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EXPERIMENTAL NEUROLOGY ARTICLE NO. 148, 367–377 (1997) EN976665 c-Jun Expression in Adult Rat Dorsal Root Ganglion Neurons: Differential Response after Central or Peripheral Axotomy E. Broude, M. McAtee, M. S. Kelley, and B. S. Bregman Georgetown University School of Medicine, Department of Cell Biology, Division of Neurobiology, 3900 Reservoir Road N.W., Washington, DC 20007 Received March 7, 1997; accepted August 5, 1997 The response of the mature central nervous system (CNS) to injury differs significantly from the response of the peripheral nervous system (PNS). Axotomized PNS neurons generally regenerate following injury, while CNS neurons do not. The mechanisms that are responsible for these differences are not completely known, but both intrinsic neuronal and extrinsic environmental influences are likely to contribute to regenerative success or failure. One intrinsic factor that may contribute to successful axonal regeneration is the induction of specific genes in the injured neurons. In the present study, we have evaluated the hypothesis that expression of the immediate early gene c-jun is involved in a successful regenerative response. We have compared c-Jun expression in dorsal root ganglion (DRG) neurons following central or peripheral axotomy. We prepared animals that received either a sciatic nerve (peripheral) lesion or a dorsal rhizotomy in combination with spinal cord hemisection (central lesion). In a third group of animals, severed dorsal roots were placed into the hemisection site along with a fetal spinal cord transplant. This intervention has been demonstrated to promote regrowth of severed axons and provides a model to examine DRG neurons during regenerative growth after central lesion. Our results indicated that c-Jun was upregulated substantially in DRG neurons following a peripheral axotomy, but following a central axotomy, only 18% of the neurons expressed c-Jun. Following dorsal rhizotomy and transplantation, however, c-Jun expression was upregulated dramatically; under those experimental conditions, 63% of the DRG neurons were c-Jun-positive. These data indicate that c-Jun expression may be related to successful regenerative growth following both PNS and CNS lesions. r 1997 Academic Press INTRODUCTION The response of mature central nervous system (CNS) neurons to injury differs significantly from that of peripherally projecting neurons (PNS). While PNS neurons are capable of axonal regeneration after injury, neurons of the CNS typically do not regenerate after axotomy (7, 30, 45, 57). Regenerative failure after CNS injury has been attributed both to intrinsic neuronal and extrinsic environmental factors. The environment of the PNS is clearly conducive for axonal regrowth of either peripheral or central axons (23, 53, 54, 68), while the mature CNS environment is refractory to axonal regrowth, due at least in part to the inhibitory influence of CNS myelin (4, 10, 15, 18, 19, 55, 57, 59). In addition, it is likely that the signals produced in the CNS and PNS neurons following disruption of target-derived influences may lead to different cell body responses to axotomy. For example, the differences in the CNS and PNS targets will determine which soluble factors are retrogradely transported to the cell body (3, 66). Retrograde transport of trophic factors from target neurons or glia is necessary to support survival and maintain normal metabolism of PNS neurons (5, 48, 61, 65). Peripheral axotomy leads to profound morphological and metabolic changes in the cell body (chromatolysis) which are absent or diminished following an equivalent injury to the central axon (2, 46). These differences in the cell body response to central and peripheral lesions may be responsible for the differences in regenerative capacity in the two types of neurons. One mechanism which may be involved in the cascade of events leading to regeneration is the induction of immediate early gene expression. Immediate early genes are known to take part in injury-related cellular mechanisms in many systems (8, 24, 29, 32–34, 41–44, 56, 64). The protein products of a number of immediate early genes act as transcription factors which regulate gene expression. These transcription factors have been shown to be important for gene regulation during both CNS development and after injury. For example, during early CNS development, increased levels of activator protein-1 (AP-1) DNA binding complex consisting of c-Jun and cyclic AMP response element binding protein (CREB) are found in developing brain regions. These levels decline as the particular brain region matures. In the CNS, AP-1 (c-Fos and Fos-related proteins and Junrelated factors) and CREB families of transcription factors have been studied extensively. The protein product of c-jun, together with Jun B, Jun D, Fra-1, 367 0014-4886/97 $25.00 Copyright r 1997 by Academic Press All rights of reproduction in any form reserved. 368 BROUDE ET AL. Fra-2, Fos B, and c-Fos proteins, belongs to the group of proteins with DNA-binding leucine zipper domains, which enable these proteins to form hetero- and homodimers, known as transcription factor AP-1, which binds to the activation site in target genes (49). Because dorsal root ganglion (DRG) neurons can be axotomized peripherally or centrally, these neurons provide a useful model for comparing the events that take place following central versus peripheral axotomy. While DRG neurons are able to regenerate axons back to their targets after peripheral lesion, central rhizotomy of dorsal roots results in only limited regeneration; the axons regrow within the Schwann cell environment, but generally fail to penetrate the CNS environment at the glia limitans of dorsal root entry zone (17, 47, 52, 60, 62). The axons show some limited regrowth into the gray matter of the dorsal horn if the cut end of the root is placed adjacent to CNS gray matter rather than white matter (60). It is clear that CNS neurons are not inherently incapable of regenerative growth, since regenerative growth of CNS neurons does occur in a peripheral nerve environment (1, 16, 23, 53, 54, 68). In fact, the injured central processes of DRG neurons have been shown to be capable of regenerating into short peripheral nerve grafts (37–40, 63, 71, 72). It is clear that it is not only the environment which restricts growth, however. The cell body response of the injured neurons also contributes to the capacity for regrowth, since a conditioning lesion of the sciatic nerve enhances regeneration of central axon of DRG neurons into peripheral nerves grafted into the dorsal column of the spinal cord (51, 53). Disrupting the contact with the peripheral target with an injury to the peripheral axon may produce changes in the cell body which are absent following an equivalent injury to the central axon (2, 46). These data suggest that the peripheral lesion may induce a cell body response that promotes regeneration, and this cell body response is able to enhance regeneration in the central process as well. When PNS neurons are connected to their targets, the c-jun gene is repressed (45). Activation of c-Jun expression appears to occur as the result of target deprivation. DRG neurons exhibit a strong and longlasting upregulation of c-Jun after section or ligation of their peripheral branch (sciatic nerve) (25, 44, 45). If the axons are prevented from reaching their targets by ligation, there is maintained expression of c-Jun in L4 and L5 DRG (25). Thus, the deprivation of trophic factors appears to be one signal capable of stimulating expression of c-Jun in sensory neurons. The suggestion that c-Jun expression is associated with conditions of axonal growth (rather than injury, per se) is also supported by studies of axonal sprouting. The axonal growth associated with sprouting of intact peripheral (saphenous) nerve into denervated territory (69) is also associated with the expression of c-Jun in neurons that contribute to the saphenous nerve (L3 DRG) (25, 44). In the present study, we evaluated the hypothesis that the induction of c-Jun is associated with successful axonal regeneration. We have examined c-Jun expression in mature DRG neurons after central or peripheral axotomy. We prepared animals that received either a sciatic nerve (peripheral) lesion or a dorsal rhizotomy in combination with spinal cord hemisection (central lesion). In a third group of animals, severed dorsal roots were placed into the hemisection site along with a fetal spinal cord transplant. This intervention has been demonstrated to promote regrowth of severed axons and provides a model to examine DRG neurons during regenerative growth after central lesion. Our results indicated that c-Jun was upregulated substantially in DRG neurons following a peripheral axotomy, but only modestly following a central axotomy. c-Jun expression was, however, upregulated dramatically following dorsal rhizotomy and transplantation. These data indicate that c-Jun expression may be related to successful regenerative growth following both PNS and CNS lesions. METHODS Experimental Animals Adult (female and male) Sprague–Dawley rats (200– 250 g) were used in this study (Zivic-Miller Laboratories, Zelienopole, PA). Animals were housed in the Georgetown University Research Resource Facility and had unlimited access to food and water throughout the duration of the experiments. All protocols were approved by the University Animal Care Committee. A total of 20 animals was used; each experimental group contained 4–6 animals. Sciatic Nerve Lesions and DRG Preparation Peripheral sciatic nerve lesions have been previously shown to induce c-Jun expression in DRG neurons (25, 44, 45). For this reason, one group of animals was prepared with sciatic nerve lesions for use as a positive control of c-Jun expression. In these animals, the right sciatic nerve was exposed by an incision in the midthigh and was transected with iridectomy scissors. Subsequently, a 1-mm-long piece of nerve was removed in order to prevent contact of the proximal and distal nerve stumps. A schematic diagram of the sciatic nerve section is shown in Fig. 1A. Following axotomy, the overlying muscles and skin were sutured together in layers. Seven days after the lesion, the animals were euthanized with an overdose of chloral hydrate, perfused intracardially with heparinized saline (0.9%), followed by 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4. Dorsal root ganglia (DRG) from both c-Jun EXPRESSION IN AXOTOMIZED DRG NEURONS 369 as a bridge between the axotomized roots and the host spinal cord. Seven days after surgery the animals were sacrificed and perfused as described above. The dorsal root ganglia and spinal cord were dissected and prepared for analysis. c-Jun expression was examined in the axotomized DRG neurons, and the spinal cord lesion and transplant were examined for axonal ingrowth from the lesioned DRG axons. DRG tissue from animals with a unilateral sciatic nerve lesion (described above) served as positive controls for c-Jun immunoreactivity. Neural Tissue Transplant Preparation FIG. 1. Schematic diagrams of the surgical paradigm. (A) Peripheral axotomy: the right sciatic nerve was axotomized and a 1-mmlong segment of nerve was removed to prevent contact of the cut ends. (B) Central lesion: a lumbar spinal cord hemisection was made, interrupting both dorsal funiculi and the right side of the spinal cord, and the central processes of three consecutive dorsal root ganglia (L4–L6) were axotomized and 1-mm-long segments of the roots were removed. (C) In the third group of animals, a hemisection was made, and the central processes of three consecutive dorsal roots (L4–L6) were axotomized, as above. In addition, the cut ends of the dorsal roots were sandwiched between two pieces of embryonic (E14) lumbar spinal cord transplants placed within the lumbar hemisection cavity. sides of the lumbar L4–L6 segments were removed and prepared for immunocytochemical analysis. In each animal, the DRG on the contralateral side served as unlesioned controls. Central Rhizotomy and Transplantation In a second group of animals, central rhizotomies were performed on four adult Sprague–Dawley rats to axotomize the central process of DRG neurons. A laminectomy was performed at the L4–L6 spinal segments. The central processes of three consecutive DRG (L4–L6) were axotomized and a 1-mm-long segment of dorsal root was removed. A schematic diagram of the dorsal rhizotomy is shown in Fig. 1B. In some of the animals, a lumbar partial spinal hemisection was made, and the cut ends of the dorsal roots were prevented from contacting spinal cord tissue by a thin piece of durafilm (Cotman, Inc.). This paradigm insured that the cut DRG process could not contact any other tissue, so any cell body responses observed could be attributed to the central rhizotomy. In other animals, the same three lumbar dorsal roots were axotomized, but in these animals the cut ends were sandwiched between two pieces of embryonic (E14) spinal cord tissue placed into the site of a lumbar hemisection. A schematic diagram of the dorsal rhizotomy plus transplant is shown in Fig. 1C. The transplant served Timed-pregnant rats were used for embryonic spinal cord transplants. Pregnant rats were anesthetized at gestation day 14 (E14). The fetuses were removed individually as donor tissue was required and maintained in sterile culture medium (DMEM). Fetal spinal cord was dissected and 1- to 3-mm3 segments of the cord were prepared for transplantation as described previously (11). Tissue Preparation One week after surgery the animals were anesthetized with an overdose of chloral hydrate (1 g/kg) and perfused with heparinized saline (0.9%) followed by 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4. The DRG and spinal cord segments of interest were blocked and equilibrated for cryoprotection in a graded series of sucrose-phosphate buffers. Tissues were frozen in OCT medium, cut on a cryostat at 20 µm, and thaw-mounted onto gelatin-subbed slides. Dorsal root ganglia and lesion sites were cut in 1:6 series and stored at 220°C for immunostaining. One set of DRG sections was stained with cresyl violet, and the expression of c-Jun was studied in the adjacent set of sections. Ten coronal sections through the ganglia from each animal were selected for c-Jun immunocytochemistry and image analysis. In each animal, the DRG on the contralateral side served as unlesioned controls. c-Jun Immunocytochemistry To assess c-Jun labeling, sections were incubated in a polyclonal antibody to c-Jun (Ab-1, Calbiochem, Cambridge, MA) at a 1:100 dilution, at room temperature for 1 h. Sections were then rinsed and incubated in a biotinylated anti-rabbit secondary antibody (1:200), rinsed, and incubated in avidin–biotin–peroxidase (ABC reagent; Vector Lab, VectaStain Elite kit) and the reaction product was visualized with diaminobenzidine with nickel intensification. CGRP Immunocytochemistry Calcitonin gene-related peptide (CGRP) immunocytochemistry was used to detect growth of sensory fibers 370 BROUDE ET AL. into the transplant. Sections were thawed, and CGRPpositive fibers were labeled by incubating the spinal cord sections in an anti-CGRP antibody (Peninsula Labs, Belmont, CA) at a 1:5000 dilution for 48 h at 4°C. Following incubation in primary antibody, the sections were washed and incubated in biotinylated secondary antibody (1:200). Last, the slides were incubated in ABC reagent as described above (VectaStain Elite kit, Vector Laboratories, Burlingame, CA). Brown reaction product was visualized by reacting the sections with DAB with 0.03% H2O2. Sections were rinsed, air-dried, dehydrated in alcohols, cleared in xylene, and coverslipped for microscopic analysis. Image Analysis and Statistics Quantitative image analysis was performed on the c-Jun immunostaining using the VayTek Image Analysis system (VayTek, Inc., Fairfield, IA) and ImagePro Plus software (Media Cybernetics, Silver Spring, MD). DRG neurons were counted in the cellular area of the ganglion in 10 20-mm-thick sections per animal. All neurons with nuclei in the plane of the section were counted; neurons were considered as c-Jun-positive if their nuclei exhibited brown c-Jun immunostaining and were considered c-Jun-negative if the staining within the nuclei was at background levels. The mean numbers of c-Jun-positive neurons were calculated in each group, and a one-way ANOVA was performed on the data. Differences between groups were examined for significance by using the Student–Newman–Keuls method of pairwise multiple comparison. RESULTS Under normal conditions, DRG neurons regenerate robustly following a peripheral (sciatic) nerve injury, but after central axotomy (dorsal root rhizotomy), while there is some minor regrowth of DRG axons within the Schwann cell environment of the central process, the axons fail to reenter the CNS environment within the spinal cord (25, 44, 69). In the present study, we have examined the expression of c-Jun in DRG neurons under conditions known to promote regeneration (sciatic nerve transection) and under conditions that should not promote regeneration (dorsal root rhizotomy). In addition, a third group of animals was prepared with central dorsal root rhizotomy and transplantation of fetal spinal cord tissue, to elicit regenerative growth of the DRG axons back into a CNS environment (9, 11, 63). c-Jun Expression Following Peripheral Axotomy The basal expression of c-Jun in control DRG is shown in Fig. 2A. In unlesioned dorsal root ganglia, very few neurons exhibited nuclear c-Jun immunostain- FIG. 2. Expression of c-Jun 7 days after peripheral axotomy. (A) L4 dorsal root ganglion from a control rat (CON). Note the overall absence of c-Jun immunoreactivity in the nuclei of the DRG neurons. (B) L4 DRG from a rat that received a unilateral sciatic nerve transection (SN) 7 days earlier. Intense c-Jun immunoreactivity is present within the nuclei of many DRG neurons (arrows); this staining was not observed in unlesioned control DRG neurons. Scale bar, 50 µm. ing. The levels of c-Jun immunostaining were comparable in unlesioned normal rat and in unlesioned DRG contralateral to central or peripheral rhizotomy. Axotomy of the sciatic nerve and the interruption of its contact with the target tissue resulted in a dramatic upregulation of c-Jun expression in DRG neurons ipsilateral to the lesion (Fig. 2B). The increase in c-Jun immunostaining was observed in small, medium, and large diameter DRG neurons. This increased expression was observed for at least 7 days after the lesion and is consistent with the observations of others (24, 25, 43). The absence of c-Jun immunostaining in a small population of DRG neurons after this peripheral c-Jun EXPRESSION IN AXOTOMIZED DRG NEURONS axotomy can be explained by the fact that not all DRG neurons send their peripheral axons through the sciatic nerve. c-Jun Expression Following Central Axotomy In animals that received dorsal root rhizotomy alone (no transplant), regrowth of dorsal root axons into the spinal cord did not occur. In contrast to the robust upregulation of c-Jun expression after peripheral axotomy, the upregulation of c-Jun expression observed after central axotomy was modest (Fig. 3A). Following central rhizotomy, approximately 18% of the axotomized DRG neurons had c-Jun-positive nuclei (Fig. 4). This subpopulation of neurons expressing c-Jun after central rhizotomy may reflect those neurons that undergo axonal regrowth within the Schwann cell environment of the central process of the dorsal root. In the animals that received a dorsal root rhizotomy plus transplant, we documented the regrowth of sensory axons into the transplant using immunohistochemical techniques to visualize calcitonin gene-related peptide (CGRP), which labels a subpopulation of sensory fibers (Figs. 5A and 5B). A camera lucida diagram of a cross-section of a spinal cord with a spinal cord lesion plus a transplant and dorsal root reapposition is shown in Fig. 5A. The lesioned dorsal root was sandwiched between two pieces of transplants (TP). Ingrowth of CGRP-positive fibers was evident within the transplant. Serial reconstruction of the transplant indicated that CGRP-positive dorsal root axons were present throughout the transplant (data not shown). A photomicrograph of the area within the box is shown in Fig. 5B. The regrowth of CGRP-positive dorsal root axons was observed in each of the animals examined with rhizotomy plus transplant. Under these conditions of axonal regrowth, in animals that received rhizotomy plus transplant, there was a substantial increase in the number of neurons exhibiting nuclear c-Jun staining (Figs. 3B and 3C). In order to quantify the increases in c-Jun expression, the numbers of c-Jun-positive and c-Jun-negative nuclei were counted in both rhizotomy alone and rhizotomy plus transplant conditions and expressed as a percentage of the total number of DRG neurons. In contrast to animals with rhizotomy alone in which only 18% of the neurons were c-Jun-positive, in animals with rhizotomy plus transplant, 63% of the neurons were c-Jun-positive (Fig. 4). There is a significant increase in c-Jun expression after central rhizotomy plus transplant compared to rhizotomy alone (P , 0.001, student’s t test). Surprisingly, the increase in c-Jun expression was not distributed uniformly across all cell sizes. While small and medium diameter DRG neurons exhibited increased c-Jun expression following rhizotomy and transplant (Figs. 3B, 3C, and 6), the largest (.1200 µm2) neurons did not upregulate c-Jun expression after 371 central rhizotomy (Figs. 3C and 6). This suggests that the signals required for the regrowth of particular populations of neurons may differ. These studies demonstrate that c-Jun was upregulated substantially in DRG neurons following a peripheral axotomy, but not following a central axotomy. c-Jun expression was, however, upregulated dramatically following dorsal rhizotomy and transplantation. Taken together, these studies indicate that after CNS lesions, interventions which lead to increased axonal elongation (transplants, neurotrophic factors) act at the level of the cell body to alter the expression of some of the same regeneration-associated genes that are increased in peripheral projecting neurons during regeneration. These data indicate that c-Jun expression may be related to successful regenerative growth following both PNS and CNS lesions. DISCUSSION Changes in c-Jun Levels Associated with Axonal Regeneration In the present study, we examined the effects of central and peripheral axotomy on c-Jun expression in DRG neurons. We have demonstrated that c-Jun expression is differentially regulated by peripheral and central axotomy of DRG neurons. Our data indicate that while central rhizotomy led to only a modest increase in the expression of c-Jun in DRG neurons, transplantation of embryonic (E14) spinal cord tissue led to increases in c-Jun immunoreactivity more than threefold higher than in rhizotomy-only controls. Increased c-Jun expression was observed in both small and medium-sized DRG neurons, but was absent in the largest neurons. Moreover, this upregulation of c-Jun immunoreactivity was concomitant with a substantial ingrowth of CGRP-positive sensory fibers from the axotomized dorsal root into the transplant. Together, these findings are consistent with the hypothesis that induction of c-Jun expression is related to regeneration. Axotomy of peripheral nerves is typically followed by axonal regeneration, whereas after central rhizotomy axonal regeneration is limited (17, 47, 52, 60, 62). After central rhizotomy, although some of the DRG axons regrow within the Schwann cell environment, the axons fail to reenter the spinal cord. Peripheral axotomy leads to metabolic and morphological changes in the cell body which do not occur following central axotomy (2, 46). The absence of the cell body response after rhizotomy has been suggested to be related to the reduced regenerative capacity of the central axon (50). Dramatic increases in c-Jun immunoreactivity in DRG neurons following sciatic lesion are associated with a successful regenerative response (25, 36, 44). Recent studies have demonstrated that peripheral axotomy leads to a marked increase in c-Jun mRNA 372 BROUDE ET AL. FIG. 4. Increase in the percentage of c-Jun-positive neurons in the DRG after rhizotomy and transplantation. The presence of c-Jun immunoreactivity in DRG neuronal nuclei is expressed as a ratio of the number of c-Jun-positive (solid bars) or c-Jun-negative (hatched bars) neurons to the total number of neurons counted, following either central rhizotomy alone or rhizotomy plus transplant. In each DRG, a total of 120–150 neuronal nuclei were counted. In animals that received a rhizotomy alone, only 18% of the DRG neurons were c-Jun-positive. However, following rhizotomy plus hemisection plus transplant, the percentage of c-Jun-positive neurons increased to 63%. The values are expressed as the mean 6 standard error. n 5 3–4 for each group. There was a significant increase in the percentage of c-Jun-positive neurons under conditions which support the regrowth of dorsal root axons (central rhizotomy plus transplant). *P , 0.001 (Student’s t test). FIG. 3. Expression of c-Jun in DRG 7 days after central axotomy. (A) There is little c-Jun expression in dorsal root ganglia after dorsal root rhizotomy and lumbar hemisection (RHIZ 1 HX). Only occasional neurons (arrows) exhibit c-Jun immunoreactivity following central rhizotomy. (B) Intense c-Jun immunostaining is observed in the nuclei of both medium (arrows) and small (arrowheads) DRG neurons after dorsal root rhizotomy plus hemisection and embryonic spinal cord transplant (RHIZ 1 HX 1 TP). (C) c-Jun immunoreactivity appears restricted to the small and medium-sized neurons, little c-Jun immunoreactivity is seen in large dorsal root ganglion cells after rhizotomy and the addition of transplant. Scale bar, 50 µm in A, B, and C. and protein in the injured dorsal root ganglion neurons and in motor neurons (25, 36, 44) in the current study. This increase in c-Jun expression is maintained if the axons are ligated to prevent target reinnervation, but returns to basal levels if the nerve is crushed and reinnervation is permitted (25). These findings support the notion that changes in c-Jun expression are related to axonal regeneration, not to injury per se. The signals that elicit changes in gene expression after injury are not clear. In the PNS, increases in c-Jun expression following axotomy have been attributed to deprivation of target factors. The finding that increases in c-Jun expression can be elicited by blockade of axonal transport in the absence of a lesion, are consistent with this hypothesis (44). In addition, sciatic nerve crush has been shown to upregulate c-Jun expression in DRG neurons; infusion of nerve growth factor (NGF) partially prevents this increase (31). In the same study, injections of NGF antiserum into the target tissue (which depletes endogenous NGF) increased c-Jun immunostaining in DRG neurons (31). Taken together, these studies suggest that target-derived factors can influence the ability of neurons to alter cellular programs associated with regenerative axonal growth. c-Jun EXPRESSION IN AXOTOMIZED DRG NEURONS 373 FIG. 5. Ingrowth of sensory axons into the transplant. (A) Camera lucida tracing of a representative lumbar hemisection plus central rhizotomy plus transplant (TP). Note the presence of a dense dorsal root rhizotomy plus transplant. Sections were reacted immunocytochemically for CGRP to visualize this subpopulation of dorsal root axons. The asterisks (*) mark one of the axotomized dorsal roots apposed to the transplant. Remnants of gelfoam (GF) are present dorsal to the root and transplant. An extensive network of CGRP-positive fibers are present within the transplant. CGRP-positive axons are also present in the contralateral intact dorsal horn (DH) and CGRP-positive cell bodies are visible in the ventral horn (VH). A photomicrograph of the boxed area is shown in B. (B) Photomicrograph of an extensive network of ingrowing CGRP fibers (arrows) within the transplant (TP). Scale bar, 50 µm. 374 BROUDE ET AL. FIG. 6. Cell size distribution of dorsal root ganglion neurons. The proportion of DRG neurons of small (100–400 µm2), medium (400–1200 µm2), and large (.1200 µm2) size categories. Only small and medium neurons were c-Jun-positive after rhizotomy and transplant. No large neurons increased their c-Jun expression. Cell size was measured using the VayTek Image Analysis system (VayTek, Inc., Fairfield, IA) and ImagePro Plus software (Media Cybernetics, Silver Spring, MD). Cell size was measured in five animals with central rhizotomy plus transplant, 10 20-µm-thick DRG sections per animal. Data is expressed as mean 6 standard error. In contrast to peripheral nerve injury, central axotomy does not elicit a marked increase in c-Jun expression. The availability of trophic factors and other targetderived influences via the peripheral process of DRG neurons may prevent the upregulation of c-Jun in these neurons after central rhizotomy. The influence of targetderived factors on the expression of regenerationassociated genes within the injured neurons is supported further by the conditioning lesion effect on regrowth of central axons in a favorable environment (51, 53). A conditioning lesion of the sciatic nerve enhances the regeneration of central axons of DRG neurons into peripheral nerves grafted into the dorsal columns of the spinal cord (51, 53). Disrupting the contact with the peripheral target may permit changes in the cell body which are absent following an equivalent injury to the central axon (2, 46), leading to enhanced regenerative growth. Relationship of c-Jun Expression to Growth-Associated Proteins Although the genes that may be upregulated by increased levels of c-Jun are not completely understood, some evidence suggests that the same manipulations that induce c-Jun expression after injury also induce expression of growth-associated proteins, such as growth-associated protein 43 (GAP-43) and T-alpha- 1-tubulin. Although the events that may link c-Jun upregulation to regeneration have not been identified, several candidate proteins have been suggested by previous studies. For example, increased expression of growth-associated protein 43 (GAP-43) following injury of peripheral axon could be one of the events initiated by c-Jun expression (6, 20, 22, 35, 67, 70). Dorsal root section, which does not upregulate c-Jun expression, does not produce the same effect on GAP-43 (21, 22, 58). Future experiments will be required to determine if dorsal rhizotomy in combination with transplants of fetal spinal cord tissue will upregulate GAP-43 expression in axotomized DRG neurons. Spinal Cord Transplants and Neurotrophic Support One factor contributing to the success of the spinal cord tissue transplants in inducing expression of c-Jun may be related to the favorable terrain provided by the embryonic CNS tissue. With rhizotomy alone, although some axons elongate in contact with the Schwann cell environment, they fail to enter the CNS environment. The favorable embryonic environment of fetal CNS tissue and the lack of inhibitory influences characteristic of the mature CNS may permit axonal regrowth by additional populations of DRG neurons. The regrowth of dorsal root axons into the transplants was accompanied by an increase in the proportion of neurons c-Jun EXPRESSION IN AXOTOMIZED DRG NEURONS expressing c-Jun. The transplants may also influence c-Jun expression in the axotomized DRG neurons by altering the availability of neurotrophic factors. Perhaps the presence of target-derived trophic support available via the peripheral process of the DRG neurons may actually restrict the capacity of these neurons for regrowth after central rhizotomy. The increased growth of central axons after a peripheral conditioning lesions (53, 54) described above is certainly consistent with this interpretation. Transplants of fetal spinal cord tissue may provide sufficient trophic and tropic support centrally to override this inhibitory influence. Previous studies in our laboratory have demonstrated that addition of neurotrophic factors can facilitate axonal growth in mature axotomized spinal cord neurons (12) and can prevent cell death in developing axotomized spinal cord neurons (27). We have also demonstrated that neurotrophic support can increase c-Jun expression in axotomized mature spinal cord neurons (13, 14). These studies indicate that exogenous neurotrophins support growth of severed axons in the CNS, and suggest that similar effects might be observed in the regrowth of the central process of dorsal root ganglion neurons as well. Surprisingly, after central rhizotomy and transplantation, the increase in c-Jun expression was restricted to the small and medium diameter neurons. Similarly, other studies using anterograde labeling techniques to examine axonal regrowth after dorsal column lesions have demonstrated that the axonal regrowth into fetal spinal cord transplants also appears to be restricted to the axons of small and medium diameter neurons (26, 28). There was no regrowth by the largest caliber axons. Thus, the response of dorsal root ganglion neurons to injury is not uniform. 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