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Axons damaged in the mammalian brain and spinal cord do notordinarily regenerate. As a result, CNS trauma, stroke or degen-erative disease leads to permanent blindness, paralysis or otherloss of function. Research over the last 20 years has identified twomajor hurdles to CNS regeneration. One is the presence on CNSglial cells of proteins and proteoglycans that can directly inhibitaxon extension1–5. Even axons that initiate effective regenerationmay be stopped when they encounter these inhibitory cues. Theother major hurdle in CNS repair is that axotomized neurons oftenfail to activate a program of gene expression adequate to supportregeneration. In particular, genes coding for protein componentsof axonal growth cones—the motile tips of extending axons—aregenerally suppressed in mature neurons, but are readily reactivat-ed by peripheral nerve injury6–8. However, following CNS injury,at least some of these growth-associated proteins (GAPs) remainsuppressed in the majority of injured neurons6,9–12.

The significance of this differential gene regulation has beentested by grafting segments of peripheral nerve into the brain orspinal cord, providing CNS axons with a supportive environ-ment for axon growth13–16. Although some CNS axons regrowfor long distances into these nerve grafts, regeneration occursonly from neurons whose cell bodies are located within a fewmillimeters of the lesion site. Such proximal lesions can activateGAP expression in a subset of the injured neurons, and regener-ating axons arise exclusively from these GAP-expressing cells17,18.

The central involvement of axotomy-induced genes in regen-eration has been demonstrated most elegantly for dorsal rootganglion neurons, which are unique in having both a long CNSaxon that ascends the spinal cord, and a second axon branch thatprojects through a peripheral nerve. Interruption of DRG spinal

axons fails to induce GAP genes11,19, and the injured axons areunable to regenerate20. However, when the spinal cord lesion iscombined with peripheral nerve injury, the neurons becomecompetent to regenerate their spinal axons into a nerve graft20.Indeed, these DRG axons can regenerate for a substantial dis-tance within the native environment of the spinal cord5,21.

These observations show that genes activated by peripheralnerve injury can be crucial in determining the success or failureof CNS axon regeneration. Which of these gene(s), then, areresponsible for triggering regeneration? The most extensivelystudied example has been the gene for GAP-43, an abundantcomponent of axonal growth cones widely correlated with suc-cessful axon regeneration7,8. Loss of GAP-43 impairs axon exten-sion in response to cell adhesion molecules22, increasessusceptibility to growth cone collapse by CNS myelin23 and dis-rupts axon guidance and synaptic organization during develop-ment24–26. In adult neurons, overexpression of GAP-43 enhancessprouting at axon terminals8,27,28. However, replacing GAP-43alone is not sufficient to trigger regeneration21,28.

Here we used an in vitro assay to search for additional genesthat can mimic the effects of peripheral nerve injury in stimu-lating axon elongation by DRG neurons. We then showed thatthese genes are sufficient to induce regeneration of spinal cordaxons in vivo.

RESULTSTo identify genes responsible for the onset of axon regeneration, weused a short-term in vitro assay that monitors a transcription-depen-dent switch in axon extension induced in DRG neurons by axoninjury29. Neurons are removed from adult animals and cultured for

Spinal axon regeneration evoked byreplacing two growth cone proteinsin adult neurons

Howard M. Bomze2, Ketan R. Bulsara3, Bermans J. Iskandar4, Pico Caroni5 and J. H. Pate Skene1

1 Department of Neurobiology, Bryan Research Building, Duke University Medical Center, Durham, North Carolina 27710-3209, USA2 Present address: Cogent Neuroscience, 4425 Ben Franklin Boulevard, Durham, North Carolina 27704, USA3 Division of Neurosurgery, Box 3807, Duke University Medical Center, Durham, North Carolina 27710, USA 4 Department of Neurological Surgery, K4/832 University of Wisconsin, Madison, Wisconsin 53792, USA 5 Friedrich Miescher Institute, CH-4058 Basel, Switzerland

Correspondence should be addressed to J.H.P.S. (skene@neuro.duke.edu)

The first two authors contributed equally to this work

In contrast to peripheral nerves, damaged axons in the mammalian brain and spinal cord rarelyregenerate. Peripheral nerve injury stimulates neuronal expression of many genes that are notgenerally induced by CNS lesions, but it is not known which of these genes are required for regener-ation. Here we show that co-expressing two major growth cone proteins, GAP-43 and CAP-23, canelicit long axon extension by adult dorsal root ganglion (DRG) neurons in vitro. Moreover, thisexpression triggers a 60-fold increase in regeneration of DRG axons in adult mice after spinal cordinjury in vivo. Replacing key growth cone components, therefore, could be an effective way to stimu-late regeneration of CNS axons.

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tical to the length of ganglia subjected to peripheral nerve injury(546 ± 49 µm). However, the small difference in branching frequencypersisted (data not shown). Thus, co-expression of GAP-43 andCAP-23 triggered a transition in axon growth that is very similar—but not identical—to that evoked by the full complement of genesinduced by peripheral nerve injury.

In vivo, peripheral nerve injury enables DRG neurons to sup-port regeneration of their axons in the spinal cord20,21. Thesedorsal column axons arise from a specific population of large,mechanosensory neurons in the DRG. Immunostaining con-firmed that the largest DRG neurons in our dissociated cultures(≥ 40 µm diameter) expressed the GAP-43 and CAP-23 trans-genes at a frequency similar to other DRG neurons (data notshown). Furthermore, a second analysis of axon outgrowth forthis subpopulation showed that the frequency of axon extensionand the mean axon length and number of axon branches for theseneurons fell within the 95% confidence interval for the overallpopulation of DRG neurons (data not shown). This suggests thatthe elongating mode of axon growth can be elicited by GAP-43and CAP-23 expression in the large mechanosensory cells, as wellas in other classes of DRG neurons. Moreover, immunostainingof cryostat sections showed that the large DRG neurons expressedthe GAP-43 and CAP-23 transgenes in vivo, and transported theproteins into their axons in the dorsal columns (Fig. 4). If expres-sion of these proteins were sufficient to mimic the effects ofperipheral nerve injury in stimulating regeneration both in vivoand in vitro, then we thought that DRG neurons in our trans-genic animals should support significant regeneration of spinalaxons in the absence of a peripheral nerve injury.

To test this possibility, we made spinal cord lesions severingthe central axons of DRG neurons in wild-type mice and intransgenic animals expressing both GAP-43 and CAP-23. Dorsal

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18–24 hours29. Because the neurons are axotomized during thisremoval, they eventually respond by inducing the full complementof growth-associated genes29. However, over the first 24 hours inculture, axon outgrowth depends only on genes that were alreadyexpressed in the neurons at the time of their removal from the ani-mal29. Neurons isolated from adult mice with no previous manip-ulation (naive neurons) supported a limited amount of outgrowth(ref. 29 and Fig. 1), characterized by the emergence of relatively shortand highly branched axons (Figs. 2a and 3). In contrast, neuronsthat had responded to a peripheral nerve lesion several days beforeremoval were much more likely to extend axons (Fig. 1), and thoseaxons were long and sparsely branched (Figs. 2e and 3). This ‘elon-gating’ growth resembled the extension required for nerve regener-ation in vivo, and reflected the expression of genes induced byperipheral nerve injury29.

To identify genes that triggered this regenerative growth, we iso-lated neurons from transgenic animals in which expression of spe-cific growth-associated proteins is maintained in adult neurons27,30.Persistent expression of GAP-43 in adult DRG neurons increasedthe propensity of naive adult neurons to extend axons in the acuteoutgrowth assay (Fig. 1), but the majority of those axons remainedrelatively short, with a modal length of 100−150 µm (Fig. 2c). Onlya small fraction of the GAP-43-expressing cells extended long (> 300 µm) axons of the sort induced by peripheral nerve injury(Fig. 2c). To ensure that this was not due to limited expression ofthe transgene, we stained the cultures with an antibody against chickGAP-43. At least 80% of the DRG neurons stained intensely fortransgene expression, and only those cells were included in theanalyses reported here. The minimal effect of GAP-43 on the exten-sion of long axons is consistent with earlier reports that GAP-43alone is not sufficient to trigger regeneration of CNS axons invivo21,28. This implies that additional genes are involved in the tran-sition from local axon arborization to elongating growth.

GAP-43 shares a number of features with another prominentgrowth cone component induced by peripheral nerve injury,CAP-23 (ref. 31). Both GAP-43 and CAP-23 are members of aMARCKS-related group of acylated membrane proteins thatinteract with calmodulin, actin filaments, protein kinase C andphosphoinositides31–33. In transgenic mice, both GAP-43 andCAP-23 enhance local sprouting at axon terminals in vivo8,30. Wetherefore tested whether CAP-23, alone or in combination withGAP-43, can contribute to the induction of axon elongation fol-lowing peripheral nerve injury. As with GAP-43, persistentexpression of CAP-23 increased the number of adult DRG neu-rons that extended axons in short-term cultures (Fig. 1), but didnot elicit extension of long axons (Fig. 2b). Combined expres-sion of GAP-43 and CAP-23, however, induced a large popula-tion of DRG neurons to extend long (> 300 µm) axons (Fig. 2d).

The effects of co-expressing GAP-43 and CAP-23 were qualita-tively different from the effects of either protein alone. Although eachprotein alone acted primarily to reduce axon branching, simultane-ous expression of these growth cone components triggered a dra-matic increase in axon length (Fig. 3). Averaged over the entirepopulation of DRG neurons, the effects of GAP-43/CAP-23 co-expression approximated the effects of peripheral nerve injury (Fig. 3), although there were small differences. Axons from the trans-genic mice tended to be slightly shorter and branched somewhatmore frequently than after peripheral nerve injury. The difference inaxon length arose from the persistence of a small population of neu-rons with short (100−150 µm) axons in ganglia from the transgenicanimals (Fig. 2). When this subpopulation was removed from theanalysis, the average axon length of the remaining neurons from GAP-43/CAP-23 expressing animals (538 ± 54 µm) was essentially iden-

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Fig. 1. GAP-43 and CAP-23 increase the propensity of adult neuronsfor axon growth in vitro. DRG neurons were isolated from control(non-transgenic) adult mice or from transgenic mice expressing highlevels of GAP-43 and/or CAP-23 in adult neurons. For comparison,neurons were isolated from non-transgenic animals that had undergonea peripheral nerve lesion four days before removal of the ganglia. Thegraph indicates the percentage of adult neurons that extended axonalprocesses by 24 hours after plating.

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column axons were transected on both sides of the spinal cord,at the level of the cervico-thoracic junction. To provide theinjured axons with an optimal environment for regrowth, weresected a segment of peripheral nerve (sciatic) on one side andgrafted the nerve segment into the spinal cord lesion site20,34. Theresection produced a peripheral nerve injury that affected DRGneurons on the same side as the lesion, but left the contralateralganglia uninjured except for the spinal cord lesion itself (Fig. 5).

After one to four months, we introduced the fluorescent axon-al tracer DiI into the distal end of the nerve graft, to label any neu-rons that had been able to regenerate their axons at least 5 mminto the graft. As expected from previous studies20,21, dorsal rootganglia subjected to the peripheral nerve injury contained numer-ous labeled neurons (63 ± 22 labeled neurons per ganglion, Fig. 5).For those ganglia, we found no difference between control (wild-type) and transgenic animals. This is not surprising, because theperipheral nerve injury induces GAP-43 and CAP-23, along withother growth-associated proteins, in the DRG neurons of bothwild-type and transgenic animals.

The retrograde labeling procedure does not account for axonsthat may be competent to regenerate, but that fail to encounter adirect tissue bridge between the spinal cord and graft tissue. Nordoes the procedure account for axons blocked from entering thegraft by inhibitory molecules at the lesion site35, or those that growaround the lesion site rather than entering the graft21. To estimatethe efficiency of our grafting procedure in identifying axons com-petent for regeneration, we applied DiI directly to the spinal cordlesion sites to label all axons transected by the lesions. This directspinal application labeled 372 ± 60 neurons per ganglion. Thus,when DRG neurons were expressing the full complement of genesinduced by peripheral nerve injury, approximately 17% (63/372)of spinal DRG axons successfully entered the graft and regener-ated for at least 5 mm.

In the absence of a peripheral nerve lesion, however, adultDRG neurons of control (non-transgenic) mice were unable toextend their spinal axons into the nerve grafts. Ganglia on theside contralateral to the peripheral nerve lesion contained a meanof 0.4 labeled neurons per ganglion (n = 5 animals), consistentwith previous observations in rats20. Expression of GAP-43 and

CAP-23 induced dramatic increase in the number of neuronsthat regenerated their spinal axons. In animals expressing bothtransgenes, 25 ± 8 cells per ganglion were labeled in the absenceof a peripheral nerve injury, more than 60 times as many as incontrols (n = 6 animals; p < 0.0001). This means that approxi-mately 7% of transected axons in the dorsal column were able toregenerate into and through the nerve graft.

As predicted by the in vitro assays, neither GAP-43 nor CAP-23alone could elicit the regeneration triggered by the two transgenestogether. Introduction of the peripheral nerve grafts into the dorsalcolumns of transgenic mice expressing either gene alone (n = 2 ani-mals each), resulted in retrograde labeling of only 1–2 cells per gan-glion in the absence of peripheral injury (data not shown). Thislabeling was not statistically distinguishable from non-transgenicanimals in the absence of peripheral injury, but was dramaticallyless than in animals expressing both transgenes (p < 0.0005). Ret-rograde labeling on the side subjected to peripheral nerve injury(55–88 cells per ganglion) confirmed that the grafting and label-ing procedures were effective in these animals. Thus, the dramaticincrease in spinal axon regeneration triggered by co-expression ofGAP-43 and CAP-23 was not supported by either gene acting alone.

In animals expressing both GAP-43 and CAP-23, immuno-histochemistry showed that almost all retrogradely labeled neu-

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Fig. 2. Combined expression of GAP-43 and CAP-23 triggers an elongat-ing mode of axon extension. DRG neurons from non-transgenic, wild-type mice (wt), or from animals expressing the indicated transgenes, wereisolated and plated as in Fig. 1. Ganglia were isolated with no previousmanipulation (a–d), or four days following a crush injury to the sciaticnerve (peripheral lesion, e). Left, phase-contrast images of representativeneurons from each type of culture. Axons from the GAP-43/CAP-23transgenic animals and from neurons that have responded to a peripheralnerve lesion extend beyond the borders of these images. The cellsdepicted here were stained with antibodies against tubulin (wild-type ani-mals) or the relevant transgene products. For the doubly transgenic cell,staining is for GAP-43. Scale bars, 100 µm. Right, length of the longestprocess for individual neurons in each culture. Naive ganglia from non-transgenic control animals (a) extend primarily short (100–200 µm)axons, whereas peripheral nerve injury (e) elicits the extension of verylong (> 300 µm) axons. Expression of CAP-23 alone (b) fails to trigger theextension of long axons comparable to those induced by peripheral nerveinjury. Although GAP-43 leads to the emergence of a small populationneurons with very long axons (> 300 µm), the majority of neurons con-tinue to extend the shorter axons (100–150 µm) characteristic of naiveadult neurons (c). In contrast to either protein alone, simultaneousexpression of GAP-43 and CAP-23 (d) triggers the extension of very longaxons by the majority of DRG neurons, which mimics the effect of periph-eral nerve injury (e).

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rons expressed both of the transgenes (Fig. 4). Quantitation ofthe results from one ganglion showed that all retrogradelylabeled neurons stained intensely for the chick CAP-23 protein,whereas 25 of 26 DiI-filled cells showed clear cell body stainingfor GAP-43 (Fig. 4). The remaining cell was surrounded byintense membrane-like staining for GAP-43, but intense stainingof axons (Fig. 4) made it difficult to determine whether thetransgene was expressed in the DiI-labeled cell or in neighboringneurons. Despite this ambiguity, the results show that theincrease in spinal axon regeneration in animals expressing bothGAP-43 and CAP-23 arises from individual neurons that expressboth transgenes within the same cell.

DISCUSSIONOur findings indicate that sustained expression of two prominentgrowth cone proteins, GAP-43 and CAP-23, can substantially mimicthe effects of peripheral axon injury in activating neuronal compe-tence for axon regeneration. In vitro analysis shows that the switchfrom a limited, arborizing mode of growth to axon elongationoccurs in two stages. GAP-43 or CAP-23 alone can enhance thepropensity to initiate axon extension and reduce the frequency ofbranching. Both proteins are required, however, to elicit the exten-sion of very long axons, the hallmark of effective regeneration.

The stepwise activation of regenerative growth explains pre-vious observations that GAP-43 alone is not sufficient to stimu-late regeneration of DRG or Purkinje cell axons in vivo21,28. It isalso relevant to the remodeling of synaptic connections in theintact nervous system. Although GAP-43, CAP-23 and othergrowth-associated proteins are generally suppressed in the adultnervous system, each of these GAPs persists in a distinct sub-population of adult neurons36–40. Our results indicate that thedifferential expression of individual GAPs provides a mechanismto modulate local remodeling of axon terminals without trigger-ing long-distance growth of primary axons.

Simultaneous expression of GAP-43 and CAP-23 in the samecell, however, activates a mode of axon growth that closely resemblesthe growth triggered by the full complement of genes induced byperipheral nerve injury. Co-expression of these proteins results in a60-fold increase in the competence of DRG neurons to regeneratetheir spinal axons in vivo. Approximately 7% of dorsal column axonstransected in the transgenic animals regenerate into a peripheral

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Fig. 3. Stepwise induction of axon elongation by GAP-43 and CAP-23. DRG neurons were analyzed for axon growth in vitro as in Fig. 2.The number of branch points and total axon length were measuredfor the longest process for individual neurons; the graph shows themean branch number and length ± 95% confidence interval for eachcondition. For non-transgenic animals (open symbols) naive neurons(�) extend relatively short, highly branched axons, whereas periph-eral nerve injury (�) induces the extension of very long, sparselybranched axons. For naive neurons isolated from transgenic animalswith no previous nerve injury (closed symbols), expression of eitherGAP-43 (�) or CAP-23 (�) reduces axonal branching, but does nottrigger axon elongation. In contrast, combined expression of thesetwo growth-associated proteins (�) mimics the effects of peripheralnerve injury in triggering the elongating mode of growth.

Fig. 4. Expression of GAP-43 and CAP-23 and regeneration of spinalaxons by large mechanosensory DRG neurons of transgenic mice invivo. (a) Immunofluorescent staining shows the presence of chickenCAP-23 (blue) or GAP-43 (green) in dorsal column axons in longitudi-nal sections of spinal cord from adult transgenic mice. Left, borderbetween the dorsal columns (left side of the image) and the gray matterof the dorsal horn. CAP-23 is present in dorsal columns axons, and alsoin neurons of the dorsal horn. Right, GAP-43 positive axons in the dor-sal columns. Bottom, control sections stained with no primary anti-body. (b) Neuron cell bodies in the dorsal root ganglion (DRG) of ananimal transgenic for both GAP-43 (green) and CAP-23 (blue). Bothproteins are expressed in many large DRG neurons. Three of thesecells also contain the retrograde axonal tracer DiI (red), indicating thatthey have regenerated their spinal axons through a peripheral nervesegment placed in the dorsal columns five weeks earlier. All three cellsdisplay strong cell body staining for both GAP-43 and CAP-23. Enlargedviews at right, separate images of GAP-43 and CAP-23 immunofluores-cence for one of these neurons.

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nerve graft in the absence of a peripheral lesion, compared to 17%after peripheral nerve injury. Furthermore, in the in vitro assays,the axon outgrowth stimulated by GAP-43 and CAP-23 expressionis similar, but not identical, to the elongation evoked by peripheralnerve injury. Thus, GAP-43 and CAP-23 expression substantiallymimics the effects of peripheral nerve injury in activating axonregeneration, but it is likely that additional genes induced by periph-eral axotomy can further enhance regeneration.

It is not yet known whether the enhanced competence forregeneration produced by prolonged expression of GAP-43 andCAP-23 is due solely to direct actions of these proteins, or to addi-tional growth-associated genes that might be activated in the trans-genic animals. GAP-43 and CAP-23 are among the most abundantproteins in axonal growth cones, and the two proteins mediateboth shared and distinct interactions with calmodulin, the actincytoskeleton, and the lipid signaling molecule phosphatidyinositol-4,5-bisphosphate (PIP2)41,42. Given the central importance of cal-cium signaling and PIP2 in growth cone signal transduction43,44,the actions of GAP-43 and CAP-23 could be sufficient to increasegrowth and to convert local arborization to elongation. On theother hand, prolonged expression of these proteins in transgenicanimals also could generate additional signals that feed back onneuron cell bodies to induce a broader set of growth-related genes.Whether GAP-43 and CAP-23 act directly, or indirectly throughinduction of additional growth-associated genes, our results indi-cate that expression of these two proteins can be effective in stim-ulating neuronal competence for axon regeneration.

The small number of proteins required to activate regenera-tion raises the prospect that direct gene replacement could be usedto enhance axon regeneration in the adult CNS. In addition, induc-tion of GAP-43 and CAP-23 could be a common mode of actionfor treatments reported to enhance axon regrowth and functionalrecovery after spinal cord injury45–47. Replacement or re-induc-tion of these neuronal growth-associated proteins, alone or com-bination with treatments that modify the inhibitory environmentof the CNS48,49, may be useful in stimulating functional recoveryfollowing spinal cord injury or other CNS damage in humans.

METHODSTransgenic mouse lines expressing chicken GAP-43 or CAP-23 under thecontrol of a neuron-specific Thy-1 promoter were derived from line wt3

(GAP-43) and line c11 (CAP-23), as previously described27,30. The avianproteins are effective in modulating phosphoinositide distribution andactin dynamics, and can stimulate axonal sprouting in mammalian neu-rons41,42. The transgenic lines were chosen so that the levels of transgeneexpression in adults were similar to the expression of endogenous GAP-43 and CAP-23 in developing neurons. Transgene expression begins atapproximately postnatal day 6 and continues through adult life. Mice weregenotyped using standard PCR methods. The primers (5´-CCAACAGCG-GAGAAAAAAGGG-3´) and (5´-TCTTCTTTCACCTCTTCCTGC-3´)amplify a 380 bp DNA fragment from the chicken GAP-43 transgene; forthe CAP-23 transgene, the primers (5´-AAGGATGCTCAGGTCTCTGC-3´)and (5´-GTCTTTTTGGCTTCCCCTTCC-3´) amplify a 317 bp fragment.Neither set of primers amplifies the corresponding endogenous gene frommouse. Mice positive for each transgene were mated to ensure heterozy-gosity in the experimental animals and to generate doubly transgenic ani-mals. Control animals were generated as littermates in the same breedings.

For the in vitro analysis, dorsal root ganglion neurons from adult mice(> 8 weeks) were dissociated essentially as described29, and centrifuged at200 × g for 10 min through a cushion of 10% Ficoll in F14 culture mediumto remove myelinated axons, cellular debris and non-neuronal cells. Neu-rons were resuspended in serum-free F14 medium containing N1 supple-ments, and plated on polylysine/laminin coated glass coverslips asdescribed29. Cells from 12–14 ganglia were plated in 12 wells of a standard24-well plate. After 18–24 h, cultures were fixed in 4% paraformaldehydefor 30 min at room temperature, washed and stained with antibodies todetect neurons expressing chicken CAP-23 (monoclonal antibody 15C1)and chicken GAP-43 (rabbit polyclonal antibody)27,30. To visualize all neu-ronal processes, cultures were stained with antibodies to βIII tubulin (MAB1637; Chemicon, Temecula, California). Cells were viewed with a CCDcamera and analyzed with IPLab 3.2 for the Macintosh (Scanalytics, Fair-fax, Virginia). Only neurons that stained strongly for the appropriate trans-gene(s) were analyzed. In control cultures, cells that stained strongly forthe neuron-specific βIII tubulin were analyzed. Cells with processes greaterthan two cell body diameters were scored. We then measured the lengthof the longest process for each cell, and the number of branches formedalong that process.

For analysis of spinal axon regeneration in vivo, DRG axons were tran-sected in the dorsal columns on both sides of the spinal cord in adult mice(> 4 weeks), at the level of the cervico-thoracic junction. A segment ofsciatic nerve on one side was resected and grafted into the spinal cordlesion site20,34. After 1–4 months, we introduced the fluorescent tracerDiI into the nerve graft, 5 mm from the spinal cord. After another 5 days,the animals were perfused transcardially with 4% paraformaldehyde, andthe dorsal root ganglia removed and post-fixed in 30% sucrose. Thirty-micron cryostat sections were evaluated under fluorescent microscopy.

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Fig. 5. Replacement of GAP-43 andCAP-23 permits regeneration of spinalsensory axons in vivo. (a) Schematic ofthe experiment. Axons ascending in thedorsal columns of the spinal cord wereinterrupted in adult non-transgenic(wild-type) mice or mice expressingboth the GAP-43 and CAP-23 trans-genes (transgenic). A segment of periph-eral nerve was removed from the leftsciatic nerve, severing the peripheralaxons of DRG neurons on one side. Thenerve segment was then grafted into thespinal cord lesion site, spanning the dor-sal columns on both sides of the midline.One to four months later, a fluorescent tracer (DiI, depicted in red) was applied to the distal end of the graft. Axons that had regenerated at least 5mm into the nerve graft were able to take up the fluorescent tracer and transport it retrogradely to the neuron cell bodies. (b) Mean number oflabeled neurons detected in the lumbar dorsal root ganglia. DRG neurons subjected to peripheral nerve injury at the same time as the dorsal columnlesion (white bars) were able to regenerate their spinal axons into the nerve grafts. In non-transgenic (wt) mice, neurons that have not responded toa peripheral nerve lesion (black bars) fail to regenerate their spinal axons. Expression of GAP-43 and CAP-23 induced a 60-fold increase in the num-ber of DRG neurons that could regenerate their spinal axons from the dorsal column lesion. tg, transgenic.

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Fluorescently labeled cells were counted, and differences due to genotypeand peripheral nerve injury were analyzed by two-way ANOVA followedby Fisher’s protected least significant difference post hoc test (StatView;SAS, Cary, North Carolina). To identify cells expressing the transgenes,cryostat sections were stained with antibodies against chicken CAP-23and GAP-43, followed by secondary antibodies labeled with Alexa Fluor488 and Alexa Fluor 350 (Molecular Probes, Eugene, Oregon). Sectionswere viewed with narrow-band filter sets for each of the labels; controlsections stained with no primary antibodies, or with only one primaryantibody, confirmed that there was no detectable cross-over of signals.

All experiments involving animals were approved by the Duke Uni-versity Animal Care and Use Committee, and conducted in accordancewith federal guidelines.

ACKNOWLEDGEMENTSWe thank R. Chesebro for technical support, N. Cant for advice and assistance

with retrograde labeling and L. Katz and J. Crowley for help with the

fluorescence imaging. We also thank B. Finch, L. Katz, D. Purves,

D. Chikaraishi, M. Nicolelis and A. Udvadia for comments on the manuscript.

Support for this work was provided by the National Eye Institute (NIH),

Novartis Pharmaceuticals and the Christopher Reeves Paralysis Foundation.

K.R.B. is the recipient of a Ruth K. Broad Foundation fellowship.

¶RECEIVED 3 OCTOBER; ACCEPTED 11 NOVEMBER 2000

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