Regeneration of immature mammalian spinal cord after injury

REVIEW Regeneration of immature mammalian spinal cord after injury John Nichollsand Norman Saunders In this reviewwe describethe growth of regeneratingfibres through lesionsin immature mammalian spinal cord. In newborn opossums and foetalrats,repairoccursrapidlyand reliably

withoutantibodies,implantsor bridgesof undamagedspinalcord.In the neonatalopossumone cancomparerecoveryfrom lesionsmade to the CNS at variousstagesof developmentin the animal and in culture.As the CNS matures,the capacityfor regenerationceasesabruptly.In particular,the extracellularmatrixand moleculesassociated withgliahavebeenshownto playa role in promotingand inhibitingregeneration.Major problemsconcernthe precisionwithwhich regeneratingaxonsbecomereconnectedto theirtargets,andthe specificity neededfor recovery of function. Trends Neurosci. (1996)19,229-234

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PINAL-CORDINJURY,which has such catastrophic consequencesfor the victim, presentsa challenging problem for neuroscientist as well as clinicians. Why is it that the adult CNS of higher vertebrates, such as birds and mammals, has little or no capacity for functionally useful regeneration? Although it has been known since the time of Ram6n y Cajall that some damagedCNSneurones can sendout sprouts,they fail to growthrough lesions and fail to make connections. By contrast, peripheralnerves regeneratesuccessfully with recovery of function after injury as do certain connections in the CNS of fish, amphibians, reptiles for example,neurones and invertebratesz-6 . In leeches, can reform synapseson their targets in the CNSwith a high degreeof precision. One approach toward understanding mechanisms that prevent neurite outgrowthin mammaIianCNSis to compare systems in which regeneration does and does not occur so as to pinpoint the key differences. Another fascinating and worthwhile comparison is with immature mammalian CNS, which Ramtm y Cajall showeddoes have some capacity to repairitself after injury. Recent experiments made on embryonic or neonatal animals including chicks7, hamsters8, opossumsand rats have begun to throw some light on why this shouldbe so in an immature CNSbut not in the adult.In particular,one clearcorrelationhasemerged between events occurringduringdevelopmentof glial cells and the loss of ability for repair. Other, as yet illdefined, factors are also likely to be involved. Repairof lesionswith grafisfrom peripheralnerve or foetalCNS

A key step in defining problems of CNS regeneration was the use of peripheralnerve grafts to bridge distant parts of the CNS, for example from the retina to the superiorcolliculus or from the thalamus to the spinal cord. Such experiments have demonstrated that: (1) adult CNS neurones still possess intrinsic mechanisms for axonal growth over long distances if they are providedwith an appropriateenvironment; and (2) adult CNS tissue is unfavorable for growth. Copyright O 1996, Elswier Science Ltd. All rights resewed. 0166-

2236/96/$15.00

Thus, axons that leave the graft to enter the CNSstop growingabruptly9.At the same time adult CNS does support growth of embryonic neurones which can sprout for long distances and form connections after they have been implanted1W13. One strategyfor promoting CNS repair in animals has been to implant foetal CNStissue into spinal-cord lesions.Forexample,fibrescan growinto, through and beyond a graft of embryonic rat CNStissue implanted into the spinalcord of a neonatal rat. Impressivestructural and functional recoverycan thereby be brought about in animals otherwise too old to regenerate14’15. Sincefibrescontinueto growcaudallybeyondthe graft, this impliesthat the morecaudalregionsof the cordare still providing a favorable environment for neurite growth,in spiteof the fact that without the foetal CNS implant no such growth occurs. This is in contrast to the situationin foetalrat or newbornopossumin which axonal growthoccurswithout the need for an implant, as discussedbelow. Furthermoresince some degreeof functional recoveryhas been reportedin the animals with lesion implants, this implies that adequate synaptic connections can be established,notwithstanding the massivedisruptionpresent at the site of injury. It remains to be determined whether the connections are normal, and if they involve regeneratingor newly growing fibres. Similar grafts with embryonic tissue or Schwann cells into adult spinal cord can induce John Nicholls is sproutingbut do not leadto comparableregenerationlG. at the Dept of Together these results indicate that immature spinal Pharmacology, cord can support axonal growth, and that the neur- Biozentrum, ofBaseZ, ones at earlier stages of development have a greater Universi~ CH-4056Basel, potential for growththan those later in life. Moleculesthat inhibitneuriteoutgrowth

In a systematic and elegant series of experiments, growth-inhibitory molecules associated with myelin from the CNS have been isolated and characterized. KnownasNI-35/250(N1standingfor ‘neuriteinhibitor’ and 35/250for the molecularweights),these molecules blockthe outgrowthof neuritesin culture17’18. Moreover, growthcones that makecontact with oligodendrocytes PII: S0166-2236(96)1

OO21-7

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Switzerland,and NormanSaunders is at the Deptof Anatomyand Physiology, Universityof Tasmania, Tasmania, Australia.

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- Spinal-cord regeneration

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R

c

Fig.1. Adult and newly born opossums. (A)The adult opossum is a friend/y creature the size of a snsa//rat. (B) The number of pups born ranges from 2–12. The pups can be removed one at a time so that siblings can be compared at different ages. The main problem with this procedureis the riskofcanniba/ism by the mother. (C) At postnata/ day2 the eyes and ears are rudimentary; the front limbs can move, a/beit ineffective/~ whereas the hind /imbs are sti//poor/y developed buds. The animal cannot right itself or craw/. It can however breathe and suckle. Scale bar, 10 mm. Savioand Schwabzlhave shown that demyelination of rat spinal cord by exposureto X-raysalso produced an altered environment through which axons could grow neurites. In two series of experiments, Steeves and colleaguesZZZ3 Showed the importance of myelin in blocking successful growth of axons following spinal-cord injury in the chick. In the first series of experiments, immunological suppressionof the onset of myelination in chick embryos extended the period during which functional repair of the injured spinal cord could occur. In the second seriesof experiments, immunologicaldisruptionof myelin in post-hatching chicks (in which the spinal-cord axons are fully myelinated) resulted .in a permissive growth of neurites following injury and some evidence of synapseformation. In additionit has been shown that myelin-associatedglycoprotein (MAG) inhibits axon growth‘WSbut, unlike the experimentsof Schwaband of Steeves, MAG inhibition has only been demonstrated s. far in vit-ro2G. The injured spinal cords of knockout mice that lack MAGstill cannot regenerate, but they can do,so in the presence of IN-1 (Ref. 27). From all these results emerges the concept that first, failure of regeneration after injury in mature mammals is correlatedwith the development of oligodendrocytes and myelin; and second, interference with myelin or associated inhibitory factors allows extensive growth following injury. In fish, the role of IN-1 in preventing regeneration is still controversial (for review,see Ref. 28). Regenerationof CNS in neonatalmarsupials

or myelin from the CNS collapselg.These inhibitory effects are in turn blocked by a monoclinal antibody (IN-l), in the presence of which neurites continue to grow and even traverseoligodendrocytesand myelin. That NI-35/250proteins play a part in preventing regeneration was shown by making spinal-cordlesions in the presence of antibody IN-1. Injured fibres grew aroundand beyondlesionsfor long distances,provided that antibody was suppliedby secretion from hybridoma cells implanted into the ventricles of adult ratszo. 230

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Marsupials have certain advantages for studying regeneration in immature mammalian CNS and for determining the stage of development at which it stops. Newborn opossums are born at a highly immature stage, particularly with respect to their CNS and limb development. In many respects they correspond to E13-E14 rat embryos29’30. Thus, the cortex consists of two layers of cells (neuroependymaand marginal zone). The cerebellum is still rudimentary. There is a more pronounced rostrocaudalgradient of maturity in opossums than in rats, so that the caudal spinal cord is very poorly developedin opossums. For the first three weeks, their behavioral repertoire consists mainly of breathing and sucking milk from the mother to whose surfacethe pupscling without a protective pouch (Monodelphis dornestica) or within the pouch (Didelphis virg”niana). Monodelphis domestica, a South American opossum, is a small rat-like animal that can be bred in the laboratory.Adultand neonatal M. domestica are shown in Fig. 1. Aswith Didelphis virfl”niana,the North American opossum, immature pups can be operated on under anesthesia whilestill attachedto the mother. Afterthe spinalcord has been partiallyor completelytransected, the main difficulty in following their progressis the

J. Nicholls

danger of cannibalism by the mother31-33.Such M. domestica or D. virginiana pups that do surviveshow an extraordinarydegreeof recovery.Spinal cords that have been completely transectedor crushed,repairso that the site of the lesion becomes barely noticeable under the dissecting microscope. In animals that are killed immediately after a crush or cut to verify the lesioning procedure,serial sections show massiveand generallycomplete destructionat the site of injury. In M. domestica pups lesioned in the same way but observedafter weeksor months, the spinal cord shows well-developedpatterns of grey and white matter as well as characteristic dorsal and ventral horns at the site of the lesionsz. Representativelongitudinal and cross-sections are shown in Fig. 2. In such animals conduction of impulsesthrough the spinal-cordlesion is re-established.Especiallyimpressiveis the degreeof functional recoveryof co-ordinatedwalking,climbing and swimming, assessed by observing the animals directlyor by recordingwith video. In adult opossums, as in other mammals, the adult CNS does not regenerate.The transition point occurs in the early days of postnatal life. Thus, lesions made in the retina of newborn M. domestica are followedby regenerationof ganglion-cellaxons providedthat the cut is made before postnatal day 12 (Ref. 33). In the spinal cord the critical period is similar but there is a lack of homogeneity in the exact transition point for each descendingspinalprojection. Martin and his colleagues have shown that rubrospinal‘fibres can continue to regenerate, even after other tracts cannot, until about postnatal day 30. However,for a complete transection of the cervical-thoracic cord the upper limit is similar (postnatal day 11)35-37.

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Regenerationor growthof newfibres?

There is an inherent interpretation problem with experiments examining the response to injury in immature spinal cord. At the time when lesions are made, major spinal-cordtracts have not developed,so some fibres might not have arrived at the site of injury. When axons subsequently grow across the lesion, it is important to know whether they are regeneratingfrom injured cells or are new, uninjured Fig.2. Regeneration and recovery of function aflerlesions of the spinal cord. Anacutecrush fibres that had not reached the site of injury at the madeunderanesthesiato opossumpupswhile still attached to the mother causes a massive time when it was made. Severalgroups have studied lesion with disruption of the vertebral column and transection of the spinal cord. (A) shows a this problem using a double-labelling technique in silver-stained longitudinal section of the spin:l cord prepared immediately following crush which one marker (for example, rhodamine dextran injury in a five-day old pup. (B) shows a similar longitudinal section prepared two hours later. amine) is injected into the spinal cord prior to injury. Bythis time, the two ends of the cord have often become apposed. (C and D) showcrossThis labels, by retrograde transport, cell bodies of sectionsof the spinal cord at the level of Tz in a normal adult opossum (C)or in an animal that axons present at the subsequent site of crushing. had been operated on at this site three months previously at postnatal day 5. Thisanimal Following a recovery period after crushing, a second couldwalk, swimand climb.Note that at the site of the lesionthe spina/cordhad repaired label (for example, cascade blue dextran amine) is itselfwithremarkablefidelity.Scalebars,0.2 mm(A andB),and0.5 mm (C andD). Adapted injected proximallyto the first label. Cell bodies origi- from Ref.32. nating from fibres traversingthe injury will again be labelled. Those labelled with both dyes were, there- cussed in detail, for example by Bates and Stelzner40, fore, both present at the crush and have since regener- and by Hasan et al.’. One mechanism that is often ated; those with only the first label failed to regener- hard to rule out is the possibility of collateral sproutate, whereas those with only the second label were ing from uninjured neurones, particularly in experinewly growingfibres which had not reached the site ments involving only a hemisection. An alternative of injury at the time when it was made. Such experi- approachuses an in vitro preparation,and is described ments have been carried out in chick embryos7’38’39in the next section. and neonatal rats with hemisected cords and foetal Regenerationof immature mammalianCNS in implants40,41. All of these experiments show double culture labellingof about 30-40V0of cell bodiesthat originate Certainadvantagesfor the studyof spinal-cordrepair from brainstem nuclei. But there are severalmethodological pitfalls in such experimentsthat havebeen dis- accrue from CNSpreparationsthat can be maintained TINSVO1. 19, N0. 6,1996

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A

Fig. 3. Thei$olatedCM of cmopossum. (A) The CIW at the time of birth is still immature and correspondsroughly to that of an El 3-E14 rat embrya. The farebrain consistsof two large vesiclessurrounded by plates af dividing cells with no mature neurones. The cerebellum is absent from the dorsal part of the medulla. At the same time the neurones that camprise respiratory circuits are mature and functioning. A gradient of development is apparent along the spinal cord such that caudal segments are less mature. In culture, the isolated CNS continues to develop and to produce reflexes as well as fictive respiration. The structure of the spinal card is well maintained in culture as shown by the cross-sections(B) showing normal spinal cord and (C) showing a section of card that had been maintained in culture for five days (Basal Medium Eagles with foetal calf serum, oxygenated with 95% 0, and 5% CO,). Some degeneration is apparent in dorsal regions af the spinal cord; in this preparation the dorsalroatganglia have been removed. If the dorsal-raotganglia are retained very little degeneration appears. Notable is the normal structure of motoneurones in ventral regions af the spinal cord which do not undergo massive cell death or chromatalysis43. Similarly the fine structure abserved using EM has not changed after five days in culture: dendrites, radial glia and synapses all retain their normal appearances. Scale bar, 0.1 mm.

Fig. 4. The critical period for regeneration in neonatal opossum spinal cord and its prolongation by the application of antibodies. (A) Profuse outgrowth of fibres occurs into, through and beyand a crushmade at the level af C6 in the spinal cord removed from a six-dayold opassum. The fibres in the living preparation were stained with the carbocyanine dye Dil. Such regeneration occurred routinely in preparations in culture taken fram young animals. Bycontrast the spinal cords removed fram 72-day-old animals showed no regeneration: Axons failed to invade the crush site. (B) Application of the antibody IN-7 to the spinal cord removed from a 73-day-aid apossum enabled fibres to grow into the site of the lesion after four days in culture. The growth was nat as profuse as that seen in spinal cords removed from animals between postnatal day three and nine. Comparable growth was never seen in any animal 12 days or older without antibody. The antibody IN-1 blocks the growth inhibitory effect af the myelin-associated protein N1-35/250.

in culture. As in the chick, fibre outgrowth can be observedin living preparations,and drugsas well as antibodies can be applieddirectly to the preparation via the culture medium. The CNS of neonatal M. domestica aged1-18 dayscan be removedin its entirety and maintained for periodsof seven days or more in suitable medium4=4.Under these conditions reflexes persist, fine structure remains surprisinglynormal in appearance, and cell division continues in the dish. (Note that cell death is minimal in cultured opossum CNS;this paperis therefore exceptional in not paying homageto apoptosis.)Althoughallcranialandperipheral nerveshave been severed,the variouspartsof the CNS been shown to growpreferentiallywhile in direct conarestillin continuity,unlikein a slicewhereconnections tact with the basal lamina under the pia mater. Later, to neighboring areashaveinevitablybeen broken.Fig- fibresgrowalong the pioneer axons; radialglia do not ure 3 showsthe isolatedCNSof a five-dayold opossum invade the crush or provide a substrate for growth48. and cross-sectionsof spinalcord immediatelyafter dis- To test whether repair is due to the regrowth of cut section and after five daysin culture.When the spinal axons or to new growth of undamagedaxons into the cord is crushedwith forceps or completely transected lesion, double-labellingexperiments have been made with scissors, repair occurs rapidly and reliably45-47. with dyesappliedbefore and after injury. Such experiWithin five days the lesion is traversedby axons that ments have confirmed that true regeneration of cut conduct impulses.Figure4A showsan example of the axons occurs49.Thus, in Fig. 5 the dorsal-rootganglion profuseoutgrowththat occurredafter five daysin cul- cells labelled by both red and green dyes had been ture in the CNS from a six-dayold opossum. Embry- lesioned and had then regenerated.In living preparonic rat CNS,at 15-17 days,showssimilarsurvivaland ations one can directly observe a fibre that had prerepairin culture3AIn . the isolatedCNSfrom Op0S5Um, viouslybeen labelledas it growsinto the lesion. Moreover,Fig. 6 showsthat impulsesin dorsal-root axons from dorsal-rootganglioncells labelledwith the carbocyanine dye DiI can be followed by video mi- axons that have grown across the lesion once again croscopyas they grow.UsingEM the same fibres have elicit discharges in ventral roots either rostrally or 232

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caudallydependingon the site of the lesion in relation to the ganglion. Whether these connections are monosynaptic has not yet been shown. As the opossum matures the scaffold of radial glial cells becomes replaced by oligodendrocytes that develop at about eight days and astrocytes that develop at about nine days. With the appearance of both these cells and myelin, the growth inhibitory protein NI-35/250 begins to increase rapidly in concentration between postnatal days 8-15 (Ref. 50). The critical period at which regeneration stops coincides with myelination and oligodendrocytedevelopment,as it does in chick. Thus, it is after postnatal day 12 in isolated CNSfrom opossum that fibres cease to enter or traverselesions of spinal cord in the cervical region. This coincides exactly with the time that retinal and spinal-cord regeneration ceases to occur in neonatal opossums animals, attached to the mother32,33,36 . A5 in operated the critical period at which regeneration stops in culture is not uniform: caudal regions of the spinal cord and dorsally located tracts of dorsal-root ganglion cells become myelinated later and continue to regenerate until 16–18 days after birth. Contrary to what one might have guessed neither oligodendrocytes nor astrocytes invade the crush site or form a scarto block regeneration48;microglialcells are rare or absent. The critical period at which growth stops can be extended by adding the antibody IN-1 to the culture medium49.Table 1 showsthat regenerationof cervical spinal cord continues beyond the critical period for several days provided that the inhibitory action of NI-35/250 has been antagonized. The outgrowth is sparserthan that in youngeranimalsbut it is unequivocal and reliable. Control IgM antibodies against peroxidase are, like IN-1, able to diffuse through the isolated CNS but they do not influence neurite outgrowth. Figure 4 shows the cessation of growth at postnatal day 12 and the effect when IN-1 antibody is appliedto the culture medium. Prospectsfor spinal-cordrepair in patients

What can the neuroscientist workingin the laboratory with experimental animals tell victims of spinalcord injury today about the chances for effective new treatments? At present, the only possible answer is that no new therapy is yet at the stage where clinical trials could be contemplated. The problems are immense. They include the heterogeneous types of lesions, ranging from compression to transection, with variable degrees of hemorrhage, tissue destruction, and variable delays between injury and treatment. The time that elapses might be of key importance: few neuroscientist making experimental lesions in animal spinal cord wouldwaitdaysor weeksbefore testing their new procedures. In the long run it might become practicable

and N. Saunders

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- Spinal-cord regeneration

DRG

Spinal cord

II 1 II

Lesion

DiO

Dil

Fig.5. Evidence far regeneration of severed axons. Sinceexperiments areperformedin immature animals, tests are required to distinguish whether the fibres thatgrow through a lesionare fibres that were damaged by the injury or are newly grown fibres that approach the lesion in the course of development. The first label, DiO, is applied prior to the crush injury and labels a population of dorsal-root ganglion cells, shown in green below. Fivedaysatier the crush, to allow time for repair to tokeplace, a second application of dye is made using Dil (red). The two dyescan be comp/ete/y separated by the use of appropriate filters. A cell that is doubly Iabelled by both dyes therefore represents a cell that was present before the injury and that has grown back into the /esion.These experiments suggest that at least 20% of the fibres that grow through a crush do indeed regenerate. Scale bar, 25pm. Adaptedfrom Ref.49.

to create bridgeswith peripheralnerve, Schwann cells or embryonic tissue, or to apply a cocktail of monoclinal antibodies against inhibitory molecules P5 opossum; six days in culture

Fig. 6. Regenerated dorsal-root axons are capable of eliciting discharges in ventral roots. Stimulation of dorsal roots gave rise to o volley in ventral roots on”the other side of a crush five days after the lesion had beenmade with the preparation iti culture. These results suggest that dorsal-root ganglion fibres become reconnected to motor neurones in the adjacentsegment. Whether such connections are monosynaptic orpolysynaptic has not been established. Adaptedfrom Ref.48.

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TABLE 1. Repairof opossum spinalcordin culture Age

(days)

Regeneration before andatlercritical period

Regeneration afterthecritical periodin thepresence ofantibody IN-I

<10 >12

30 out of 66 0 out of 59

Control —

27 out of 46

0 out of 8

The IN- I antibodyblocksthe attion of the growth inhibitoryprotein (N1-35/250) associatedwith myelin.All animalsin these serieswere comparedin simultaneoustrials. The isolatedCNS was crushedat the level of C6 and allowed to remainin culture for five days before testing for regeneration.Sincepublicationof Refs49 and50 numerousother trialshaveconfirmedtheseresults.

together with neurotrophic molecules that promote growth. Promising structural and behavioral results have been achieved in rats by chronically infusing IN-1, neurotrophin-3 or both together5’-53. Apartfrom the purelytechnical problemsof obtaining sufficient material and applying it, one major hurdle has been the paucity and slow rate of growth into tissue on either side of the graft. Another is our inadequate knowledge of how many growthinhibitory and growth-promoting molecules exist, and how their receptors change during development and following lesions. Equallyimportant is the specificity of connections that are made by regenerating axons. Presumablyeven inaccurate connections could be corrected for by experience - except in the disastrous eventuality of ascendingfibres becoming reconnected so as to allow perception of pain. It is sad but true that we have little encouragement to offer the paralysed patient of today. And yet the preceding paragraph shows that new strategies can at least be contemplated. This wouldnot have been possiblejust a few years ago. Concludingremarks

A major recent advance is that pieces of the puzzle are becoming availableeven though their place in the picture as a whole is not yet known. Thus, adult CNS neurones can indeed grow for long distances and make connections if they are providedwith an appropriate environment; and the spinal cord in an immature mammal can repair as effectively as that in a Acknowledgements We aregratefil to frog or a fish. Particularly encouraging for future our colleagues, research is the abrupt transition that occurs in just a W. Adams, the late few daysin earlylife. One can imagineperformingdifS. Ercclkar, ference screeningof mRNAsin two agesof spinal cord J. Femandez, that are separatedby only 2-3 days of development. G. Knott, That one factor on its own prevents regeneration is M. Schwab and unlikely. After all, unmyelinated nerve fibres in Z. Vargawho mature mammalian chick spinal cord are distant from andmyelinbut they donot regenerate. collaborated in the oligodendrocytes As in other systems, it could be that essential new experimentsreported here. We also wish information will transform problems such as these. to thankP. Battig For example, one simply could not have invented the forphotography. concept of nerve growth factors without the experiWe thankthe Swiss ments of Rita Levi-Montalcini54,or conceived of NationalFundsand columnar processingof information in the visual cortheInternational tex without the experimentsof Hubeland Wiesels5.In ResearchInstitute a recent authoritative book entitled Principles and forParaplegiafor Practice of RestorativeNeurologya discussionof how to grantstoJGN, and bridge the gap between theory and practice by Young concludeswith: ‘We appearto be at the theAustralian and SarkaratisG ResearchCouncilfor threshold of exciting new developments in techgrantsto NRS. nology transfer, functional neurosurgeryand pharma234

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cotherapy. Neurobiologicalresearch will provide the miracles of tomorrow’. One’s hope is that this might turn out to be true. Selectedreferences 1

Ram6ny Cajal,S. (1928)Degeneration and Regeneration of the

NervousSystem; tmrrsl. (1959),Hafner 2 Sperry,R.W. (1963)FYoc.Natl Acad.Sci.USA50, 703-710 3 Zottoli, S.J. et aL (1994)Prog.BrainRes.103,219-228 4 Rovainen, C.M. (1976) J. Comp. Neurol. 168, 545-554 72, 847-860 5 McClellan, A.D. (1994) J. Neurophysiol. 6 Von Bemhardi, R. and Muller, K.J. (1995) ]. NeurobioL27,

353-366 7 Hasan, S.J. et al. (1993) J. Neurosci. 13, 492-507 8 Keifer,J. and Kalil, K. (1991) fixp.NeuroL 111, 98-105 9 Carter, D.A., Bray, G.M. and Aguayo, A.J. (1994) J. Neurosci.

—-,-. - -. —

14. 590-s98

10 Wictorin,

K. and Bjmklund, A. (1992) NeuroReport3,

1045-1048 11 Li, Y. and Raismen, G. (1993) Brain Res. 629, 115-127 12 Rossi, F., Borsello, T. and Strata, P. (1992) Eur. J. Nearosci.4, 589-593 13 Sotelo, C. and Alvarado-Mallart, R.M. (1991) TrendsNeurosci.

14.350-355 14 Bregnran, B.S. et al. (1993)Exp. NeuroL123,3-16 15 Iwashita, Y., Kawaguchi,S. and Murata, M. (1994) Nature367, 167-170 16 Xu, X.M. et al. (1995) J. Comp. NeuroL351, 145-160 17 Caroni, P. and Schwab, M.E. (1988) J. CellBiol.106,1281-1288 18 Caroni, P. and Schwab, M.E. (1988) Neuron 1, 85-96 19 Bandtlow, C.E. et al. (1993) Science 259, 8@83 20 Schnell, L. end Schwab, M.E. (1990) Nature 343, 269-272 21 Savio, T. and Schwab, M.E. (1990) Proc.Natl Acad. Sci. USA87, 4130-4133 22 Keirstead, H.S. et aL (1992) Proc. NatZAcad. Sci. USA 89, 11664-11668 23 Keirstead,H.S. et al. (1995) J. Neurosci. 15, 6963-6974 24 McKerracher,L. et aL (1994) Neuron13, 805-811 25 Mukhopadhyay, G. et al. (1994) Neuron 13, 757-767 26 Filbin, M.T. (1995) Curr. Opin. NeccrobioL5, 588-595 27 Bartsch, M. et aL (1995) Neuron 15, 1375-1381 28 Sivron, T. and Schwartz, M. (1994) Trends Neurosci. 17, 277-281 29 Kraus, D.B. and Fadem, B.H. (1987) Lab. Anim. Sci. 37, 478-482 30 Saunders,N.R. et al. (1989) Anat. Errtbryol.173, 81-94 31 Xu, X.M. and Martin, G.F. (1991) J. Comp. NeuroL 313, 103-112 32 Saunders, N.R. et al. (1995) Clin. Exp.Pharmacol.Physiol.22, 518-S26 33 McLaren, R. and Taylor, J.A. (1995) Eur. J. Neurosci. 7, 2111-2118 34

Saunders, N.R. et al. (1992) Proc. R. Soc. LondonSer. B 150,

171-180 35 Xu, X.M. and Martin, G.F. (1992) J. Nercrotrauma 9, 93-105 36 Wang, X.M., Tennan, J.R. and Martin, G.F. J. Comp. Neurol. (in press) 37 Martin, G.F. et aL (1994) Prog.Brain Res. 103, 175-201 38 Shimizu, L. et al. (1990)J. NeurobioL21, 918-937 39 Steeves,J.D. et al. (1994)Prog,Brain Res. 103, 243-262 40 Bates, C.A. and Stelzner, D.J. (1993) Exp. NeuroL 123, 106-117 41 Bemstein-Goral, H. and Bregrrsan,B.S. (1993) Exp. NeuroL 123,

118-132 42 Nicholls, J.G. et aL (1990) J. Exp. BioL 152, 1-15 43 Stewart, R.R. et al. [1991) J. Exp. BioL161, 25-41 44 Mallg&d, K. et al. (1994) J. NeurocytoL 23, 151-165 45 Treheme, J.M. et al. (1992) Proc. Nat/ Acad. Sci. USA 89, 431-434 46 Woodward, S.K.A.et aZ.(1993) J. Exp. BioL 176, 77-88 47 Vischer, H.A. (1994) Soc.Neurosci.Abstr. 20, 1270 48 Varga, Z.M. et al. (1996)J. Comp. Neurol. 366, 600-612 49 Varga, Z.M., Schwab, M.E. and Nicholls, J.G. (1995) Proc.Nat2 Acad. Sci. USA92, 10959-10963 50 Varga, Z.M. et aL (1995) Eur. J. Neurosci.7, 2119-2129 51 Diener, P.S. and Bregman, B.S. (1994) NeuroReport 5, 1913-1917 52 Schnell, L. et al. (1994)Nature 367, 170-173 53 Bregman, B.S. et al. (1995) Nature 378, 498-501 54 Levi-Montalcini, R. (1987) EMBOJ. 6, 1145-1154 55 Hubel, D.H. (1988) in Eye,Brain and Vision, ScientificAmerican

Library 56 Young, R.R. and Sarkarati, M. (1992) in Principles and Practice of Restorative Neurology (Young,R.R.and Delwaide, P.J., eds), pp. 125-135,Butte~orth-Heinemann