are expressed at the site of CNS injury, and semaphorin 3A is repellent to adult DRG neuron growth cones (17Reza J.N. Gavazzi I. Cohen J Mol. Cell. Neurosci. 1999; 14: 317-326Crossref PubMed Scopus (58) Google Scholar). Other contributors may be extracellular matrix molecules like the proteoglycans phosphacan, versican, brevican, and neurocan and some adhesion molecules such as tenascin-R, acting as a growth cone glue (reviewed by 6Fawcett J.W. Asher R.A Brain Res. Bull. 1999; 49: 377-391Crossref PubMed Scopus (1473) Google Scholar). It is interesting that if neurons are primed for regeneration at the time of injury (12Neumann S. Woolf C.J Neuron. 1999; 23: 83-91Abstract Full Text Full Text PDF PubMed Scopus (545) Google Scholar) or if myelin inhibitors are neutralized immediately after injury (8Huang D.W. McKerracher L. Braun P.E. David S Neuron. 1999; 24: 639-647Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar), axons regenerate before the glial scar forms, and the scar forms around the regenerated axons. Positive signals in the environment can augment regeneration. The addition of neurotrophic factors, for example, improves CNS regeneration, which may be accomplished by ensuring survival, acting as positive growth cues, as well as priming the injured cells to produce the molecular machinery necessary for growth. McMahon and colleagues have recently shown that adult DRG axons grow across the dorsal root entry zone into the spinal cord only after intrathecal administration of neurotrophins (15Ramer M.S. Priestley J.V. McMahon S.B Nature. 2000; 403: 312-316Crossref PubMed Scopus (465) Google Scholar). A different approach to overcome the negative CNS environment has been to use cell and tissue transplants to provide a physical growth-promoting substrate. Such grafts have included peripheral nerves, Schwann cells, and embryonic stem cells (10McDonald J.W. Liu X.Z. Qu Y. Liu S. Mickey S.K. Turetsky D. Gottlieb D.I. Choi D.W Nat. Med. 1999; 5: 1410-1412Crossref PubMed Scopus (1078) Google Scholar). Recently, Ramon-Cueto and colleagues using olfactory ensheathing glial cell (OEG) transplants into the injured spinal cord (16Ramon-Cueto A. Cordero M.I. Santos-Benito F.F. Avila J Neuron. 2000; 25: 425-435Abstract Full Text Full Text PDF PubMed Scopus (759) Google Scholar) substantially improved recovery of function. In addition to the environment, the state of the injured neuron is critical. One imperative is to prevent cell death; if this has occurred, stem cell transplants offer the possibility of replacing lost neurons (10McDonald J.W. Liu X.Z. Qu Y. Liu S. Mickey S.K. Turetsky D. Gottlieb D.I. Choi D.W Nat. Med. 1999; 5: 1410-1412Crossref PubMed Scopus (1078) Google Scholar). Another is to ensure that the injured neuron is capable of growth. Preinjury to the peripheral branch of DRG neurons, which induces an increase in intrinsic growth capacity, has recently been shown to significantly improve the regenerative response of the central branch in the dorsal columns after spinal cord injury (12Neumann S. Woolf C.J Neuron. 1999; 23: 83-91Abstract Full Text Full Text PDF PubMed Scopus (545) Google Scholar). For a presently unknown reason, injuring one axon of sensory neurons (the peripheral) and not another (the central) switches the cell into an actively growing state such that it can grow into environments normally hostile for regeneration. We need to find out why and replicate this without injuring the neuron. Two other studies have also shown that increasing the intrinsic growth state improves the regenerative response. Inosine, acting via a direct intraneuronal mechanism, improves the growth response of intact neurons (collateral sprouting) after spinal cord injury (1Benowitz L.I. Goldberg D.E. Madsen J.R. Soni D. Irwin N Proc. Natl. Acad. Sci. USA. 1999; 96: 13486-13490Crossref PubMed Scopus (119) Google Scholar), while inhibition of Rho, a small G-coupled protein involved in signaling to the growth cone cytoskeleton, improves regeneration of the optic nerve (9Lehmann M. Fournier A. Selles-Navarro I. Dergham P. Sebok A. Leclerc N. Tigyi G. McKerracher L J. Neurosci. 1999; 19: 7537-7547Crossref PubMed Google Scholar). New insights into signal transduction pathways in the developing growth cone suggest that repulsive signals can be inverted into positive cues (18Song H.J. Poo M.-M Curr. Opin. Neurobiol. 1999; 9: 355-363Crossref PubMed Scopus (406) Google Scholar). One indication of this is that conditioning stimuli such as neurotrophins that raise intracellular cAMP invert the response to MAG (2Cai D. Shen Y. De Bellard M. Tang S. Filbin M.T Neuron. 1999; 22: 89-101Abstract Full Text Full Text PDF PubMed Scopus (466) Google Scholar). Therefore, targeting the intracellular signal transduction cascade or activating enzymes downstream from such cascades may be a useful therapeutic approach to promote regeneration without having to change the local environment. Most recent studies in the regeneration field tend to emphasize the increased axonal growth that can be achieved by different manipulations. Axonal growth, although necessary, is not by itself sufficient for functional recovery; however, retrieval of the appropriate pathways, target recognition, orderly reinnervation, and reestablishment of functioning synapses are essential. These are areas where we know considerably less about the signal determinants, although developmental studies are likely to help substantially. A risk inherent in promoting growth without controlling target connections is that maladaptive function may develop. Several neurological conditions, including neuropathic pain, are actually an expression of such maladaptive structural plasticity. We do not want axonal growth at any price; targeted growth is required. Demonstration of successful spinal cord regeneration in experimental models is nontrivial. It requires unambiguous proof that injured and not neighboring intact neurons have grown, that novel functional synapses have been established, and that these are responsible for any improvement in functional recovery and that any such recovery is not accompanied by maladaptive function. Very few studies fulfill all these criteria, and consequently there is often uncertainty as to how much regeneration has occurred and its functional significance. The rate of recent progress indicates, though, that we are likely to learn soon why regeneration fails in the adult CNS and may be able to devise technical approaches to use this knowledge to further enhance regeneration. The therapeutic strategies necessary to achieve successful spinal cord regeneration are shown in Table 1. Given the complexity and diverse response of the CNS to injury, it is extremely unlikely that a single approach will suffice. Multiple interventions will be required at coordinated times to initiate, maintain, and guide regeneration of injured neurons from their site of injury to the appropriate target neurons, and to reestablish functional synaptic contact. Although cautious optimism is beginning to be appropriate, there are great scientific challenges that still need to be met before it is time to focus on or initiate clinical therapies.Table 1Strategies for Promoting Spinal Cord RegenerationObjectivesneuronal death/atrophyglial cell reaction/extracellular matrix formationdecrease inhibitory environment provide permissive environmentincrease growth response decrease response to inhibitory influences provide axon guidance cues ensure contact with appropriate target neuron promote synaptic differentiation prevent maladaptive connectivityTechniquesanti-inflammatory agents—methyl prednisoloneneuroprotection—glutamate receptor antagonists/ion channel blockers/growth factorstarget glial cell reactionvaccination against myelin proteins neutralizing antibodies/receptor bodies targeted at inhibitory proteins (Nogo, etc.) receptor antagonistsabort growth cone collapse—intracellular signal transduction modifierssupporting cell bridges—peripheral nerve grafts embryonic tissue/stem cell grafts Schwann cells/olfactory ensheathing glia acellular matrix conduitsadminister molecules that increase intrinsic growth capacity activate growth cone signal transduction cascades potassium channel blockers—optimizing conduction replace neurons—stem cell transplantsprovide guidance cues and ensure specific target recognition Open table in a new tab