Download c-Jun Expression in Adult Rat Dorsal Root

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,
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Copyright r 1997 by Academic Press
All rights of reproduction in any form reserved.
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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
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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
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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.
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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. This suggests that
particular subpopulations of neurons may have differing requirements for regrowth or may differ in their
capacity to upregulate cellular programs associated
with regrowth. Preliminary data (Bregman et al., unpublished results) suggest that after central rhizotomy,
when exogenous NT-3 is supplied in combination with
transplant, there is an increase the number of large
neurons expressing c-Jun.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
ACKNOWLEDGMENTS
This work was supported by NIH Grant NS 19259 to B.S.B. We are
grateful to Hai Ning Dai for his excellent technical support.
17.
18.
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