Fetal Spinal Cord Transplants and Exogenous Neurotrophic Support Enhance c-Jun Expression in Mature Axotomized Neurons after Spinal Cord Injury

Experimental Neurology 155, 65–78 (1999) Article ID exnr.1998.6964, available online at http://www.idealibrary.com on

Fetal Spinal Cord Transplants and Exogenous Neurotrophic Support Enhance c-Jun Expression in Mature Axotomized Neurons after Spinal Cord Injury 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 October 8, 1998; accepted October 14, 1998

43, 56). These responses are not observed in axotomized neurons of the mature central nervous system (CNS), where regeneration does not occur. In the CNS, axotomized neurons typically atrophy (3, 4, 11, 45). However, regeneration-associated genes (i.e., ␣ and ␤-tubulin, actin, and growth-associated protein-43) tend to increase transiently in these neurons after axotomy close to the cell body, but not more distally (59). These observations suggest that prolonged induction of regeneration-associated genes may be required for regeneration to occur in axotomized CNS neurons and that particular interventions may increase the ability of neurons to initiate a regrowth response. Although the cascade of events that leads to these changes in cell morphology and regenerative capacity has not been identified, it will likely involve the induction of specific gene expression. One group of genes that has been suggested to play a role in axonal regrowth is the immediate early genes (IEGs). Immediate early genes have been shown to take part in injury-related cellular mechanisms in many different systems, including axotomy, neuronal transport blockade, neuronal differentiation, and cell death (9, 19, 20, 27–30, 34, 37, 40, 42, 54, 58). In this study, we have assessed the expression of the protein product of the IEG c-Jun in axotomized CNS neurons. c-Jun is a member of a family (including jun B, jun D, etc.) of closely related proteins which can homodimerize or heterodimerize via a leucine zipper domain. These dimers can serve as transcription factors by binding to the activator protein-1 (AP-1) site in target genes (47). The c-Jun transcription factor may play a role in the transcription of mRNAs that code for proteins important for neuronal structure and/or synapse function reestablishment, including T␣1 tubulin and growth-associated protein 43 (GAP-43), as well as principal constituents of the cytoskeleton (neurofilament proteins and ␣-tubulin) (46, 58). Thus, c-Jun may be involved in the axonal growth responses following axotomy in some types of neurons. For example, periph-

The responses of the central (CNS) and peripheral (PNS) nervous system to axotomy differ in a number of ways; these differences can be observed in both the cell body responses to injury and in the extent of regeneration that occurs in each system. The cell body responses to injury in the PNS involves the upregulation of genes that are not upregulated following comparable injuries to CNS neurons. The expression of particular genes following injury may be essential for regeneration to occur. In the present study, we have evaluated the hypothesis that expression of the inducible transcription factor c-Jun is associated with regrowth of axotomized CNS neurons. In these experiments, we compared c-Jun expression in axotomized brainstem neurons after thoracic spinal cord hemisection alone (a condition in which no regrowth occurs) and in groups of animals where hemisections were combined with treatments such as transplants of fetal spinal cord tissue and/or application of neurotrophic factors to the lesion site. The latter conditions enhance the capacity of the CNS for regrowth. We have demonstrated that hemisections alone do not upregulate expression of c-Jun, indicating that this particular cell body response is not a direct result of axotomy. However, c-Jun expression is upregulated in animals that received application of transplants and neurotrophins. Because these interventions also promote sprouting and regrowth of CNS axons after spinal cord lesions, we suggest that transplants and exogenous neurotrophic factor application activate a cell body response consistent with a role for c-Jun in axonal growth. r 1999 Academic Press Key Words: c-Jun; axotomy; neurotrophins; red nucleus; raphe nucleus; locus coeruleus; BDNF; NT-3.

INTRODUCTION

Axotomy of neurons in the peripheral nervous system (PNS), frequently leads to the upregulation of a number of regeneration-associated genes (8, 9, 23, 25, 65

0014-4886/99 $30.00 Copyright r 1999 by Academic Press All rights of reproduction in any form reserved.

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eral axotomy in vivo results in chronic activation of c-Jun amino-terminal kinase-1 (JNK) in the dorsal root ganglion neurons (40). This activation is associated with c-Jun amino-terminal phosphorylation in neurons and lasting AP-1 binding activity with both c-Jun and JunD participating in DNA binding complexes (40). In the present study, we test the hypothesis that induction of c-Jun expression is associated with the initiation of axonal regrowth in CNS neurons after injury, rather than with axotomy alone, and that transplants and neurotrophic factors increase c-Jun expression. If adult rats receive a thoracic spinal cord lesion, axotomized brainstem-spinal neurons fail to regenerate (49, 63) and fail to express increased levels of c-Jun (39). In this study, we have examined the expression of c-Jun in axotomized brainstem-spinal neurons under conditions which promote axonal growth (12, 14). Specifically, fetal spinal cord transplants with or without exogenous neurotrophic support were applied to adult spinal cord lesions to promote axonal regrowth. Using this approach, we have demonstrated that the induction of c-Jun is associated with regrowth and/or activation of a cell body reaction response, rather than with axotomy alone and that interventions that increase axonal growth also increase c-Jun expression in the injured neurons. METHODS

37, 40, 42). For this reason, one group of animals (N ⫽ 4) 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 and a 1-mm segment of nerve was removed. 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 sides of the lumbar L4–L6 segments were removed and prepared for immunocytochemical analysis. Preparation of Spinal Cord Transplants Timed-pregnant rats (Zivic Miller, Zelienople, PA) were used for embryonic spinal cord transplants. Pregnant female rats were anesthetized at 14 days of gestation (E14). The fetuses were removed individually as donor tissue was required and maintained in sterile culture medium (Dulbecco’s Modified Eagles Medium, DMEM). Fetal spinal cords were dissected and 1–3 mm3 segments of the cord were prepared for transplantation. Details of transplant preparation have been described elsewhere (13).

Experimental Animals

Spinal Cord Hemisections

Adult Sprague–Dawley rats (male and female, 200– 250 g) were used in this study (Zivic-Miller Laboratories, Zelienople, 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. Final data analysis was based on data from 4–6 animals in each experimental group which met all of the criteria for inclusion in this study. Initially 56 animals with spinal cord lesions were prepared; of these, 30 were examined at 1 week after the hemisection and 26 at 4 weeks after the lesion. The following groups of experimental animals were used: hemisection plus saline (HX), hemisection plus brain-derived neurotrophic factor (HX ⫹ BDNF), hemisection plus neurotrophin-3 (HX ⫹ NT-3), hemisection plus transplant plus saline (HX ⫹ TP), hemisection plus transplant plus BDNF (HX ⫹ TP ⫹ BDNF), and hemisection plus transplant plus NT-3 (HX ⫹ TP ⫹ NT-3). An additional 5 unlesioned control animals were used for analysis of brainstem nuclei.

Adult rats were anesthetized with 4% chloral hydrate (400 mg/kg body weight, intraperitoneal, IP). The surgical techniques were modified from those described previously (6, 10) and are shown schematically in Fig. 1A. Briefly, iridectomy scissors were used to create spinal cord overhemisections at the T6 spinal cord level in all animals. This lesion destroys the right side of the cord plus the dorsal columns bilaterally. Following the hemisection (HX), the retrograde neuronal tracer FluoroGold (FG; Fluorochrome, Inc., Englewood, CO), was introduced into the lesion cavity via gelfoam pledgets soaked in 4% FG in 0.9% saline. Fluoro-Gold applied at the time of the lesion is transported retrogradely to the cell bodies of axotomized neurons. This tracer has been shown in previous studies to be a reliable marker for this type of labeling because it remains in the cell bodies for extended periods of time after application at the lesion site (35). The animals were maintained under sedation, and 2–3 h later the spinal cord was reexposed and the gelfoam containing the FG was removed. The lesion site was aspirated gently with a fine blunt-ended pipette to remove all of the FG in all animals prior to the placement of segments of E14 spinal cord tissue (transplant, TP) and/or pieces of gelfoam soaked with saline or neurotrophic factor (NTF) (either brain-derived neurotrophic factor,

Sciatic Nerve Lesions and DRG Preparation Peripheral sciatic nerve lesions have been previously shown to induce c-Jun expression in DRG neurons (20,

EXPRESSION OF c-JUN IN AXOTOMIZED CNS NEURONS

BDNF, or neurotrophin-3, NT-3; approximately 5 µl of a 1 mg/ml solution for each) into the lesion site. Human recombinant BDNF and NT-3 was generously supplied by Regeneron Pharmaceuticals (Tarrytown, NY). A piece of durafilm was placed over the lesion site and the muscles and skin were sutured together in layers. Following surgery, the bladders were expressed manually until reflex voiding was established. Rats received a course of antibiotics in the event of bladder inflammation and were given food and water ad libitum. Immunocytochemistry and FluoroGold Tracing Seven and 28 days 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 brain and spinal cord segments of interest were blocked and equilibrated in a graded series of sucrose-phosphate buffers, tissues frozen in OCT medium (Miles, Inc., Elkhart, IN), cut on a cryostat at 20 µm, and thaw-mounted onto gelatinsubbed slides. The brain was sectioned in the coronal plane, and the lesion site (about 0.5 cm in both the rostral and caudal direction from the center of the lesion) was cut transversely or longitudinally. Both brain and spinal cord were cut in 1:6 series and stored at ⫺20°C for immunostaining. Sections used for FluoroGold analysis were stored at 4°C and were photographed on a fluorescent microscope (Axioscope, Zeiss) to assess the distribution of FluoroGold-labeled neurons. c-Jun Immunocytochemistry Sections adjacent to those used for FG were stained with cresyl violet, and the expression of c-Jun was studied immunocytochemically in a third set of adjacent sections. To assess c-Jun labeling, sections were incubated in a polyclonal antibody to c-Jun (Ab-1, Calbiochem, Oncogene Research Products, 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 dilution), rinsed, and incubated in avidin–biotin–peroxidase (Vector Lab, VectaStain Elite kit, Burlingame, CA). The reaction product was visualized with diaminobenzidine with nickel intensification. Double Immunolabeling with c-Jun and FG Following labeling for the c-Jun protein and visualization with DAB/nickel, selected sections were washed again in PBS and preincubated in 5% NGS for 30 min followed by incubation with a polyclonal antibody to FG (Chemicon International, Inc., Temecula, CA), diluted 1:1000, for 1 h at room temperature. Sections were rinsed and incubated in a biotinylated anti-rabbit IgG

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(1:200) and the ABC reagent as described above (Vector, VectaStain Elite kit). Double-immunostained sections were visualized with DAB for 5 min, which resulted in a brown cytoplasmic precipitate, which was distinct from the dark nuclear c-Jun staining. Data Analysis Quantitative image analysis was performed on sections immunostained for c-Jun using the VayTek Image Analysis system (VayTek, Inc., Fairfield, IA) and ImagePro Plus software, (Version 1.3, Media Cybernetics, Silver Spring, MD). Red nucleus (RN) neurons were examined on every 6th section through the nucleus, for a total of 10 20-µm sections for each of the 5–6 animals in each treatment group. Axotomized RN neurons were identified by the presence of retrogradely immunolabeled FG cell bodies and were counted as c-Jun positive if a darkly stained nucleus was present. Computer images of every c-Jun immunostained section of interest were acquired with a color digital videocamera KP-D50 (Hitachi, Japan) and the Imascan image capturing program (Imagraph Corp., Chelnsford, MA). An original macro was written to distinguish between positively labeled cells and cells with background staining, so every section throughout the entire study was evaluated under uniform conditions. Means and standard errors of each intervention group were calculated, and a one-way ANOVA was performed. Differences between every two groups were examined for significance by using the Student–Newman–Keuls method of pairwise multiple comparison. Neuronal cell counts in unlesioned animals were obtained from the cresyl violet-stained material. RESULTS

Criteria for Inclusion The transverse and longitudinal sections through the lesion site were stained with cresyl violet in order to evaluate the extent of the lesion, the transplant size and apposition, and the presence of neurotrophin or saline-soaked gelfoam in the lesion site. Only those animals that met all of the following criteria were included in this study: (1) the lesion was at the T6 spinal cord level and interrupted the intended transverse extent of the cord; (2) the gelfoam with saline or neurotrophins and transplant were present and in good apposition with the host spinal cord; (3) there were no remnants of FG label at the lesion site. It is difficult to assess the acute spread of FluoroGold at the survival times of 7 and 30 days. It is possible that some diffusion into more cranial segments of the cord may label a few nonaxotomized rubrospinal neurons. This does not constitute a major problem in interpretation of the data, however, since such spurious labeling will occur

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control animals. These regions included a number of brainstem motor, sensory, and autonomic nuclei, such as oculomotor nucleus, trigeminal motor nucleus, abducens nucleus, facial nucleus, nucleus ambiguus, dorsal motor nucleus, and hypoglossal nucleus. The hippocampus showed moderate levels of c-Jun expression. Scattered neurons throughout the brainstem also showed weak, but detectable levels of immunostaining. Labeling was also detected in a small number of neurons scattered throughout additional brain regions; in most cases these neurons were few in number and expressed a very low level of immunostaining. The pattern of c-Jun expression observed in these control animals is consistent with previous reports (17, 31, 33, 62) and will not be further described except in reference to expression in spinally projecting neurons. Unlike the brain regions listed above, most neurons projecting to the spinal cord show very low or undetectable levels of c-Jun expression in control animals. These regions included the red nucleus, locus coeruleus, vestibular complex, and raphe nuclei. Control red nucleus neurons are shown in Fig. 2; few control RN neurons exhibit c-Jun staining (Fig. 2B). Expression of c-Jun Following Thoracic Hemisection

FIG. 1. Schematic diagram of the surgical procedure. (A) Adult rats received thoracic spinal cord hemisection (HX) at the T6 vertebral level, interrupting the right side of the cord plus the dorsal columns bilaterally. In a subset of animals, the retrograde neuronal tracer Fluoro-Gold (FG) was introduced into the lesion cavity, and pieces of embryonic spinal cord and/or gelfoam pledgets soaked with saline or neurotrophic factors (NTF): NT-3 (1 mg/ml) or BDNF (1 mg/ml) were placed into the lesion site. (B) Photomicrograph of a cross-section through the site of the hemisection plus transplant. Note the close apposition (arrowheads) between the host and the transplant (TP) tissues. The transplant grows and survives in the lesion site. Undisturbed host ventral horn (VH) and central canal (CC) are indicated. Gelfoam (GF) which had been soaked in neurotrophic factor is seen just dorsal to the transplant. Scale bar, 500 µm.

in all experimental and control groups and the population of neurons so affected should be small. A representative example of a lesion meeting the criteria for inclusion is shown in Fig. 1B. Basal Expression of c-Jun Detectable levels of c-Jun immunostaining were observed in a number of brain regions in unoperated

The expression of c-Jun in rubrospinal neurons has been described previously (39) in animals following high cervical (C3) axotomy, but has not been observed previously in animals 8 days following the lesion at the thoracic (T10) level. Consistent with these previous reports, we also found extremely low levels of c-Jun immunoreactivity in the red nucleus, locus coeruleus, lateral vestibular nucleus, and raphe nucleus neurons of rats following midthoracic hemisection at spinal cord level T6 (Fig. 3A). The presence of intrinsic c-Jun staining in other brain regions within the same tissue sections (i.e., oculomotor nucleus, cortex, dentate gyrus, for example) indicated that the absence of c-Jun was specific to distinct populations of neurons, such as RN neurons. As an additional positive control for c-Jun immunostaining, we evaluated the expression of c-Jun in RN neurons after cervical lesion and dorsal root ganglion neurons after sciatic (peripheral nerve) lesion. These lesions have been shown previously to induce expression of c-Jun (30, 37). As predicted, these lesions induced substantial upregulation of c-Jun immunostaining in the respective populations of neurons. Expression of c-Jun in Red Nucleus Following Hemisections Plus Transplants and Neurotrophic Support In contrast to the lack of c-Jun expression after spinal cord hemisection alone, c-Jun expression in axotomized brainstem-spinal neurons was elicited by the addition of transplants and neurotrophic factors at

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Quantitative Analysis of c-Jun Expression

FIG. 2. Normal RN. (A) Cresyl violet (CV) staining of the red nucleus from a control unoperated animal. (B) c-Jun immunoreactivity (C-JUN) in an unlesioned control red nucleus. In most of the neurons, there is only background staining and no basal expression of c-Jun, although a few of the neurons exhibit c-Jun nuclear staining (arrows). Scale bar for A and B, 50 µm.

the lesion site. The addition of embryonic spinal cord transplants at the site of axotomy increased the number of c-Jun immunopositive neurons in the red nucleus (Fig. 3B) and other brainstem-spinal nuclei, compared to hemisection alone (Fig. 3A). The exogenous application of neurotrophic factors (BDNF or NT-3) (Figs. 3C and 3E) at the lesion site also induced c-Jun expression in the axotomized neurons. Induction of c-Jun expression appeared greatest (as indicated by the number of c-Jun-positive cells within the caudal part of the red nucleus) in animals which received a combination of both embryonic spinal cord transplant and exogenous neurotrophic support at the site of hemisection (Figs. 3D and 3F).

The red nucleus was selected for quantitative analysis of c-Jun expression because it exhibited changes in c-Jun expression that were representative of all of the brainstem-spinal neurons examined, its borders are easily defined, and the pathway is crossed. The total number of c-Jun-positive neurons in the RN was counted for all treatment groups and is shown in Fig. 4A. As suggested by the qualitative observations above, the lowest levels of c-Jun expression were observed in the group receiving a HX only. Moderate increases (approximately four- to fivefold) were observed in groups receiving either a transplant (TP) or addition of either BDNF or NT-3. The greatest increases, however, in total number of c-Jun-positive neurons (approximately sevento eightfold) were observed in animals that had received a combination of transplant plus either BDNF or NT-3. The combination of transplants and neurotrophic factors had an additive effect on c-Jun expression in the axotomized neurons. A posthoc Student–Neuman– Keuls pairwise comparison was performed; the results of the multiple comparisons are shown in Table 1. The number of c-Jun-positive neurons in each of the treatment groups was significantly different from the HX plus saline group (P ⬍ 0.01). The number of c-Junpositive neurons in groups that received a combination of transplant plus neurotrophin (HX ⫹ TP ⫹ BDNF or HX ⫹ TP ⫹ NT-3) was significantly greater (P ⬍ 0.01) than that in animals receiving lesion and the respective neurotrophin alone (HX ⫹ BDNF, HX ⫹ NT-3). The expression of the inducible transcription factor c-Jun was present at both 7 and 28 days after the lesion and transplant and neurotrophins. The pattern and extent of c-Jun expression was similar at both 7 and 28 days after injury (Fig. 4B), suggesting that transplants and neurotrophins may support long term plasticity within these neuronal populations. All neurons that expressed c-Jun following hemisection and a given experimental treatment were also labeled with FluoroGold; however, not all FluoroGold-labeled neurons were c-Jun-positive. c-Jun Expression in Other Brainstem Spinal Neurons The levels of c-Jun expression and changes in cell morphology in axotomized lateral vestibular, raphe, and locus coeruleus neurons were similar to the levels and changes in axotomized red nucleus neurons. Colocalization of FluoroGold and c-Jun immunoreactivity is shown in Fig. 5 for an animal that had received a hemisection, transplant, and BDNF 7 days earlier. Axotomized red nucleus neurons are shown in Fig. 5A by Nissl staining and in 5B by FluoroGold immunofluorescence. A camera lucida drawing of the neurons in Fig. 5B is shown in Fig. 5C. Neurons double-labeled with both c-Jun and FluoroGold are shown in Fig. 5D.

FIG. 3. Expression of c-Jun in axotomized red nucleus neurons. A number of different treatments resulted in the upregulation of c-Jun in the axotomized red nucleus neurons 7 days after injury as compared to unlesioned control red nucleus neurons (compare Figs. 4B–4F to 2B). Coronal sections of the red nucleus at comparable levels in six different treatment groups after spinal cord hemisection: (A) Hemisection-only (HX). (B) Hemisection plus transplant (TP). (C) Hemisection plus NT-3 (NT-3); (D) Hemisection plus transplant plus NT-3 (TP ⫹ NT-3); (E) Hemisection plus BDNF (BDNF); (F) Hemisection plus transplant plus BDNF (TP ⫹ BDNF). In each of the treatment groups (B–F) note the darkly stained nuclei surrounded by pale cytoplasm. Note the paucity of nuclear c-Jun immunostaining in HX only animals (A) compared with the relative abundance

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TABLE 1 Effect of Various Interventions on c-Jun Immunoreactivity in Axotomized RN Neurons HX HX ⫹ HX ⫹ TP NT-3 HX HX ⫹ TP HX ⫹ NT-3 HX ⫹ BDNF HX ⫹ TP ⫹ NT-3 HX ⫹ TP ⫹ BDNF

X ** ** ** ** **

** X * * NO **

** * X NO ** **

HX ⫹ HX ⫹ TP HX ⫹ TP BDNF ⫹ NT-3 ⫹ BDNF ** * NO X ** **

** NO ** ** X NO

** ** ** ** NO X

Note. A posthoc Student–Neuman–Keuls pairwise multiple comparison showed each of the treatment groups to be significantly different from the HX only group, indicating that interventions that increase axonal regeneration (transplants and neurotrophic factors) lead to increases in c-Jun expression in the injured CNS neurons. Additionally, a number of the different treatment groups were significantly different from one another, * P ⬍ 0.05; ** P ⬍ 0.01. NO, no significant difference between treatments; X, no comparison possible; HX, hemisection; TP, transplant; NT-3, neurotrophin-3; BDNF, brainderived neurotrophic factor.

FIG. 4. Number of c-Jun positive cells in the RN. The expression of c-Jun inducible transcription factor was assessed at 7 and 28 days after the lesion and addition of embryonic spinal cord transplant and neurotrophins. A total of 300–500 neuronal nuclei were counted in each group. An one-way ANOVA was performed on the means in each treatment group, and these means are expressed ⫾ standard error. Note that the number of c-Jun positive cells increased approximately four- to fivefold with a hemisection plus transplant or neurotrophic factor. However, in animals that received a hemisection plus a transplant plus neurotrophic factor, the number of c-Jun positive cells increased seven- to eightfold. Thus, combinations of interventions appear to have an additive influence on c-Jun expression in the injured neurons at short survival times (7 days, 5A) and this expression was sustained for a long time after injury (28 days, 5B). A posthoc multiple pairwise comparison was performed on these data; the results of this analysis are presented in Table 1. N ⫽ 5–6 animals for each treatment group. HX, hemisection; TP, transplant; NT-3, neurotrophin-3; BDNF, brain-derived neurotrophic factor.

The large arrows in Figs. 5B–5D indicate the same neuron with FluoroGold labeling alone (Fig. 5B), camera lucida (5C), and double-labeling (5D). The inset in Fig. 5D shows an enlarged view of the same cell in which both the punctate cytoplasmic FluoroGold immunostaining and dark nuclear c-Jun immunostaining are apparent. The heavy nuclear c-Jun immunolabeling in

other brainstem neurons axotomized by the lesion, including coeruleospinal, raphespinal, and vestibulospinal neurons, was similar to that observed in the rubrospinal neurons. Neurons in the raphe nucleus from the same animal are shown in Figs. 5E and 5F. Figure 5E shows FluoroGold labeling of axotomized raphe neurons prior to staining with c-Jun and 5F shows c-Jun labeling of neurons from the same animal. Levels of c-Jun expression in lateral vestibular nucleus and locus coeruleus are shown in Fig. 6 (lateral vestibular nucleus) and Fig. 7 (raphe and locus coeruleus). In control vestibulospinal neurons, levels of c-Jun expression were low (Fig. 6A). Following a hemisection and addition of neurotrophic support (either NT-3, Fig. 6B; or BDNF, Fig. 6C), c-Jun expression increased. As observed in the red nucleus neurons, however, the most robust c-Jun expression was observed in lesioned animals that received a combination of transplant and neurotrophic support (Fig. 6D, HX ⫹ TP ⫹ BDNF). Similarly, c-Jun expression increased in both raphe neurons (Figs. 7A and 7B) and locus coeruleus neurons (Figs. 7C and 7D) when transplant, neurotrophin, or both were present at the lesion site. Although the increase in the number of c-Junpositive neurons was not always apparent in individual 20-mm-thick sections from locus coeruleus, raphe, or vestibular nuclei, serial analysis of sections throughout these nuclei confirmed the observations documented quantitatively for the red nucleus.

of neurons which increased c-Jun expression in the presence of transplants and/or neurotrophins (B–F).Sections were taken from similar levels of the magnocellular portion of the red nucleus in each figure. The apparently smaller neuronal size in the HX group is a reflection of the atrophy of these neurons after axotomy (11). We have shown recently that this atrophy is prevented by transplants and neurotrophins (11). Scale bar, 50 µm, A–F.

FIG. 5. Colocalization of Fluoro-Gold and c-Jun immunoreactivity. (A) Cresyl violet staining (CV) of the axotomized red nucleus 7 days after injury in a rat that received a hemisection and transplant plus BDNF. (B) Nearby adjacent section showing the fluorescent tracer FluoroGold (FG) in the axotomized RN neurons (arrows). Neurons axotomized at the time of lesion take up the FG and retrogradely transport it back to their cell bodies. This image is 40 µm rostral to that in Fig. 2A. (C) Camera lucida drawing of the same section shown in B and D. (D) The same section in B shows cells (arrows) that were double immunostained for c-Jun (dark nuclei) and Fluoro-Gold (punctate cytoplasmic staining) (FG ⫹ C-JUN). For comparison, one representative cell (large arrow) is shown in B (fluorescence), C (camera lucida tracing), and D (double-labeling for c-Jun and FluoroGold immunocytochemistry). Inset, higher magnification of the neuron indicated by the large arrow in D. Note the punctate cytoplasmic staining (FluroGold immunocytochemical labeling), coupled with the darkly stained nucleus (c-Jun immunocytochemical labeling). (E) FluroGold labeling in the raphe nucleus of the same animal, photographed before c-Jun immunostaining. Arrows indicate axotomized neurons, as shown by retrograde labeling with FG. (F) Higher power of subsequent c-Jun (C-JUN) immunostaining of the same brain section. Note: the same cells (indicated by the arrows in E and F) that are marked with the FG tracer (Fig. 3E) have dark c-Jun immunostained nuclei (arrows). Scale bar, 50 µm. 72

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FIG. 6. Expression of c-Jun in lateral vestibular nucleus. Coronal sections through the lateral vestibular nucleus of adult rats in unlesioned control and 7 days after thoracic hemisection and hemisection combined with fetal spinal cord transplant and neurotrophic support. (A) Control (CON). There is little c-Jun immunostaining in unlesioned control vestibulospinal neurons. (B) Hemisection plus NT-3 (NT-3); (C) Hemisection plus BDNF (BDNF). In both (B) and (C), increases in the number of c-Jun immunostained neurons were apparent. (D) Hemisection plus transplant plus BDNF (TP ⫹ BDNF). The combination of transplant plus neurotrophic support led to the greatest increases in c-Jun expression and the best preservation of cell morphology in lateral vestibular nucleus neurons, as was seen in red nucleus neurons (shown above) and raphe-spinal and coeruleospinal neurons (see below). Scale bar, A–D, 50 µm.

Taken together, these findings indicate that interventions that lead to increases in axonal regrowth after CNS injury lead to increases in the expression of regeneration-associated cellular programs. The increase in c-Jun immunostaining seen at both 7 and 28 days after the injury suggests that once initiated, the upregulation of c-Jun, and presumably the associated axonal growth, continue for relatively long intervals. DISCUSSION

In this study we describe the pattern of expression of the inducible transcription factor c-Jun in the adult rat

brain after thoracic spinal cord injury and the effects of exogenous neurotrophins and transplantation of embryonic spinal cord tissue into the lesion site on its expression. Control animals show very little intrinsic c-Jun expression in brainstem-spinal neurons; following thoracic hemisection; levels of c-Jun immunostaining in the axotomized brainstem-spinal neurons remain exceedingly low. In animals that received a hemisection plus a fetal spinal cord transplant and/or addition of exogenous neurotrophic support (BDNF or NT-3), c-Jun expression was dramatically upregulated. Our laboratory has previously demonstrated that the interventions that increase c-Jun expression in the

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FIG. 7. Expression of c-Jun in raphe spinal and locus coeruleus nuclei. (A, B) Sections through the axotomized raphe spinal nucleus of adult rats 7 days after thoracic hemisection and hemisection combined with neurotrophic support. (A) Hemisection plus BDNF (BDNF). (B) Hemisection plus NT-3 (NT-3). The exogenous application of neurotrophic factor (BDNF or NT-3) at the site of the spinal cord lesion leads to an increase in c-Jun expression in the axotomized neurons. (C, D) Sections through the axotomized locus coeruleus after hemisection and transplant (C) or transplant and neurotrophic support (D). Both interventions increase in c-Jun immunostaining in axotomized locus coeruleus neurons. Scale bar for A–D, 50 µm.

present study also enhance the extent of axonal elongation within transplants of fetal spinal cord tissue (12, 14). Therefore, our observations that c-Jun expression is low in axotomized brainstem-spinal neurons that do not regenerate or sprout after injury, but high under conditions where some regrowth does occur, is consistent with the hypothesis that c-Jun is associated with axonal growth rather than axotomy alone. c-Jun Expression and Neurotrophic Factor Support In the PNS, increases in c-Jun expression following axotomy have been attributed to deprivation of target

factors. Findings that prolonged increases in c-Jun expression occur in dorsal root ganglion (DRG) neurons after axotomy or ligation of the peripheral axons in the sciatic nerve (20, 40, 42) are consistent with this hypothesis. If the axons are prevented from reaching their targets (by ligation), expression of c-Jun in L4 and L5 DRG is maintained (20, 38, 40). In addition, sciatic nerve crush has been shown to upregulate c-Jun expression in DRG neurons; infusion of NGF partially prevents this increase (26). Sprouting of intact peripheral (saphenous) nerve into denervated territory (61) is also associated with the expression of c-Jun in neurons that

EXPRESSION OF c-JUN IN AXOTOMIZED CNS NEURONS

contribute to this nerve (L3 DRG) (20, 42). Thus, the capacity of PNS neurons to increase c-Jun expression is associated with conditions of axonal regrowth. After central rhizotomy, only about 20% of DRG neurons increase c-Jun expression. Under conditions that increase axonal regrowth of the DRG neurons (fetal spinal cord transplants), however, the proportion of c-Jun immunopositive DRG neurons also increased to 80% (15). Taken together, these studies suggest that DRG neurons increase c-Jun expression during axonal regrowth after either central (15) or peripheral axotomy (40). It is unclear, however, whether the same mechanisms operate in CNS neurons. Although axotomy of rubrospinal neurons at the cervical level does lead to a transient increase in c-Jun expression (39), lesions of the thoracic spinal cord fail to produce an increase in c-Jun expression. These data suggest that axotomy alone is not sufficient to produce an increase in c-Jun expression. Similarly, in the present study, we have shown that levels of c-Jun do not increase in brainstemspinal neurons following axotomy alone. Thus, our data are not consistent with the interpretation that c-Jun induction is solely the result of target factor deprivation. Rather, in our studies the upregulation of c-Jun in axotomized CNS neurons is associated with conditions that support axonal growth following the lesion. Transplants and Neurotrophic Factors Increase Axonal Growth of Axotomized CNS Neurons The response of mature CNS neurons to injury is paradoxical in several ways. Mature CNS neurons axotomized at a distance from the cell body are more likely to survive the lesion than are those axotomized close to the cell body. They are, however, also less likely upregulate cellular programs associated with axonal growth or to regenerate into a permissive environment (1, 2, 5, 11, 15, 18, 44, 49, 51, 53, 59, 60). For example, after optic nerve lesions and PNS grafts, retinal ganglion cells regenerate into the permissive PNS environment after axotomy close to the optic disc, but as the distance increases, the number of neurons which regenerate decreases (5, 50). Similarly, after peripheral nerve grafts (48, 49, 51, 52) or transplants of fetal spinal cord tissue in the spinal cord (36), it is the neurons closest to the graft that extend axons into the transplant. After distal axotomy, mature CNS neurons are more likely to survive the injury, but are less likely to regenerate. This suggests that cues from the intact axon proximal to the lesion or from collateral projections to other targets may repress the regenerative capacity of the neuron. The lack of an upregulation of c-Jun after thoracic but not cervical axotomy is consistent with this interpretation (11, 14, 20, 39). Our data suggest that transplants and neurotrophic factors are able to overcome this distance effect on the capacity of CNS neurons for regrowth. The availability of either

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transplants or neurotrophins at the injury site in the immediate postinjury period increases the capacity of injured CNS neurons for regrowth (14) and these increases are associated with increases in c-Jun expression in the axotomized neurons. In addition, these same interventions reverse the lesion-induced atrophy of CNS neurons (11). The lack of c-Jun expression after thoracic spinal cord injury suggests that signals derived from intact collaterals may suppress regenerative cellular programs. Transplants and neurotrophins may be able to overcome these suppressive signals to elicit c-Jun expression in the cell body and axonal growth from the axotomized distal projections. Recent studies suggest that expression of the immediate early genes such as c-Jun is associated with a neuronal growth response, not simply a response to damage (16, 32, 40, 57). c-Jun as a transcription factor may be in a position to influence the expression of downstream genes required for axonal regrowth (11, 12, 14, 15, 20, 39, 40). The precise down-stream targets for c-Jun during axonal regrowth are not yet completely understood. It is interesting to note that neurotrophins alone, or in combination with transplants, increased c-Jun expression in axotomized RN neurons and that the combination of both was additive. Either BDNF or NT-3 was able to increase c-Jun expression in axotomized red nucleus neurons. After axotomy at birth, both BDNF and NT-3 rescue immature axotomized red nucleus neurons from retrograde cell death transiently (21). Only BDNF, however, rescued these neurons permanently (21). Mature red nucleus neurons have high affinity receptors (trk B) for BDNF (41). The response of these neurons to exogenously applied BDNF may be mediated by the interactions with the trk B receptors. Although NT-3 acts primarily through the trk C receptor, it may also interact through trk B receptors to influence the neurons. The magnitude of the increase in c-Jun expression in response to either of the neurotrophins was similar. The application of transplants and neurotrophins recruits c-Jun expression in additional axotomized neurons. The mechanism underlying this recruitment is not clear. We have predicted that if transplants and neurotrophins altered c-Jun expression acutely (7 days after axotomy), these changes would be down-regulated by 28 days after axotomy. To the contrary, c-Jun expression remained upregulated 28 days after axotomy plus transplants and neurotrophin application, suggesting that these interventions may elicit a relatively long window of plasticity in these brainstem-spinal projections. Recent studies of axotomized PNS neurons have also demonstrated a surprisingly long lasting upregulation of c-Jun expression (40). The long-term expression of c-Jun in these neurons may reflect a long-term capacity for plasticity in the central or peripheral projection of the injured neurons. Alternatively, the

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maintenance of c-Jun expression in these neurons may indicate that sufficient reconnections with the target to down-regulate c-Jun may not have been established. The extent to which neurons positive for c-Jun are regenerating or sprouting cannot be determined from the present studies. Other studies in this laboratory indicate that after spinal cord lesions and transplants, both regenerating and sprouting neurons contribute to the axonal regrowth observed (7). Furthermore, these two forms of axonal growth appear to be regulated independently both in vivo (7) and in vitro (55). It is clear, however, that after either CNS or PNS injury, an increase in c-Jun is associated with increased axonal elongation (15, 32, 40), and current study. c-Jun Expression and Growth-Associated Proteins Although neurotrophic factors have not been directly linked to the activation of an immediate early gene cascade, some evidence is consistent with this possibility. BDNF can activate binding of transcription factors to various regulatory elements, including the serum responsive element, the AP1-like element, and cyclic AMP-responsive element sequences (22, 24). Although the genes that may be upregulated by increased levels of c-Jun are not completely known, some evidence suggests that the same manipulations that induce c-Jun expression in the CNS also induce expression of growth-associated proteins, such as growth-associated protein 43 (GAP43) and T␣1 tubulin. For example, following lesion of the cervical spinal cord, both GAP43 and T␣1 tubulin mRNAs are increased in axotomized rubrospinal neurons (58). Increases in c-Jun expression have also been observed in rubrospinal neurons following cervical axotomy (39); interestingly, these increases are observed before increases in GAP43 and T␣1 tubulin mRNAs appear to increase. Together, these data are consistent with the possibility that early increases in c-Jun may play a role in the upregulation of growthassociated genes such as GAP43 and T␣1 tubulin, although this association has not yet been tested directly. Our data provide a distinct link between c-Jun expression and the recovery and regrowth of injured adult neurons. c-Jun expression is low in axotomized brainstem-spinal neurons that do not regenerate, but is high and sustained when lesions are coupled with interventions such as fetal transplants and exogenous neurotrophic support. c-Jun expression is related to the cell-body response to injury, and not axotomy per se. It seems clear that c-Jun expression is not simply associated with the activation of suicide programs, since the early onset of c-Jun expression may also be directly associated with initiating or maintaining a regeneration response in the injured neurons (32). The current observations that transplants and neurotrophic factor administration can modulate c-Jun expression in axoto-

mized CNS neurons and that these same interventions are associated with increases in axonal regeneration suggests that these interventions may overcome suppressive signals that restrict axonal growth in mature axotomized CNS neurons after distal axotomy and support long-term plasticity in these injured CNS neurons. ACKNOWLEDGMENTS This work was supported by NIH Grant NS 19259. We are extremely grateful to HaiNing Dai for expert technical assistance. Brain-derived neurotrophic factor and neurotrophin-3 were generously supplied by Regeneron Pharmaceuticals, Tarrytown, NY.

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