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Keeping in touch: sensory neurone regeneration in the CNS
REVIEW
Keeping in touch: sensory neurone regeneration in the CNS Elizabeth J. Bradbury, Stephen B. McMahon and Matt S. Ramer Adult neurones fail to regenerate when injured in the CNS, which leads to severe and irreversible functional deficits. Several important advances in understanding the reasons for this failure have been gained from the use of primary sensory neurones as a model system. The peripherally and centrally projecting branches of sensory neurones are differentially capable of regeneration, which is why these cells are ideally situated to elucidate the mechanisms that underlie regeneration failure. Such mechanisms include both a hostile environment within the spinal cord and a poor growth response following injury. For successful functional regeneration to occur, it is likely that both of these barriers will have to be surmounted, along with the challenge of guiding regrowing axons to appropriate postsynaptic targets. The contribution that the study of primary sensory neurones has made to the attainment of this goal will be reviewed. The failure of regeneration of the mature mammalian CNS has been recognized and studied for more than a century. Although much has been learned, there are no effective clinical treatments for injuries such as brachial plexus avulsion and spinal cord damage, beyond limiting the immediate effects of trauma with steroids1. However, there is now well-founded optimism that novel treatments might soon emerge. This stems from our rapidly increasing knowledge of the reasons why regeneration fails and from new experimental strategies that promote significant and functionally useful regeneration in animal models. This review will describe these new developments in one particular context, namely damage to the axons of primary sensory neurones. The cell bodies of these neurones lie in the dorsal root ganglia (DRG) and have axonal branches in the PNS and CNS. The central and peripheral projections of these neurones have differential regenerative capacities after injury. Therefore, the primary sensory neurone system has provided important insights into the mechanisms that usually prevent CNS regeneration. Regenerative capacities of damaged sensory neurones
The axons of primary afferents can be damaged at several sites, namely within the peripheral nerve, the dorsal root and the dorsal columns (Figs 1,2). Although the portion of the axon that is isolated from the cell body undergoes Wallerian degeneration in each case, the regenerative outcome is dramatically different. Peripheral nerve lesions
Axotomy of peripheral nerves is followed by regeneration and target re-innervation under optimal conditions (e.g. crush or freeze lesions, which leave basal laminae intact). Within a day or two, sprouts issue from the truncated axons and begin to grow along the degenerating nerve at a rate of several millimetres per day, following their original trajectory until they reach their peripheral targets. Axon calibre and receptor sensitivity mature over the ensuing weeks and months. Even under sub-optimal conditions
(e.g. following complete section of a nerve, which disrupts basal laminae), many axons can navigate across the lesion site and successfully re-innervate peripheral targets, although this innervation is often topographically inappropriate. Dorsal root lesions (rhizotomy)
Crush of a dorsal root is also followed by sprouting of the damaged axon and regeneration towards the original target (in this case, the spinal cord), although regrowth is slower than that which follows peripheral nerve lesions2. Crucially, however, regeneration stops precisely upon contact with the CNS at the dorsal root entry zone (DREZ) (Fig. 2d). The axons form club-like endings (retraction bulbs) or synapse-like structures3 and invariably fail to enter the spinal cord. Dorsal column lesions
Lesioning the dorsal column projection of sensory axons results in a complete lack of regeneration. When damaged in the CNS, the axons form retraction bulbs and cannot elongate to re-innervate target cells in the dorsal column nuclei (Fig. 2f ). Why should the same neurones exhibit such distinctly different capacities for regeneration in these different conditions? There are two categories of explanation, which we will examine below. First, injuring the axons at different sites might induce different degrees of inherent regenerative capacity. Second, the environments in which regeneration takes place might be differentially permissive. Modulation of inherent regenerative capacity Responses to injury
One of the most striking differences between lesions that are peripheral as opposed to central to the DRG is the response of the cell body. Peripheral nerve injury changes the function of DRG neurones from neurotransmission to regeneration, which is associated with dramatic changes in gene expression. These changes include the upregulation
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M.S. Ramer*, Postdoctoral Fellow, E-mail: [email protected] E.J. Bradbury, Postdoctoral Fellow, E-mail: elizabeth.bradbury@ kcl.ac.uk and S.B. McMahon, Sherrington Professor of Physiology, Sensory Function Group, Centre for Neuroscience Research, Hodgkin Building, King’s College London, Guy’s Campus, London Bridge, London, UK SE1 1UL. E-mail: stephen.mcmahon@ kcl.ac.uk *Also at: Neuroscience Section, Division of Biomedical Science, St Bartholomew’s and the Royal London School of Medicine and Dentistry, Queen Mary and Westfield College, Mile End Road, London, UK E1 4NS.
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trkB trkA C Aβ CGRP NF200 C P2X3
The association of a robust cell body response with regeneration has led to studies in which ‘conditioning’ peripheral nerve lesions have been used in attempts to augment central regrowth following dorsal root5 and dorsal column6 lesions. Such lesions can double the rate of axonal growth within the dorsal root7 and produce some regeneration into the cord when performed simultaneously with a rhizotomy5. The timing of the conditioning lesion appears to be important, because if peripheral nerve injury precedes dorsal column injury, regeneration of the injured ascending afferents is augmented6.
trkC RET GFRα1 Dorsal columns
RET, GFRα1/2 Peripheral nerve
Dorsal root
Neurotrophic factors
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Fig. 1. Organization of primary sensory neurones. The cell bodies of most primary afferent neurones are located in dorsal root or cranial ganglia. The axon that emerges from the cell body bifurcates to send one branch peripherally and another towards the CNS in the dorsal root. In both the nerve and root, axons are associated with Schwann cells. At the dorsal root entry zone (DREZ), an abrupt transition occurs where Schwann cells give way to astrocytes and oligodendrocytes. Within the spinal cord, sensory axons have extensive axonal terminations in the dorsal grey matter. Some have a rostrally projecting branch in the dorsal columns, which terminates in the ipsilateral dorsal column nuclei (DCN). Sensory neurone cell bodies are phenotypically heterogeneous (top left). Largediameter neurones have myelinated (Ab-fibre) axons (Ab), innervate sensitive mechanoreceptors peripherally (and therefore subserve tactile and proprioceptive functions) and have terminations in both the deep laminae of the spinal cord and the DCN (see Fig. 2). Small-diameter sensory neurones mostly have unmyelinated (C-fibre) axons (C), innervate nociceptors peripherally and terminate in superficial laminae of the dorsal horn. The neurotrophins, including nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and neurotrophin 3 (NT3) are essential for the development and maintenance of the different subclasses of primary sensory neurones. NGF acts through the tyrosine kinase neurotrophin receptor trkA, which is expressed on half of all smalldiameter dorsal root ganglia (DRG) neurones (~40% of all cells). BDNF signals via trkB and NT3 signals via trkC, which is expressed almost exclusively by large-diameter neurones. Glial cell line-derived neurotrophic factor (GDNF) receptor components [RET and GDNF family receptor a 1/2 (GFRa1/2)] are expressed in ~50% of all small-diameter neurones and 40% of large-diameter neurones15,16. Different subclasses of DRG neurones express characteristic neurochemical markers such as calcitonin gene-related peptide (CGRP), P2X3 receptors and the heavy-chain neurofilament NF200.
of the regeneration-associated immediate-early gene Jun in all injured neurones, as well as induction of the growth associated protein GAP-43, which is expressed constitutively in only a small subpopulation of primary sensory neurones4. These changes are muted following lesions that are central to the DRG (Ref. 2) and, consequently, the average rate of regeneration of dorsal root axons is only half that of their peripheral counterparts (except for those that constitutively express GAP-43, which regenerate at a normal rate4). This occurs despite the permissive Schwann cell environment of the dorsal root. In a similar manner, the weak intrinsic regenerative capacity of dorsal column axons contributes to their failure to regenerate after axotomy. Regeneration strategies that focus on the growth responses of damaged neurones have included substitutive measures in which adult neurones are replaced with embryonic neurones (Box 1). However, another approach involves boosting the inherent growth potential of the existing cells.
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An alternative treatment option is the delivery of exogenous neurotrophic factors. These molecules have been shown to promote the outgrowth of sensory neurites in vitro8, even in the presence of CNS-derived myelinassociated glycoprotein9. The potential regenerationpromoting effects of neurotrophic factors have also been investigated in vivo. Anatomical studies have shown that neurotrophic factors promote the regeneration of dorsal root axons into the spinal cord, whether delivered in fibrin glue10, via adenoviral vectors11 or intrathecally12,13. Intrathecal treatment with different neurotrophic factors resulted in the selective ingrowth of appropriate subclasses of axons13. Specifically, nerve growth factor (NGF) promoted the ingrowth of axons that express calcitonin generelated peptide (CGRP), but not axons that express NF200, which is a large-calibre fibre marker. By contrast, neurotrophin 3 (NT3) promoted ingrowth of NF200expressing axons, but not axons that express the P2X3 receptor [i.e. glial-derived neurotrophic factor (GDNF)sensitive axons]. GDNF promoted the regeneration of large- and small-calibre axons, which was expected based on the expression patterns of neurotrophic factor receptors14,15 (Fig. 1). Encouragingly, anatomically demonstrable regrowth was accompanied by synaptic re-connection, again in a selective fashion, such that NGF and GDNF treatments resulted in re-activation of dorsal horn neurones by slow-conducting sensory axons, and NT3 and GDNF treatments facilitated the recovery of postsynaptic potentials that are evoked by fast-conducting sensory fibres13. Most importantly, NGF- and GDNF-treated animals regained the ability to sense mechanically and heat-induced pain, indicating that appropriate spinal circuits were being activated by sensory input. Neurotrophic factors also promote regeneration following dorsal column lesions. For example, NT3 and NGF promote extension of CGRP-expressing sensory neurones into pre-degenerated nerve grafts that have been implanted into injured dorsal columns12. Furthermore, intrathecal administration of NT3 alone can induce sprouting and several millimetres of regeneration in the lesioned dorsal columns16 (Fig. 2g). However, an important difference between neurotrophic-factor-induced regeneration after dorsal column and dorsal root injury is that regenerating dorsal column axons do not reach their normal targets12,16.
REVIEW (a)
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(b) Gracile fasciculus Midline
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Fig. 2. The effects of axotomy and treatment with neurotrophin on the population of large-diameter centrally projecting dorsal root ganglia (DRG) afferents can be investigated by using cholera toxin b subunit (CTB) tracing. CTB injected into a sciatic nerve is transported transganglionically and labels central terminals in the lumbar dorsal horn (as well as motoneurones in the ventral horn) (a) and the axon collaterals that ascend in the dorsal columns (b) to terminate in the dorsal column nuclei in the brainstem (c). Dorsal root injury (d) results in the regeneration of axons, which stops at the dorsal root entry zone (DREZ; broken line). Intrathecal administration of neurotrophin 3 (NT3) results in the regeneration of dorsal root axons across the DREZ (e). Dorsal column injury (f) prevents regeneration and causes retraction of injured axons (asterisk indicates the site of a 4-week-old crush lesion). Intrathecal administration of NT3 results in the regeneration of dorsal column axons through the lesion site (g). Scale bar equivalent to 300 mm in (a), 200 mm (b), 50 mm (d,e) and 100 mm (c,f,g). Arrows indicate regenerating fibres.
This suggests that the central injury induces a barrier to growth that is insurmountable with neurotrophic factor treatment. The permissiveness of different cellular environments for regeneration Altering the CNS environment
Since landmark experiments in which CNS axons were found to grow into peripheral nerve grafts17,18, various methods have been employed to alter the non-permissive CNS environment to stimulate regeneration. Regeneration of central sensory axons has occurred across grafts of peripheral nerves and foetal spinal cord19–21. A major problem with grafting experiments is that although axons can grow across the grafts, they rarely re-enter host tissue at the graft–host interface. However, olfactory ensheathing cells (OECs)
offer the prospect of more extensive regeneration because they are highly migratory. For example, OEC transplants can promote regeneration of dorsal root projections through the DREZ into the spinal cord22. In addition, OECs can also promote the regeneration of corticospinal tract axons through the glial scar and into their appropriate pathway23. These studies have revealed that central DRG axons are capable of regeneration when the injured CNS environment is altered from being non-permissive to being more permissive to growth. Therefore, identifying the factors that are responsible for the inhibitory properties of the injured CNS is essential. Inhibitory molecules
A crucial difference between the PNS and CNS environments is the presence of peripheral Schwann cells in the
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Box 1. Taking cues from development Foetal nervous tissue is not only permissive to the regeneration of adult axons, but is also capable of growth within adult CNS. How is axonal growth in embryonic CNS possible and can the properties of embryonic tissue be exploited in adult CNS repair?
2 Spinal cord penetration (boundary cap cells) 1 Root formation (semaphorin 3A)
Bridging the gap
In vitro experiments have shown that embryonic spinal cord supports the regeneration of adult dorsal root ganglia (DRG) neuronesa. The CNS only becomes non-permissive to axonal regeneration after the first postnatal week, because dorsal root axons will regenerate into the spinal cord if there is an injury before this timeb. Embryonic spinal cord grafts are permissive to primary afferent regeneration, as demonstrated in studies by Tessler and co-workersc,d. The axons that invade the graft include all known classes of DRG neurones, become myelinated and form permanent functional synapses.
5 Terminal pattern formation (semaphorin 3A)
6 Formation of inhibitory barrier (CS-PGs)
An elevated propensity for growth
Embryonic neurones have a greater inherent capacity for growth than adult neurones. Neurones in embryonic spinal cord grafts send axons into the adult host cord and primary afferent inputs can cause induction of Fos in both transplant and host neurones, indicating that the grafts integrate primary afferent input into host circuitryd. Embryonic DRG neurones are capable of traversing the adult dorsal root entry zone (DREZ) in vitroa and in vivoc,d. This is indicated by the fact that when human or rat embryonic DRGs are placed into the cavity left after adult DRG removal, axons grow both peripherally and centrally, circumventing the DREZ and re-entering the spinal corde. A drawback to the application of these techniques is their complexity. In embryonic cord transplant experiments, adult sensory neurones must make contact with embryonic neurones, which in turn need to connect with host spinal neurones, and in DRG transplantation studies, the grafted cells must connect with appropriate peripheral and central targets.
trends in Pharmacological Sciences
Fig. I. Developmental cellular and molecular processes that are involved in spinal cord afferentation. (1) After migrating away from the neural tube, dorsal root ganglia (DRG) neurones send axons peripherally and centrally. The repulsive guidance molecule, semaphorin 3A, might direct the axons towards the entry zoneh. (2) Neural-crest-derived ‘boundary cap’ cells aid the ingrowth of sensory axons into the CNS and impede outward extension of astrocytic processesi. These might be a source of axonal chemoattractants. (3) Once within the cord, axons that are destined to convey mechanosensory or proprioceptive information ramify, sending out rostral, caudal and ventral branches, a process that is thought to be mediated by the diffusible molecule Slit-2 (Ref. j). (4) Contact-inhibiting B-class ephrins might contribute to the formation of longitudinally oriented axon bundles, such as the dorsal columnsk. (5) The termination pattern of nociceptive and proprioceptive axons might be determined by ventrally expressed semaphorin 3A, which repels small-diameter nerve growth factor (NGF)-sensitive axons but has no such effect on neurotrophin 3 (NT3)-sensitive large-diameter axonsh. (6) In the first postnatal week, inhibitory chondroitin sulfate proteoglycan (CS-PG) expression begins at the midline and spreads laterally towards the entry zone, coinciding with the end of the permissive period of the immature dorsal root entry zone (DREZ)l.
Homing signals
Several lines of evidence suggest that regenerating axons are capable of reinnervating appropriate laminae in the spinal cord. For example: (1) olfactory ensheathing cell transplants allow the ingrowth of calcitonin generelated peptide (CGRP)-expressing axons, which stop in laminae I and II (Ref. f ); (2) olfactory gliaf and neurotrophic factorsg promote re-connection with spinal neurones in such a way as to mediate spinal reflexes; and (3) intrathecal neurotrophic factor treatment promotes the growth of axons deep into the grey matterg. How do these axons find their appropriate targets? Part of the answer might be found in embryonic transplant studies in which grafts of brain were innervated in a more random manner by injured dorsal root axons in comparison with grafts of spinal cord, which caused the ingrowing axons to branch extensively and form bundlesd. The developmental program that normally guides dorsal root axons into the CNS might operate not only in transplanted embryonic cord, but also in denervated adult cord (Fig. I). If this is true, it suggests that adult neurones possess (or re-express) the ligands and/or receptors that are needed to interpret guidance cues. By understanding the process of sensory innervation of the spinal cord during development, we might be able to understand and eventually manipulate the molecules that are responsible and thereby improve sensory regeneration into and within the adult spinal cord after injury. Selected references a Golding, J.P. et al. (1999) Behaviour of DRG sensory neurites at the intact and injured adult rat dorsal root entry zone: postnatal neurites become paralysed, whilst injury improves the growth of embryonic neurites. Glia 26, 309–323 b Carlstedt, T. (1997) Nerve fibre regeneration across the peripheral–central transitional zone. J. Anat. 190, 51–56
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3,4 Branching and dorsal column formation (Slit-2, B-class ephrins)
c Fraher, J.P. (2000) The transitional zone and CNS regeneration. J. Anat. 196, 137–158 d Ramer, M.S. et al. Axon regeneration across the dorsal root entry zone. Prog. Brain Res. (in press) e Kozlova, E.N. et al. (1997) Human dorsal root ganglion neurones from embryonic donors extend axons into the host rat spinal cord along laminin-rich peripheral surroundings of the dorsal root transitional zone. J. Neurocytol. 26, 811–822 f Navarro, X. et al. (1999) Ensheathing glia transplants promote dorsal root regeneration and spinal reflex restitution after multiple lumbar rhizotomy. Ann. Neurol. 45, 207–215 g Ramer, M.S. et al. (2000) Functional regeneration of sensory axons into the adult spinal cord. Nature 403, 312–316 h Wright, D.E. et al. (1995) The guidance molecule semaphorin III is expressed in regions of spinal cord and periphery avoided by growing sensory axons. J. Comp. Neurol. 361, 321–333 i Golding, J.P. and Cohen, J. (1997) Border controls at the mammalian spinal cord: late-surviving neural crest boundary cap cells at dorsal root entry sites may regulate sensory afferent ingrowth and entry zone morphogenesis. Mol. Cell. Neurosci. 9, 381–396 j Wang, K.H. et al. (1999) Biochemical purification of a mammalian slit protein as a positive regulator of sensory axon elongation and branching. Cell 96, 771–784 k Imondi, R. et al. (2000) Complementary expression of transmembrane ephrins and their receptors in the mouse spinal cord: a possible role in constraining the orientation of longitudinally projecting axons. Development 127, 1397–1410 l Pindzola, R.R. et al. (1993) Putative inhibitory extracellular-matrix molecules at the dorsal-root entry zone of the spinal-cord during development and after root and sciatic nerve lesions. Dev. Biol. 156, 34–48
REVIEW former and central myelin in the latter. Several studies have investigated the inhibitory actions of CNS myelin on regeneration24,25. Neutralization of one constituent of CNS myelin with the antibody IN-1 promoted axonal regeneration, plasticity and some functional recovery after corticospinal tract lesions24,25. However, the observed regeneration was limited and functional synaptic contacts have not been demonstrated. The antigen that is recognized by IN-1 has recently been identified as Nogo-A, which is a potent inhibitor of neurite outgrowth of adult DRG neurones in vitro26,27. The development of reagents that target Nogo-A, Nogo-A knockout animals and mice that overexpress Nogo-A should give further insight into the role of this protein in inhibiting CNS regeneration in vivo. Several in vitro experiments have demonstrated that myelin-associated glycoprotein (MAG) can dramatically inhibit neurite outgrowth of adult DRG neurones. Myelin from MAG-null mice supported growth of longer neurites compared with wild-type myelin28. When normally permissive Schwann cells were induced to express MAG, both neurite outgrowth and branching were dramatically inhibited28. Another obstacle to regeneration is the formation of an astrocytic glial scar around the lesion site. Cells that are associated with the glial scar form a physical barrier to regenerating axons and also secrete inhibitory factors, such as chondroitin sulfate proteoglycans (CS-PGs)29. In vitro experiments have correlated the inhibition of neurite outgrowth with the expression of CS-PGs (Refs 30,31) and enzymatic degradation of CS-PGs has enabled neurite outgrowth from DRG neurones cultured on a substrate that is normally nonpermissive32. One important study used primary sensory neurones to investigate the relative importance of myelin-associated inhibition versus the inhibitory glial scar. In vivo regeneration of cultured adult DRG neurones was demonstrated when they were microtransplanted into the degenerating dorsal columns. Axons were observed to regrow until they reached the lesion site, where CS-PGs were present33. This showed that the glial scar is an important barrier to regeneration and that primary sensory neurones are capable of growing in degenerating white matter tracts, which are classically thought to be a hostile CNS environment. However, because intrinsic DRG neurones do not normally regenerate after injury, it is possible that culturing the neurones before transplantation altered their regenerative capacity, perhaps by downregulating receptors for inhibitory CNS ligands or increasing the axotomy-related cell body response. Another explanation for growth of adult DRG neurones through degenerating white matter is that although the microtransplantation technique avoids inducing a scar at the transplant site, it might also avoid damaging myelin. Thus, the myelin encountered by the regenerating DRG neurones in the degenerating dorsal columns might not express inhibitory factors, unlike the myelin that is damaged at a lesion site. Several groups of extracellular matrix molecules mediate the restrictive boundary-like properties that are ascribed
to glial cells during development34 (Box 1). These molecules might be re-expressed in the adult following injury and cause potent inhibition of growth. One such molecule, semaphorin 3A, is expressed in the ventral spinal cord during development and repels developing NGF-dependent DRG axons, restricting them to superficial laminae of the dorsal horn35. Adult sensory neurones retain their ability to respond to semaphorin 3A (Refs 36,37) and semaphorin 3A is upregulated following lesions to different areas of the adult CNS, including injury to the spinal dorsal columns, where it is primarily located in the fibroblast-like cells that are associated with the glial scar38. The uncontrolled expression of this or other chemorepulsive axon guidance molecules might play a key role in mechanisms that inhibit growth in the CNS. Treatment with neurotrophic factors following different injuries
An important point that is illustrated by primary sensory neurone injury models is that the ability of a treatment to promote regeneration might differ among models. For example, we have used neurotrophic factors to promote the regeneration of axons following both dorsal root13 and dorsal column16 injury and find that NT3 induces regeneration in both cases (Fig. 2e,g), but GDNF is only effective following rhizotomy. One reason for this difference might be that the scar and cavitation39 that are necessarily associated with the dorsal column lesion (and that are not associated with rhizotomy) form a barrier to axonal growth that is surmountable with NT3, but not GDNF treatment. Another possibility is that the same factors might have different effects on damaged neurones that depend on whether the neurones are in contact with CNS myelin before or after initiation of treatment. In vitro experiments have shown that prior treatment with neurotrophic factors allowed sensory neurones to overcome MAG-induced inhibition9. In the case of rhizotomy, the lesion is made outside the spinal cord. If treatment follows immediately, the growing axons receive exogenous neurotrophic factors before they encounter CNS tissue. This is not the case with dorsal column lesions, in which the lesion is within the CNS. Concluding remarks
Achieving successful regeneration following adult CNS injury will almost certainly require a combination of treatments in which both the intrinsic neuronal growth capacity and the inhibitory CNS environment are targeted. In sensory systems, there have already been some attempts to combine growth-promoting neurotrophic factors or conditioning lesions with growth-permissive grafts or foetal transplants21. However, although boosting the regenerative capacity of DRG neurones and reducing inhibition might allow sensory regeneration within the spinal cord, this will have little significance if growing axons do not activate useful postsynaptic pathways. Therefore, it will be necessary to guide regenerating axons to their appropriate targets. Although our knowledge of developmental axon guidance mechanisms is increasing rapidly (Box 1), it is not
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REVIEW clear to what extent these mechanisms might be re-activated following CNS injury in the adult. Although we are still some way from achieving the ultimate goal of regeneration, appropriate reconnection and functional recovery following adult CNS injury, many important advances have been made in understanding the mechanisms that underlie neuronal repair and regeneration. The unique anatomical organization of primary sensory neurones and their projections offers a valuable tool to further differentiate such mechanisms. References
Acknowledgements We thank Tim Boucher for critical evaluation of the manuscript and Pedran Badakhchani for illustrations. E.J.B. is supported by the Wellcome Trust. M.S.R. is supported by the EU and the Canadian MRC.
1 Short, D. et al. High-dose methylprednisolone in the management of acute spinal cord injury – a systematic review from a clinical perspective. Spinal Cord (in press) 2 Chong, M.S. et al. (1996) Intrinsic versus extrinsic factors in determining the regeneration of the central processes of rat dorsal root ganglion neurones: the influence of a peripheral nerve graft. J. Comp. Neurol. 370, 97–104 3 Carlstedt, T. (1997) Nerve fibre regeneration across the peripheralcentral transitional zone. J. Anat. 190, 51–56 4 Andersen, L.B. and Schreyer, D.J. (1999) Constitutive expression of GAP-43 correlates with rapid, but not slow regrowth of injured dorsal root axons in the adult rat. Exp. Neurol. 155, 157–164 5 Chong, M.S. et al. (1999) Axonal regeneration from injured dorsal roots into the spinal cord of adult rats. J. Comp. Neurol. 410, 42–54 6 Neumann, S. and Woolf, C.J. (1999) Regeneration of dorsal column fibers into and beyond the lesion site following adult spinal cord injury. Neurone 23, 83–91 7 Richardson, P.M. and Verge, V.M. (1986) The induction of a regenerative propensity in sensory neurones following peripheral axonal injury. J. Neurocytol. 15, 585–594 8 Gavazzi, I. et al. (1999) Growth responses of different subpopulations of adult sensory neurones to neurotrophic factors in vitro. Eur. J. Neurosci. 11, 3405–3414 9 Cai, D. et al. (1999) Prior exposure to neurotrophins blocks inhibition of axonal regeneration by MAG and myelin via a cAMP-dependent mechanism. Neurone 22, 89–101 10 Iwaya, K. et al. (1999) Neurotrophic agents in fibrin glue mediate adult dorsal root regeneration into spinal cord. Neurosurgery 44, 589–595 11 Zhang, Y. et al. (1998) NT-3 delivered by an adenoviral vector induces injured dorsal root axons to regenerate into the spinal cord of adult rats. J. Neurosci. Res. 54, 554–562 12 Oudega, M. and Hagg, T. (1999) Neurotrophins promote regeneration of sensory axons in the adult rat spinal cord. Brain Res. 818, 431–438 13 Ramer, M.S. et al. (2000) Functional regeneration of sensory axons into the adult spinal cord. Nature 403, 312–316 14 Bennett, D.L.H. et al. (1998) A distinct subgroup of small DRG cells express GDNF receptor components and GDNF is protective for these neurones after nerve injury. J. Neurosci. 18, 3059–3072 15 Bennett, D.L.H. et al. (2000) The glial cell line-derived neurotrophic factor family receptor components are differentially regulated within sensory neurones after nerve injury. J. Neurosci. 20, 427–437 16 Bradbury, E.J. et al. (1999) NT-3 promotes growth of lesioned adult rat sensory axons ascending in the dorsal columns of the spinal cord. Eur. J. Neurosci. 11, 3873–3883
17 Richardson, P.M. et al. (1980) Axons from CNS neurones regenerate into PNS grafts. Nature 284, 264–265 18 David, S. and Aguayo, A.J. (1981) Axonal elongation into peripheral nervous system ‘bridges’ after central nervous system injury in adult rats. Science 214, 931–933 19 Fawcett, J.W. (1998) Spinal cord repair: from experimental models to human application. Spinal Cord 36, 811–817 20 Ramer, M.S. et al. Progress in spinal cord research: a refined strategy for the International Spinal Research Trust. Spinal Cord (in press) 21 Ramer, M.S. et al. Axon regeneration across the dorsal root entry zone. Prog. Brain Res. (in press) 22 Navarro, X. et al. (1999) Ensheathing glia transplants promote dorsal root regeneration and spinal reflex restitution after multiple lumbar rhizotomy. Ann. Neurol. 45, 207–215 23 Li, Y. et al. (1998) Regeneration of adult rat corticospinal axons induced by transplanted olfactory ensheathing cells. J. Neurosci. 18, 10514–10524 24 Bandtlow, C.E. and Schwab, M.E. (2000) NI-35/250/nogo-a: a neurite growth inhibitor restricting structural plasticity and regeneration of nerve fibers in the adult vertebrate CNS. Glia 29, 175–181 25 Qiu, J. et al. (2000) Glial inhibition of nerve regeneration in the mature mammalian CNS. Glia 29, 166–174 26 Chen, M.S. et al. (2000) Nogo-A is a myelin-associated neurite outgrowth inhibitor and an antigen for monoclonal antibody IN-1. Nature 403, 434–439 27 GrandPre, T. et al. (2000) Identification of the Nogo inhibitor of axon regeneration as a Reticulon protein. Nature 403, 439–444 28 Shen, Y.J. et al. (1998) Myelin-associated glycoprotein in myelin and expressed by Schwann cells inhibits axonal regeneration and branching. Mol. Cell. Neurosci. 12, 79–91 29 Fawcett, J.W. and Asher, R.A. (1999) The glial scar and central nervous system repair. Brain Res. Bull. 49, 377–391 30 Smith-Thomas, L.C. et al. (1994) An inhibitor of neurite outgrowth produced by astrocytes. J. Cell Sci. 107, 1687–1695 31 Fidler, P.S. et al. (1999) Comparing astrocytic cell lines that are inhibitory or permissive for axon growth: the major axon-inhibitory proteoglycan is NG2. J. Neurosci. 19, 8778–8788 32 Zuo, J. et al. (1998) Degradation of chondroitin sulfate proteoglycan enhances the neurite-promoting potential of spinal cord tissue. Exp. Neurol. 154, 654–625 33 Davies, S.J. et al. (1999) Robust regeneration of adult sensory axons in degenerating white matter of the adult rat spinal cord. J. Neurosci. 19, 5810–5822 34 Faissner, A. and Steindler, D. (1995) Boundaries and inhibitory molecules in developing neural tissues. Glia 13, 233–254 35 Goodman, C.S. (1996) Mechanisms and molecules that control growth cone guidance. Annu. Rev. Neurosci. 19, 341–377 36 Tanelian, D.L. et al. (1997) Semaphorin III can repulse and inhibit adult sensory afferents in vivo. Nat. Med. 3, 1398–1401 37 Reza, J.N. et al. (1999) Neuropilin-1 is expressed on adult mammalian dorsal root ganglion neurones and mediates semaphorin3a/collapsin-1induced growth cone collapse by small-diameter sensory afferents. Mol. Cell Neurosci. 14, 317–326 38 Pasterkamp, R.J. et al. (1999) Expression of the gene encoding the chemorepellent semaphorin III is induced in the fibroblast component of neural scar tissue formed following injuries of adult but not neonatal CNS. Mol. Cell. Neurosci. 13, 143–166 39 Fitch, M.T. et al. (1999) Cellular and molecular mechanisms of glial scarring and progressive cavitation: in vivo and in vitro analysis of inflammation-induced secondary injury after CNS trauma. J. Neurosci. 19, 8182–8198
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Keeping in touch: sensory neurone regeneration in the CNS Elizabeth J. Bradbury, Stephen B. McMahon and Matt S. Ramer Adult neurones fail to regenerate when injured in the CNS, which leads to severe and irreversible functional deficits. Several important advances in understanding the reasons for this failure have been gained from the use of primary sensory neurones as a model system. The peripherally and centrally projecting branches of sensory neurones are differentially capable of regeneration, which is why these cells are ideally situated to elucidate the mechanisms that underlie regeneration failure. Such mechanisms include both a hostile environment within the spinal cord and a poor growth response following injury. For successful functional regeneration to occur, it is likely that both of these barriers will have to be surmounted, along with the challenge of guiding regrowing axons to appropriate postsynaptic targets. The contribution that the study of primary sensory neurones has made to the attainment of this goal will be reviewed. The failure of regeneration of the mature mammalian CNS has been recognized and studied for more than a century. Although much has been learned, there are no effective clinical treatments for injuries such as brachial plexus avulsion and spinal cord damage, beyond limiting the immediate effects of trauma with steroids1. However, there is now well-founded optimism that novel treatments might soon emerge. This stems from our rapidly increasing knowledge of the reasons why regeneration fails and from new experimental strategies that promote significant and functionally useful regeneration in animal models. This review will describe these new developments in one particular context, namely damage to the axons of primary sensory neurones. The cell bodies of these neurones lie in the dorsal root ganglia (DRG) and have axonal branches in the PNS and CNS. The central and peripheral projections of these neurones have differential regenerative capacities after injury. Therefore, the primary sensory neurone system has provided important insights into the mechanisms that usually prevent CNS regeneration. Regenerative capacities of damaged sensory neurones
The axons of primary afferents can be damaged at several sites, namely within the peripheral nerve, the dorsal root and the dorsal columns (Figs 1,2). Although the portion of the axon that is isolated from the cell body undergoes Wallerian degeneration in each case, the regenerative outcome is dramatically different. Peripheral nerve lesions
Axotomy of peripheral nerves is followed by regeneration and target re-innervation under optimal conditions (e.g. crush or freeze lesions, which leave basal laminae intact). Within a day or two, sprouts issue from the truncated axons and begin to grow along the degenerating nerve at a rate of several millimetres per day, following their original trajectory until they reach their peripheral targets. Axon calibre and receptor sensitivity mature over the ensuing weeks and months. Even under sub-optimal conditions
(e.g. following complete section of a nerve, which disrupts basal laminae), many axons can navigate across the lesion site and successfully re-innervate peripheral targets, although this innervation is often topographically inappropriate. Dorsal root lesions (rhizotomy)
Crush of a dorsal root is also followed by sprouting of the damaged axon and regeneration towards the original target (in this case, the spinal cord), although regrowth is slower than that which follows peripheral nerve lesions2. Crucially, however, regeneration stops precisely upon contact with the CNS at the dorsal root entry zone (DREZ) (Fig. 2d). The axons form club-like endings (retraction bulbs) or synapse-like structures3 and invariably fail to enter the spinal cord. Dorsal column lesions
Lesioning the dorsal column projection of sensory axons results in a complete lack of regeneration. When damaged in the CNS, the axons form retraction bulbs and cannot elongate to re-innervate target cells in the dorsal column nuclei (Fig. 2f ). Why should the same neurones exhibit such distinctly different capacities for regeneration in these different conditions? There are two categories of explanation, which we will examine below. First, injuring the axons at different sites might induce different degrees of inherent regenerative capacity. Second, the environments in which regeneration takes place might be differentially permissive. Modulation of inherent regenerative capacity Responses to injury
One of the most striking differences between lesions that are peripheral as opposed to central to the DRG is the response of the cell body. Peripheral nerve injury changes the function of DRG neurones from neurotransmission to regeneration, which is associated with dramatic changes in gene expression. These changes include the upregulation
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M.S. Ramer*, Postdoctoral Fellow, E-mail: [email protected] E.J. Bradbury, Postdoctoral Fellow, E-mail: elizabeth.bradbury@ kcl.ac.uk and S.B. McMahon, Sherrington Professor of Physiology, Sensory Function Group, Centre for Neuroscience Research, Hodgkin Building, King’s College London, Guy’s Campus, London Bridge, London, UK SE1 1UL. E-mail: stephen.mcmahon@ kcl.ac.uk *Also at: Neuroscience Section, Division of Biomedical Science, St Bartholomew’s and the Royal London School of Medicine and Dentistry, Queen Mary and Westfield College, Mile End Road, London, UK E1 4NS.
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trkB trkA C Aβ CGRP NF200 C P2X3
The association of a robust cell body response with regeneration has led to studies in which ‘conditioning’ peripheral nerve lesions have been used in attempts to augment central regrowth following dorsal root5 and dorsal column6 lesions. Such lesions can double the rate of axonal growth within the dorsal root7 and produce some regeneration into the cord when performed simultaneously with a rhizotomy5. The timing of the conditioning lesion appears to be important, because if peripheral nerve injury precedes dorsal column injury, regeneration of the injured ascending afferents is augmented6.
trkC RET GFRα1 Dorsal columns
RET, GFRα1/2 Peripheral nerve
Dorsal root
Neurotrophic factors
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Fig. 1. Organization of primary sensory neurones. The cell bodies of most primary afferent neurones are located in dorsal root or cranial ganglia. The axon that emerges from the cell body bifurcates to send one branch peripherally and another towards the CNS in the dorsal root. In both the nerve and root, axons are associated with Schwann cells. At the dorsal root entry zone (DREZ), an abrupt transition occurs where Schwann cells give way to astrocytes and oligodendrocytes. Within the spinal cord, sensory axons have extensive axonal terminations in the dorsal grey matter. Some have a rostrally projecting branch in the dorsal columns, which terminates in the ipsilateral dorsal column nuclei (DCN). Sensory neurone cell bodies are phenotypically heterogeneous (top left). Largediameter neurones have myelinated (Ab-fibre) axons (Ab), innervate sensitive mechanoreceptors peripherally (and therefore subserve tactile and proprioceptive functions) and have terminations in both the deep laminae of the spinal cord and the DCN (see Fig. 2). Small-diameter sensory neurones mostly have unmyelinated (C-fibre) axons (C), innervate nociceptors peripherally and terminate in superficial laminae of the dorsal horn. The neurotrophins, including nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and neurotrophin 3 (NT3) are essential for the development and maintenance of the different subclasses of primary sensory neurones. NGF acts through the tyrosine kinase neurotrophin receptor trkA, which is expressed on half of all smalldiameter dorsal root ganglia (DRG) neurones (~40% of all cells). BDNF signals via trkB and NT3 signals via trkC, which is expressed almost exclusively by large-diameter neurones. Glial cell line-derived neurotrophic factor (GDNF) receptor components [RET and GDNF family receptor a 1/2 (GFRa1/2)] are expressed in ~50% of all small-diameter neurones and 40% of large-diameter neurones15,16. Different subclasses of DRG neurones express characteristic neurochemical markers such as calcitonin gene-related peptide (CGRP), P2X3 receptors and the heavy-chain neurofilament NF200.
of the regeneration-associated immediate-early gene Jun in all injured neurones, as well as induction of the growth associated protein GAP-43, which is expressed constitutively in only a small subpopulation of primary sensory neurones4. These changes are muted following lesions that are central to the DRG (Ref. 2) and, consequently, the average rate of regeneration of dorsal root axons is only half that of their peripheral counterparts (except for those that constitutively express GAP-43, which regenerate at a normal rate4). This occurs despite the permissive Schwann cell environment of the dorsal root. In a similar manner, the weak intrinsic regenerative capacity of dorsal column axons contributes to their failure to regenerate after axotomy. Regeneration strategies that focus on the growth responses of damaged neurones have included substitutive measures in which adult neurones are replaced with embryonic neurones (Box 1). However, another approach involves boosting the inherent growth potential of the existing cells.
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An alternative treatment option is the delivery of exogenous neurotrophic factors. These molecules have been shown to promote the outgrowth of sensory neurites in vitro8, even in the presence of CNS-derived myelinassociated glycoprotein9. The potential regenerationpromoting effects of neurotrophic factors have also been investigated in vivo. Anatomical studies have shown that neurotrophic factors promote the regeneration of dorsal root axons into the spinal cord, whether delivered in fibrin glue10, via adenoviral vectors11 or intrathecally12,13. Intrathecal treatment with different neurotrophic factors resulted in the selective ingrowth of appropriate subclasses of axons13. Specifically, nerve growth factor (NGF) promoted the ingrowth of axons that express calcitonin generelated peptide (CGRP), but not axons that express NF200, which is a large-calibre fibre marker. By contrast, neurotrophin 3 (NT3) promoted ingrowth of NF200expressing axons, but not axons that express the P2X3 receptor [i.e. glial-derived neurotrophic factor (GDNF)sensitive axons]. GDNF promoted the regeneration of large- and small-calibre axons, which was expected based on the expression patterns of neurotrophic factor receptors14,15 (Fig. 1). Encouragingly, anatomically demonstrable regrowth was accompanied by synaptic re-connection, again in a selective fashion, such that NGF and GDNF treatments resulted in re-activation of dorsal horn neurones by slow-conducting sensory axons, and NT3 and GDNF treatments facilitated the recovery of postsynaptic potentials that are evoked by fast-conducting sensory fibres13. Most importantly, NGF- and GDNF-treated animals regained the ability to sense mechanically and heat-induced pain, indicating that appropriate spinal circuits were being activated by sensory input. Neurotrophic factors also promote regeneration following dorsal column lesions. For example, NT3 and NGF promote extension of CGRP-expressing sensory neurones into pre-degenerated nerve grafts that have been implanted into injured dorsal columns12. Furthermore, intrathecal administration of NT3 alone can induce sprouting and several millimetres of regeneration in the lesioned dorsal columns16 (Fig. 2g). However, an important difference between neurotrophic-factor-induced regeneration after dorsal column and dorsal root injury is that regenerating dorsal column axons do not reach their normal targets12,16.
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Fig. 2. The effects of axotomy and treatment with neurotrophin on the population of large-diameter centrally projecting dorsal root ganglia (DRG) afferents can be investigated by using cholera toxin b subunit (CTB) tracing. CTB injected into a sciatic nerve is transported transganglionically and labels central terminals in the lumbar dorsal horn (as well as motoneurones in the ventral horn) (a) and the axon collaterals that ascend in the dorsal columns (b) to terminate in the dorsal column nuclei in the brainstem (c). Dorsal root injury (d) results in the regeneration of axons, which stops at the dorsal root entry zone (DREZ; broken line). Intrathecal administration of neurotrophin 3 (NT3) results in the regeneration of dorsal root axons across the DREZ (e). Dorsal column injury (f) prevents regeneration and causes retraction of injured axons (asterisk indicates the site of a 4-week-old crush lesion). Intrathecal administration of NT3 results in the regeneration of dorsal column axons through the lesion site (g). Scale bar equivalent to 300 mm in (a), 200 mm (b), 50 mm (d,e) and 100 mm (c,f,g). Arrows indicate regenerating fibres.
This suggests that the central injury induces a barrier to growth that is insurmountable with neurotrophic factor treatment. The permissiveness of different cellular environments for regeneration Altering the CNS environment
Since landmark experiments in which CNS axons were found to grow into peripheral nerve grafts17,18, various methods have been employed to alter the non-permissive CNS environment to stimulate regeneration. Regeneration of central sensory axons has occurred across grafts of peripheral nerves and foetal spinal cord19–21. A major problem with grafting experiments is that although axons can grow across the grafts, they rarely re-enter host tissue at the graft–host interface. However, olfactory ensheathing cells (OECs)
offer the prospect of more extensive regeneration because they are highly migratory. For example, OEC transplants can promote regeneration of dorsal root projections through the DREZ into the spinal cord22. In addition, OECs can also promote the regeneration of corticospinal tract axons through the glial scar and into their appropriate pathway23. These studies have revealed that central DRG axons are capable of regeneration when the injured CNS environment is altered from being non-permissive to being more permissive to growth. Therefore, identifying the factors that are responsible for the inhibitory properties of the injured CNS is essential. Inhibitory molecules
A crucial difference between the PNS and CNS environments is the presence of peripheral Schwann cells in the
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Box 1. Taking cues from development Foetal nervous tissue is not only permissive to the regeneration of adult axons, but is also capable of growth within adult CNS. How is axonal growth in embryonic CNS possible and can the properties of embryonic tissue be exploited in adult CNS repair?
2 Spinal cord penetration (boundary cap cells) 1 Root formation (semaphorin 3A)
Bridging the gap
In vitro experiments have shown that embryonic spinal cord supports the regeneration of adult dorsal root ganglia (DRG) neuronesa. The CNS only becomes non-permissive to axonal regeneration after the first postnatal week, because dorsal root axons will regenerate into the spinal cord if there is an injury before this timeb. Embryonic spinal cord grafts are permissive to primary afferent regeneration, as demonstrated in studies by Tessler and co-workersc,d. The axons that invade the graft include all known classes of DRG neurones, become myelinated and form permanent functional synapses.
5 Terminal pattern formation (semaphorin 3A)
6 Formation of inhibitory barrier (CS-PGs)
An elevated propensity for growth
Embryonic neurones have a greater inherent capacity for growth than adult neurones. Neurones in embryonic spinal cord grafts send axons into the adult host cord and primary afferent inputs can cause induction of Fos in both transplant and host neurones, indicating that the grafts integrate primary afferent input into host circuitryd. Embryonic DRG neurones are capable of traversing the adult dorsal root entry zone (DREZ) in vitroa and in vivoc,d. This is indicated by the fact that when human or rat embryonic DRGs are placed into the cavity left after adult DRG removal, axons grow both peripherally and centrally, circumventing the DREZ and re-entering the spinal corde. A drawback to the application of these techniques is their complexity. In embryonic cord transplant experiments, adult sensory neurones must make contact with embryonic neurones, which in turn need to connect with host spinal neurones, and in DRG transplantation studies, the grafted cells must connect with appropriate peripheral and central targets.
trends in Pharmacological Sciences
Fig. I. Developmental cellular and molecular processes that are involved in spinal cord afferentation. (1) After migrating away from the neural tube, dorsal root ganglia (DRG) neurones send axons peripherally and centrally. The repulsive guidance molecule, semaphorin 3A, might direct the axons towards the entry zoneh. (2) Neural-crest-derived ‘boundary cap’ cells aid the ingrowth of sensory axons into the CNS and impede outward extension of astrocytic processesi. These might be a source of axonal chemoattractants. (3) Once within the cord, axons that are destined to convey mechanosensory or proprioceptive information ramify, sending out rostral, caudal and ventral branches, a process that is thought to be mediated by the diffusible molecule Slit-2 (Ref. j). (4) Contact-inhibiting B-class ephrins might contribute to the formation of longitudinally oriented axon bundles, such as the dorsal columnsk. (5) The termination pattern of nociceptive and proprioceptive axons might be determined by ventrally expressed semaphorin 3A, which repels small-diameter nerve growth factor (NGF)-sensitive axons but has no such effect on neurotrophin 3 (NT3)-sensitive large-diameter axonsh. (6) In the first postnatal week, inhibitory chondroitin sulfate proteoglycan (CS-PG) expression begins at the midline and spreads laterally towards the entry zone, coinciding with the end of the permissive period of the immature dorsal root entry zone (DREZ)l.
Homing signals
Several lines of evidence suggest that regenerating axons are capable of reinnervating appropriate laminae in the spinal cord. For example: (1) olfactory ensheathing cell transplants allow the ingrowth of calcitonin generelated peptide (CGRP)-expressing axons, which stop in laminae I and II (Ref. f ); (2) olfactory gliaf and neurotrophic factorsg promote re-connection with spinal neurones in such a way as to mediate spinal reflexes; and (3) intrathecal neurotrophic factor treatment promotes the growth of axons deep into the grey matterg. How do these axons find their appropriate targets? Part of the answer might be found in embryonic transplant studies in which grafts of brain were innervated in a more random manner by injured dorsal root axons in comparison with grafts of spinal cord, which caused the ingrowing axons to branch extensively and form bundlesd. The developmental program that normally guides dorsal root axons into the CNS might operate not only in transplanted embryonic cord, but also in denervated adult cord (Fig. I). If this is true, it suggests that adult neurones possess (or re-express) the ligands and/or receptors that are needed to interpret guidance cues. By understanding the process of sensory innervation of the spinal cord during development, we might be able to understand and eventually manipulate the molecules that are responsible and thereby improve sensory regeneration into and within the adult spinal cord after injury. Selected references a Golding, J.P. et al. (1999) Behaviour of DRG sensory neurites at the intact and injured adult rat dorsal root entry zone: postnatal neurites become paralysed, whilst injury improves the growth of embryonic neurites. Glia 26, 309–323 b Carlstedt, T. (1997) Nerve fibre regeneration across the peripheral–central transitional zone. J. Anat. 190, 51–56
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3,4 Branching and dorsal column formation (Slit-2, B-class ephrins)
c Fraher, J.P. (2000) The transitional zone and CNS regeneration. J. Anat. 196, 137–158 d Ramer, M.S. et al. Axon regeneration across the dorsal root entry zone. Prog. Brain Res. (in press) e Kozlova, E.N. et al. (1997) Human dorsal root ganglion neurones from embryonic donors extend axons into the host rat spinal cord along laminin-rich peripheral surroundings of the dorsal root transitional zone. J. Neurocytol. 26, 811–822 f Navarro, X. et al. (1999) Ensheathing glia transplants promote dorsal root regeneration and spinal reflex restitution after multiple lumbar rhizotomy. Ann. Neurol. 45, 207–215 g Ramer, M.S. et al. (2000) Functional regeneration of sensory axons into the adult spinal cord. Nature 403, 312–316 h Wright, D.E. et al. (1995) The guidance molecule semaphorin III is expressed in regions of spinal cord and periphery avoided by growing sensory axons. J. Comp. Neurol. 361, 321–333 i Golding, J.P. and Cohen, J. (1997) Border controls at the mammalian spinal cord: late-surviving neural crest boundary cap cells at dorsal root entry sites may regulate sensory afferent ingrowth and entry zone morphogenesis. Mol. Cell. Neurosci. 9, 381–396 j Wang, K.H. et al. (1999) Biochemical purification of a mammalian slit protein as a positive regulator of sensory axon elongation and branching. Cell 96, 771–784 k Imondi, R. et al. (2000) Complementary expression of transmembrane ephrins and their receptors in the mouse spinal cord: a possible role in constraining the orientation of longitudinally projecting axons. Development 127, 1397–1410 l Pindzola, R.R. et al. (1993) Putative inhibitory extracellular-matrix molecules at the dorsal-root entry zone of the spinal-cord during development and after root and sciatic nerve lesions. Dev. Biol. 156, 34–48
REVIEW former and central myelin in the latter. Several studies have investigated the inhibitory actions of CNS myelin on regeneration24,25. Neutralization of one constituent of CNS myelin with the antibody IN-1 promoted axonal regeneration, plasticity and some functional recovery after corticospinal tract lesions24,25. However, the observed regeneration was limited and functional synaptic contacts have not been demonstrated. The antigen that is recognized by IN-1 has recently been identified as Nogo-A, which is a potent inhibitor of neurite outgrowth of adult DRG neurones in vitro26,27. The development of reagents that target Nogo-A, Nogo-A knockout animals and mice that overexpress Nogo-A should give further insight into the role of this protein in inhibiting CNS regeneration in vivo. Several in vitro experiments have demonstrated that myelin-associated glycoprotein (MAG) can dramatically inhibit neurite outgrowth of adult DRG neurones. Myelin from MAG-null mice supported growth of longer neurites compared with wild-type myelin28. When normally permissive Schwann cells were induced to express MAG, both neurite outgrowth and branching were dramatically inhibited28. Another obstacle to regeneration is the formation of an astrocytic glial scar around the lesion site. Cells that are associated with the glial scar form a physical barrier to regenerating axons and also secrete inhibitory factors, such as chondroitin sulfate proteoglycans (CS-PGs)29. In vitro experiments have correlated the inhibition of neurite outgrowth with the expression of CS-PGs (Refs 30,31) and enzymatic degradation of CS-PGs has enabled neurite outgrowth from DRG neurones cultured on a substrate that is normally nonpermissive32. One important study used primary sensory neurones to investigate the relative importance of myelin-associated inhibition versus the inhibitory glial scar. In vivo regeneration of cultured adult DRG neurones was demonstrated when they were microtransplanted into the degenerating dorsal columns. Axons were observed to regrow until they reached the lesion site, where CS-PGs were present33. This showed that the glial scar is an important barrier to regeneration and that primary sensory neurones are capable of growing in degenerating white matter tracts, which are classically thought to be a hostile CNS environment. However, because intrinsic DRG neurones do not normally regenerate after injury, it is possible that culturing the neurones before transplantation altered their regenerative capacity, perhaps by downregulating receptors for inhibitory CNS ligands or increasing the axotomy-related cell body response. Another explanation for growth of adult DRG neurones through degenerating white matter is that although the microtransplantation technique avoids inducing a scar at the transplant site, it might also avoid damaging myelin. Thus, the myelin encountered by the regenerating DRG neurones in the degenerating dorsal columns might not express inhibitory factors, unlike the myelin that is damaged at a lesion site. Several groups of extracellular matrix molecules mediate the restrictive boundary-like properties that are ascribed
to glial cells during development34 (Box 1). These molecules might be re-expressed in the adult following injury and cause potent inhibition of growth. One such molecule, semaphorin 3A, is expressed in the ventral spinal cord during development and repels developing NGF-dependent DRG axons, restricting them to superficial laminae of the dorsal horn35. Adult sensory neurones retain their ability to respond to semaphorin 3A (Refs 36,37) and semaphorin 3A is upregulated following lesions to different areas of the adult CNS, including injury to the spinal dorsal columns, where it is primarily located in the fibroblast-like cells that are associated with the glial scar38. The uncontrolled expression of this or other chemorepulsive axon guidance molecules might play a key role in mechanisms that inhibit growth in the CNS. Treatment with neurotrophic factors following different injuries
An important point that is illustrated by primary sensory neurone injury models is that the ability of a treatment to promote regeneration might differ among models. For example, we have used neurotrophic factors to promote the regeneration of axons following both dorsal root13 and dorsal column16 injury and find that NT3 induces regeneration in both cases (Fig. 2e,g), but GDNF is only effective following rhizotomy. One reason for this difference might be that the scar and cavitation39 that are necessarily associated with the dorsal column lesion (and that are not associated with rhizotomy) form a barrier to axonal growth that is surmountable with NT3, but not GDNF treatment. Another possibility is that the same factors might have different effects on damaged neurones that depend on whether the neurones are in contact with CNS myelin before or after initiation of treatment. In vitro experiments have shown that prior treatment with neurotrophic factors allowed sensory neurones to overcome MAG-induced inhibition9. In the case of rhizotomy, the lesion is made outside the spinal cord. If treatment follows immediately, the growing axons receive exogenous neurotrophic factors before they encounter CNS tissue. This is not the case with dorsal column lesions, in which the lesion is within the CNS. Concluding remarks
Achieving successful regeneration following adult CNS injury will almost certainly require a combination of treatments in which both the intrinsic neuronal growth capacity and the inhibitory CNS environment are targeted. In sensory systems, there have already been some attempts to combine growth-promoting neurotrophic factors or conditioning lesions with growth-permissive grafts or foetal transplants21. However, although boosting the regenerative capacity of DRG neurones and reducing inhibition might allow sensory regeneration within the spinal cord, this will have little significance if growing axons do not activate useful postsynaptic pathways. Therefore, it will be necessary to guide regenerating axons to their appropriate targets. Although our knowledge of developmental axon guidance mechanisms is increasing rapidly (Box 1), it is not
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REVIEW clear to what extent these mechanisms might be re-activated following CNS injury in the adult. Although we are still some way from achieving the ultimate goal of regeneration, appropriate reconnection and functional recovery following adult CNS injury, many important advances have been made in understanding the mechanisms that underlie neuronal repair and regeneration. The unique anatomical organization of primary sensory neurones and their projections offers a valuable tool to further differentiate such mechanisms. References
Acknowledgements We thank Tim Boucher for critical evaluation of the manuscript and Pedran Badakhchani for illustrations. E.J.B. is supported by the Wellcome Trust. M.S.R. is supported by the EU and the Canadian MRC.
1 Short, D. et al. High-dose methylprednisolone in the management of acute spinal cord injury – a systematic review from a clinical perspective. Spinal Cord (in press) 2 Chong, M.S. et al. (1996) Intrinsic versus extrinsic factors in determining the regeneration of the central processes of rat dorsal root ganglion neurones: the influence of a peripheral nerve graft. J. Comp. Neurol. 370, 97–104 3 Carlstedt, T. (1997) Nerve fibre regeneration across the peripheralcentral transitional zone. J. Anat. 190, 51–56 4 Andersen, L.B. and Schreyer, D.J. (1999) Constitutive expression of GAP-43 correlates with rapid, but not slow regrowth of injured dorsal root axons in the adult rat. Exp. Neurol. 155, 157–164 5 Chong, M.S. et al. (1999) Axonal regeneration from injured dorsal roots into the spinal cord of adult rats. J. Comp. Neurol. 410, 42–54 6 Neumann, S. and Woolf, C.J. (1999) Regeneration of dorsal column fibers into and beyond the lesion site following adult spinal cord injury. Neurone 23, 83–91 7 Richardson, P.M. and Verge, V.M. (1986) The induction of a regenerative propensity in sensory neurones following peripheral axonal injury. J. Neurocytol. 15, 585–594 8 Gavazzi, I. et al. (1999) Growth responses of different subpopulations of adult sensory neurones to neurotrophic factors in vitro. Eur. J. Neurosci. 11, 3405–3414 9 Cai, D. et al. (1999) Prior exposure to neurotrophins blocks inhibition of axonal regeneration by MAG and myelin via a cAMP-dependent mechanism. Neurone 22, 89–101 10 Iwaya, K. et al. (1999) Neurotrophic agents in fibrin glue mediate adult dorsal root regeneration into spinal cord. Neurosurgery 44, 589–595 11 Zhang, Y. et al. (1998) NT-3 delivered by an adenoviral vector induces injured dorsal root axons to regenerate into the spinal cord of adult rats. J. Neurosci. Res. 54, 554–562 12 Oudega, M. and Hagg, T. (1999) Neurotrophins promote regeneration of sensory axons in the adult rat spinal cord. Brain Res. 818, 431–438 13 Ramer, M.S. et al. (2000) Functional regeneration of sensory axons into the adult spinal cord. Nature 403, 312–316 14 Bennett, D.L.H. et al. (1998) A distinct subgroup of small DRG cells express GDNF receptor components and GDNF is protective for these neurones after nerve injury. J. Neurosci. 18, 3059–3072 15 Bennett, D.L.H. et al. (2000) The glial cell line-derived neurotrophic factor family receptor components are differentially regulated within sensory neurones after nerve injury. J. Neurosci. 20, 427–437 16 Bradbury, E.J. et al. (1999) NT-3 promotes growth of lesioned adult rat sensory axons ascending in the dorsal columns of the spinal cord. Eur. J. Neurosci. 11, 3873–3883
17 Richardson, P.M. et al. (1980) Axons from CNS neurones regenerate into PNS grafts. Nature 284, 264–265 18 David, S. and Aguayo, A.J. (1981) Axonal elongation into peripheral nervous system ‘bridges’ after central nervous system injury in adult rats. Science 214, 931–933 19 Fawcett, J.W. (1998) Spinal cord repair: from experimental models to human application. Spinal Cord 36, 811–817 20 Ramer, M.S. et al. Progress in spinal cord research: a refined strategy for the International Spinal Research Trust. Spinal Cord (in press) 21 Ramer, M.S. et al. Axon regeneration across the dorsal root entry zone. Prog. Brain Res. (in press) 22 Navarro, X. et al. (1999) Ensheathing glia transplants promote dorsal root regeneration and spinal reflex restitution after multiple lumbar rhizotomy. Ann. Neurol. 45, 207–215 23 Li, Y. et al. (1998) Regeneration of adult rat corticospinal axons induced by transplanted olfactory ensheathing cells. J. Neurosci. 18, 10514–10524 24 Bandtlow, C.E. and Schwab, M.E. (2000) NI-35/250/nogo-a: a neurite growth inhibitor restricting structural plasticity and regeneration of nerve fibers in the adult vertebrate CNS. Glia 29, 175–181 25 Qiu, J. et al. (2000) Glial inhibition of nerve regeneration in the mature mammalian CNS. Glia 29, 166–174 26 Chen, M.S. et al. (2000) Nogo-A is a myelin-associated neurite outgrowth inhibitor and an antigen for monoclonal antibody IN-1. Nature 403, 434–439 27 GrandPre, T. et al. (2000) Identification of the Nogo inhibitor of axon regeneration as a Reticulon protein. Nature 403, 439–444 28 Shen, Y.J. et al. (1998) Myelin-associated glycoprotein in myelin and expressed by Schwann cells inhibits axonal regeneration and branching. Mol. Cell. Neurosci. 12, 79–91 29 Fawcett, J.W. and Asher, R.A. (1999) The glial scar and central nervous system repair. Brain Res. Bull. 49, 377–391 30 Smith-Thomas, L.C. et al. (1994) An inhibitor of neurite outgrowth produced by astrocytes. J. Cell Sci. 107, 1687–1695 31 Fidler, P.S. et al. (1999) Comparing astrocytic cell lines that are inhibitory or permissive for axon growth: the major axon-inhibitory proteoglycan is NG2. J. Neurosci. 19, 8778–8788 32 Zuo, J. et al. (1998) Degradation of chondroitin sulfate proteoglycan enhances the neurite-promoting potential of spinal cord tissue. Exp. Neurol. 154, 654–625 33 Davies, S.J. et al. (1999) Robust regeneration of adult sensory axons in degenerating white matter of the adult rat spinal cord. J. Neurosci. 19, 5810–5822 34 Faissner, A. and Steindler, D. (1995) Boundaries and inhibitory molecules in developing neural tissues. Glia 13, 233–254 35 Goodman, C.S. (1996) Mechanisms and molecules that control growth cone guidance. Annu. Rev. Neurosci. 19, 341–377 36 Tanelian, D.L. et al. (1997) Semaphorin III can repulse and inhibit adult sensory afferents in vivo. Nat. Med. 3, 1398–1401 37 Reza, J.N. et al. (1999) Neuropilin-1 is expressed on adult mammalian dorsal root ganglion neurones and mediates semaphorin3a/collapsin-1induced growth cone collapse by small-diameter sensory afferents. Mol. Cell Neurosci. 14, 317–326 38 Pasterkamp, R.J. et al. (1999) Expression of the gene encoding the chemorepellent semaphorin III is induced in the fibroblast component of neural scar tissue formed following injuries of adult but not neonatal CNS. Mol. Cell. Neurosci. 13, 143–166 39 Fitch, M.T. et al. (1999) Cellular and molecular mechanisms of glial scarring and progressive cavitation: in vivo and in vitro analysis of inflammation-induced secondary injury after CNS trauma. J. Neurosci. 19, 8182–8198
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