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Normal and injury-induced sympathetic innervation of rat dorsal root ganglia increases with age

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THE JOURNAL OF COMPARATIVE NEUROLOGY 394:38–47 (1998)
Normal and Injury-Induced Sympathetic
Innervation of Rat Dorsal Root Ganglia
Increases With Age
MATT S. RAMER AND MARK A. BISBY*
Department of Physiology, Queen’s University, Kingston, Ontario, Canada K7L 3N6
ABSTRACT
In rats, partial injury to a peripheral nerve often leads to sympathetically maintained
pain (SMP). In humans, this condition is especially apparent in the elderly. Nerve injury also
causes perivascular sympathetic axons to sprout into the dorsal root ganglion (DRG), forming
a possible anatomical substrate for SMP. Here, we describe the effects of chronic sciatic nerve
constriction injury (CCI) in young (3 months) and old (16 months) rats on neuropathic pain
behavior and on sympathetic sprouting in DRG. Behavioral tests assessed changes in thermal
allodynia and hyperalgesia and in mechanical allodynia. We found that 1) sympathetic
innervation of the DRG increased naturally with age, forming pericellular baskets mainly
around large DRG neurons, and that sympathetic fibers were often associated with myelinated sensory axons; 2) sympathetic fiber density following CCI was also greater in old than in
young rats; and 3) in old rats, thermal allodynia was less pronounced than in young rats,
whereas thermal hyperalgesia and mechanical allodynia were more pronounced. These
results highlight the possibility that sympathetic sprouting in the DRG is responsible for the
sympathetic generation or maintenance of pain, especially in the elderly. J. Comp. Neurol.
394:38–47, 1998. r 1998 Wiley-Liss, Inc.
Indexing terms: neuropathic pain; adrenergic, hyperalgesia; allodynia
Neuropathic pain is a condition that can result from
disease (e.g., diabetes, herpes zoster) or trauma to nervous
tissue (thalamic infarct, spinal cord, or peripheral nerve
injury) and that differs from ‘normal pain,‘ in that it can be
evoked at much lower stimulus thresholds and is and often
of greater severity. A recent review indicates that a significant number of elderly humans experience chronic pain
generally, and this remains underdiagnosed and undertreated (Gagliese and Melzack, 1997). Neuropathic pain
(one form of chronic pain) is especially apparent in the
elderly (Gibson et al., 1994), particularly in cases of
postherpetic or trigeminal neuralgia (Devor, 1991). It is
interesting that the more painful conditions are a result of
incomplete nerve injuries, which leave an intact connection to the periphery (Jänig, 1985). Several animal models
of neuropathic pain have been employed in the search for
underlying mechanisms, three of which are similar, in that
they all involve partial injury to a peripheral nerve
(Bennett and Xie, 1988; Seltzer et al., 1990; Kim and
Chung, 1992). Painful sensations due to normally nonnoxious stimulation (allodynia) and an abnormally intense
perception of pain due to noxious stimulation (hyperalgesia) are symptoms of neuropathic pain both in humans and
in animal models. Also common to both animals and
humans is the fact that stimulation of the sympathetic
r 1998 WILEY-LISS, INC.
nervous system often exacerbates the pain (Bennett, 1991),
whereas surgical or pharmacological sympathectomy often
relieves it, albeit to differing extents (Kim and Chung,
1991; Neil et al., 1991; Seltzer and Shir, 1991; Kim et al.,
1993; Desmeules et al., 1995). With respect to sympathetically linked pain in the elderly, as Gagliese and Melzack
(1997) point out, although a ‘significant majority of the
elderly experience pain which may interfere with normal
functioning,’ the study of pain in the elderly has been
neglected. We have found no references in the literature to
suggest that the proportion of elderly sympathetically
maintained pain (SMP) patients differs from the general
population. However, the fact that a majority of the elderly
suffer from chronic pain may be a reflection of an increase
in the prevalence of all types of pain.
In rats (Chung et al., 1993, 1996; McLachlan et al., 1993)
and in mice (Davis et al., 1994, Ramer et al., 1997), a
Grant sponsor: Medical Research Council of Canada; Grant number:
MT-5198.
*Correspondence to: Mark A. Bisby, Department of Physiology, Queen’s
University, Kingston, Ontario, Canada K7L 3N6.
E-mail: bisbym@post.queensu.ca
Received 1 July 1997; Revised 30 October 1997; Accepted 8 December
1997
ADRENERGIC SPROUTING IN DRG IN AGING
possible anatomical substrate for the interaction between
the sympathetic and sensory nervous systems has been
described: in the dorsal root ganglia (DRG) of nerveinjured animals, sympathetic fibers can be found among
DRG neurons and sometimes form baskets around largediameter neuronal profiles. Although there are conflicting
reports on the amount of contact between sympathetic
axons and DRG neurons following nerve injury (Devor et
al., 1995; Chung et al., 1997), electrophysiological evidence
supports this site as a point of communication between
these neuronal types (McLachlan et al., 1993; Devor et al.,
1994; Michaelis et al., 1996). The sprouting phenomenon
occurs after both complete and partial sciatic nerve injuries. After partial nerve injury, sprouting is well established 2 weeks postoperatively, develops with a time
course similar to that of allodynia and hyperalgesia (Chung
et al., 1996; Ramer and Bisby, 1997), and lasts as long as
10 weeks following a complete nerve injury (McLachlan et
al., 1993).
Because of the prevalence of chronic pain in elderly
humans (Gagliese and Melzack, 1997) and the potential
importance of this sympathetic sprouting in neuropathic
pain states, our objectives were to determine 1) whether
there were changes in the sympathetic innervation of the
normal, uninjured DRG with aging; 2) whether partial
nerve injuries made in young and aging rats resulted in
differences in sympathetic sprouting; and 3) whether there
were changes in the behavioral responses to partial nerve
injuries made in young and old rats. We have used the
chronic constriction injury (CCI) method of Bennett and
Xie (1988), because, in our hands, this model produces
easily reproducible behavioral results and sympathetic
sprouting in the DRG (Ramer and Bisby, 1997). There is
also evidence that this model produces pain behaviors
involving the sympathetic nervous system (Neil et al.,
1991; Wakisaka et al., 1991; Desmeules et al., 1995; Kim et
al., 1997). We show here that the invasion of the DRG by
sympathetic axons occurs naturally with age in rats, and
sympathetic sprouting is increased in aged rats along with
increased thermal hyperalgesia and mechanoallodynia.
MATERIALS AND METHODS
Animal preparation
These experiments were carried out in accordance with
the guidelines of the Canadian Council on Animal Care,
and the experimental procedures were approved by the
Queen’s University Animal Care Committee. Twenty-eight
female Sprague-Dawley rats were used, which were separated into five groups to study the effects of age and partial
nerve injury on behavior and on sympathetic sprouting in
the DRG. The groups differed in their starting age or
whether or not they were injured: Group 3/0, for example,
consisted of 3-month-old uninjured rats, whereas group
16/1 consisted of 16-month-old rats that had a partial
nerve injury for 1 month. It should be noted that the large
size difference between young and old rats precluded a
blinded study.
To determine the effect of age alone on pain-related
behavior and sympathetic sprouting in the DRG, six
3-month-old rats (group 3/0), five 10-month-old rats (group
10/0), and six 16-month-old rats (group 16/0) were left
unoperated. To determine what effect age had on chronic
constriction injury-induced behavior and sympathetic
sprouting, six 3-month-old rats (group 3/1) and five 16-
39
month old rats (group 16/1) were given a CCI, in accordance with the work of Bennett and Xie (1988; see below),
and these rats survived for 1 month.
The rats were housed in pairs on a 12-hour dark/12-hour
light cycle and had access to food and water ad libitum. For
the partial nerve injuries, rats were anesthetized with
sodium pentobarbital (48 mg/kg body weight, i.p.), an
incision was made in the upper left thigh, and the nerve
injuries occurred where the sciatic nerve passes over the
fused gemelli and obturator tendons. The CCI consisted of
tying four loose 4-0 nonabsorbable silk ligatures around
the sciatic nerve approximately 1 mm apart. The ligatures
were slowly tightened until the first visible twitch in the
leg or foot was observed. The incision was closed in layers
with sutures, and the skin was closed with clips. Rats
survived for 1 month before killing.
Tissue preparation and histofluorescence
The rats were deeply anesthetized with sodium pentobarbital and transcardially perfused with ice-cold phosphatebuffered saline, pH 7.5. The injured (left) and uninjured
(right) L4 and L5 DRG were removed and immediately
frozen on the same block. Sixteen-micrometer-thick frozen
sections were cut, thaw-mounted, and dipped five times (1
second/dip) in sucrose-potassium phosphate-glyoxylic acid
(SPG) solution (10.2 g sucrose, 4.8 g potassium phosphate
monobasic, 1.5 g glyoxylic acid monohydrate per 150 ml
H2O, pH 7.5). The slides were dried under cool air blowers,
baked at 95°C for 2.5 minutes, and coverslipped with
mineral oil. Sympathetic fibers were visualized under
incident ultraviolet light with a 395–446 nm exciter filter
and a .470 nm barrier filter. For each animal, four to eight
randomly selected whole DRG sections were inspected for
sympathetic fibers: In uninjured animals, L4 and L5 DRG
sections were sampled; and, in nerve-injured animals, only
L5 DRG sections were sampled. Images of the entire DRG
sections were captured at 803 by using a digital-imaging
camera (World Precision Instruments, Inc., Sarasota, FL),
and all fields in the cell layers (that is, areas occupied
mainly by neuronal profiles and not fiber bundles) of the
DRG sections containing fluorescent fibers were captured
at 3203. The total length of sympathetic axons in the
high-power images was measured by manually tracing the
on-screen image with a computer mouse, using an imageanalysis software package (Mocha; Jandel Inc., San Rafael, CA). It should be noted that this method consistently
underestimated the total sympathetic axon length, because invading sympathetic fibers often ran in bundles of
several axons. Care was taken not to include sympathetic
fibers obviously surrounding blood vessels. These were
rare in the cell layer in adult animals and more commonly
occurred between fiber bundles or in the DRG capsule.
Sympathetic sprout density was calculated by dividing the
total sympathetic axon length in the DRG section by the
area occupied by DRG neurons in that section. The sprout
density was averaged for each animal, and uninjured
groups (3/0, 10/0, and 16/0) were compared for significant
differences by using a one-way analysis of variance
(ANOVA) followed by Bonferroni’s t-test for pairwise differences. Significant differences in sprout density in nerveinjured rats were assessed by using paired t-tests to
investigate left-right differences and by using a one-way
ANOVA to investigate differences from rats killed preoperatively (again, followed by Bonferroni’s t-test).
40
M.S. RAMER AND M.A. BISBY
Behavioral tests
Three behavioral tests were carried out on groups 3/0,
16/0, 3/1, and 16/1. The first was an assessment of thermal
nociceptive threshold or thermal allodynia (allodynia is
defined as pain due to a stimulus that is not normally
painful): Rats were placed on a glass floor, and latency to
withdrawal from a radiant heat source applied to the
sciatic territory of the hindpaws was measured (Hargreaves et al., 1988). This is a well-characterized method of
detecting changes in sensitivity of hindpaw skin in which
decreased time to withdrawal reflects decreased thermal
nociceptive thresholds. We calibrated the intensity of the
radiant heat source in a preliminary study, so that the
average withdrawal latency for young uninjured rats was
10–12 seconds. Every rat was tested five times alternately
on each hindpaw, allowing at least 2 minutes between
tests, and withdrawal latency measurements were then
averaged. Left and right withdrawal latencies in uninjured groups (3/0 and 16/0) were averaged, and the averaged values were compared by using a t-test. Paired t-tests
were used to compare 1) postoperative (groups 3/1 and
16/1) ipsilateral to contralateral withdrawal latencies, and
2) ipsilateral and contralateral postoperative values to the
corresponding preoperative values.
We also measured the duration of the response to a
painful thermal stimulus or thermal hyperalgesia. Hyperalgesia is defined as increased pain due to a normally
noxious stimulus. Increased withdrawal durations are
presumed to reflect longer lasting pain sensation following
the thermal stimulus, and these measurements are useful,
in that they provide information on a different aspect of
thermal pain (duration) than measurements of withdrawal latency (threshold). The same paradigm was used
as that used in the threshold test, except that the duration
of withdrawal from a more intense stimulus (double the
intensity of the thermal allodynia calibration) was measured, and each rat was tested three times per hindpaw
with at least 15 minutes between tests. It should be noted
that the duration of the stimulus required to elicit hindpaw withdrawals at this increased intensity (approximately 4 seconds) was not significantly different between
any groups. This is important, because a comparison of
duration of response between groups requires that the
same suprathreshold stimulus be delivered. A flick of the
hindpaw (i.e., without a sustained elevation) was assigned
a duration of 0.5 seconds. The same statistical analysis
that was used for thermal allodynia was also used here.
A third test assessed the rats’ mechanical nocifensive
(defensive reaction to painful stimulation) response frequency. An increase in response frequency to repetitive
mechanical stimulation can be interpreted as a decrease in
the threshold of mechanically induced pain (mechanoallodynia): The more often a defined mechanical stimulus
elicits a nocifensive withdrawal response, the more painful
that stimulus is for the animal. To carry out the test, rats
were placed in a cage with a wire mesh floor, and a 45-g von
Frey hair was applied to the sciatic territory of alternate
hindpaws (ten times per hindpaw at a frequency of 1 Hz).
If the animal lifted its paw in response to the von Frey hair,
then the testing was suspended until the paw was placed
back on the cage floor. The number of sustained responses
(defined as maintained elevation for .2 seconds, or licking
the hindpaw, or vocalization) per ten trials was recorded.
We chose to quantify sustained responses, because these
served as better indicators of nociception than brisk paw
flicks, which may have reflected either pain or a nonnocifensive response to mechanical stimulation (e.g., tickle). The
same tests described above were used to compare groups
for significant differences. All statistical measures are
presented as means 6 S.E.M.
Photomicrographs
Fluorescent sections were photographed and processed
as 35-mm color slides (Kodak EL 135-24 film; Eastman
Kodak, Rochester, NY). These were scanned (Agfa Arcus II
scanner; Agfa-Gevaert N.V., Mortsel, Belgium), and the
color of the digital images was corrected to match that of
the scanned slide by using a Macintosh computer (Apple
Computers, Cupertino, CA) and Adobe Illustrator software
(Adobe Systems, Mountain View, CA). The images were
not manipulated in any other way. Composites were
assembled and printed either in color (Fig. 1) or in black
and white (Fig. 2; Kodak xls 8600 PS Printer; Eastman
Kodak).
RESULTS
Histofluorescence
After killing the rats, the sciatic nerves were exposed to
check the condition of the CCI. All ligatures were still in
place, surrounded by fibrous connective tissue. There were
no obvious differences in the appearance of the injured
nerves between group 3/1 and group 16/1. The left and
right L4 and L5 DRG from all rats were removed and
processed for glyoxylic acid-induced fluorescence of monoamines. This technique is particularly useful, because
orange autofluorescent, lipofuscin-containing DRG neurons; pale green, fluorescent myelin, and bright bluegreen, varicose sympathetic axons can all be easily distinguished.
Sympathetic sprouting in uninjured
and nerve-injured rats
In 3-month-old uninjured rats, there were rarely any
fluorescent monoamine-positive (sympathetic) fibers within
the cell layer of the DRG. Rather, they were located mainly
between fiber layers and around the perimeter of the DRG,
almost always associated with blood vessels (Fig. 1A,
arrowhead). When, on occasion, sympathetic fibers were
found in the cell body layer, they took on a ‘railroad-track’
appearance or formed a ring around an empty space (Fig.
1C,E, arrowheads), both of which are characteristic of
perivascular fibers when the blood vessel is cut in longitudinal or transverse section, respectively.
In group 10/0 (n 5 5) and group 16/0 animals (n 5 6),
lipofuscin granules were more numerous (Fig. 1), and the
number of varicose sympathetic fibers in the cell body
layer of the DRG increased compared to group 3/0 (n 5 6)
rats (compare Fig. 1A with Fig. 1B). The increase in
fluorescent fiber density was significantly different from
group 3/0 animals for both group 10/0 and group 16/0 rats,
and the sprout density was greater in the group 16/0
animals compared with the group 10/0 animals (Fig. 3).
The fluorescent fibers in both injured and aged uninjured
animals often formed basket-like structures around largediameter DRG neurons (Figs. 1C,D, 2C, arrows), as described previously (Chung et al., 1993, 1996; McLachlan et
al., 1993). The distribution of sympathetic axons in the
ganglia was not even: rather, there were often clusters of
ADRENERGIC SPROUTING IN DRG IN AGING
41
Fig. 1. Glyoxylic acid-induced fluorescence of sympathetic axons in
dorsal root ganglia (DRG) of 3-month-old and 16-month-old uninjured
rats (A,B) and 1 month following partial sciatic nerve injury in
3-month-old (C) and 16-month-old (D) rats. Large-diameter DRG
neurons surrounded by sympathetic axons at high power are also
shown from 3-month-old (E) and 16-month-old (F) nerve-injured
animals. Note the increased density of lipofuscin granules in the DRG
neurons of the older animals (B,D,F). Perivascular sympathetic axons
are rare in the cell body layer of 3-month-old DRG (A) but can be found
often in the fiber layer (A, arrow). Blood vessel-associated sympathetic
axons are indicated by thick arrows in A, C, and E. In aging animals,
fluorescent fibers can be seen invading from the DRG perimeter (B,
arrow). DRG neurons surrounded by sympathetic axons are indicated
by thin arrows in C and D. Sympathetic sprout density increases with
age. Scale bars 5 100 µm in A–D, 20 µm in E,F.
large-diameter DRG neurons with sympathetic baskets
near blood vessels or (more frequently) near the perimeter
of ganglion sections, from which the sympathetic axons
appeared to have grown (see, e.g., Fig. 1B). Many fluorescent fibers could be seen that extended from the vasculature or the DRG capsule deep into the parenchyma of the
ganglion. Often, fibers invaded the DRG in association
with myelinated axons, as shown in Figure 2A,B (from a
group 10/0 rat and a group 16/1 rat, respectively). At the
light microscope level, these abnormal arborizations, when
basketing DRG neurons, often appeared to be associated
more intimately with the very pale fluorescent glia sur-
42
M.S. RAMER AND M.A. BISBY
Fig. 3. Quantitation of sympathetic sprouting in dorsal root ganglia (DRG) of the six groups of animals (mean and S.E.M.). Vertical
axis indicates the sprout density expressed as length of fluorescent
fibers per unit area of DRG occupied by cell bodies (for details, see
Materials and Methods). On the left (unoperated groups), o indicates a
statistically significant difference from all younger groups (P , 0.05;
one-way analysis of variance; ANOVA). On the right (nerve-injured
groups), an asterisk indicates significant differences from the corresponding unoperated groups (t-test), a plus sign indicates significant
left-right differences (P , 0.05; paired t-test), and a cross indicates
significant difference between nerve-injured young and aging animals
(t-test). CCI, chronic constriction injury.
Postoperatively, the sympathetic fiber density in the
ipsilateral and contralateral DRG was compared with
age-matched control values for both sets of animals (young
and old; Fig. 3). In both age groups, a 1-month CCI induced
significant sympathetic sprouting into ipsilateral and, to a
lesser extent, contralateral ganglia (P , 0.001 for all
groups relative to age-matched control sprout densities;
one-way ANOVA). Sprout density was significantly increased in both the ipsilateral and contralateral ganglia of
group 16/1 rats compared with the respective ganglia in
group 3/1 rats (same test). In both young and old nerveinjured ganglia, many basketed DRG neurons could be
found, and, as with uninjured animals, these occurred
more frequently in aged animals (compare Fig. 1C with
Fig. 1D).
Behavior
Fig. 2. Sympathetic axons associated with myelinated sensory
axons in dorsal root ganglia (DRG) visualized histochemically with
glyoxylic acid-induced fluorescence. At low power (A; from a 10-monthold, uninjured rat) and at higher power (B; from a 16-month-old,
nerve-injured rat), sympathetic axons run along the outer surface of
myelinated axons (node of Ranvier indicated by arrow in A). When
sympathetic baskets form around large-diameter DRG neurons, these
are often intimately associated with weakly autofluorescent satellite
glia (arrows in C; from a 16-month-old, nerve-injured rat). Scale
bars 5 100 µm in A, 20 µm in B,C.
rounding the DRG neurons than with the neurons themselves (Fig. 2C). This was true both pre- and postoperatively.
All nerve-injured rats were of normal weight for age, and
their coats were well groomed. There were very slight
motor deficits in all of the nerve-injured rats when they
were observed at the end of the behavioral testing periods,
including a greater extension of the injured hind limb
when the rats were lifted by the tail, and the toes of the
ipsilateral hindpaw were slightly flexed compared with the
contralateral side. None of the nerve-injured rats showed
the hind limb paralysis that is typical of total axotomy,
indicating that the CCI-induced injury was indeed incomplete. Three of the six aging, nerve-injured rats self
mutilated the injured hind limb. In one, this was so severe
that it was killed early and was not included in the
analysis.
The results of the behavioral tests are shown in Figure
4. Preoperatively, both young (group 3/0; n 5 12) and aging
(group 16/0; n 5 12) rats had a withdrawal latency to
radiant heat of about 11–12 seconds (Fig. 4A). One month
ADRENERGIC SPROUTING IN DRG IN AGING
43
following CCI, young rats (group 3/1; n 5 6) showed a
marked decrease in withdrawal latency ipsilateral to the
injury compared with preoperative values (P , 0.05;
paired t-test), but not contralateral to the injury. There
was a significant left-right difference as well (P , 0.05;
paired t-test). Aging (group 16/1; n 5 5) rats showed a
slight (but not significant) reduction in withdrawal latency
ipsilateral to the injury 1 month following CCI. It should
be noted that, although the data presented are those for
rats at 1 month postoperative, intermittent testing at
shorter postoperative intervals showed that aging rats
never exhibited hyperalgesia within that time.
Once rats responded to the radiant heat by withdrawal,
aging, uninjured rats consistently held their hindpaws
away from the floor longer than younger uninjured rats
(Fig. 4B; 4.1 6 0.7 seconds for group 16/0, left side; 1.3 6
0.3 seconds for group 3/0, left side; P , 0.05; t-test).
Postoperatively, the ipsilateral withdrawal duration was
increased significantly in young rats (4.5 6 0.9 seconds),
but it increased much more dramatically in older rats
(17 6 4 seconds). We saw no contralateral increases in
response duration.
The frequency of withdrawal from a 45-g von Frey hair
depended on age (Fig. 4C), with older, uninjured rats
withdrawing on average more than younger uninjured
rats (P , 0.05; t-test). Postoperatively, withdrawal frequency was elevated ipsilaterally in both young and old
rats (P , 0.05; paired t-test). In old rats, withdrawal
frequency (both ipsilaterally and contralaterally) was increased compared with control values (paired t-tests) and
also compared with younger rats with the same injury (P ,
0.05; ANOVA).
DISCUSSION
Fig. 4. A–C: Behavioral assessment of uninjured and nerveinjured (chronic constriction injury; CCI) young (3 months old; circles)
and aging (16 months old; squares) rats. All points represent the mean
and S.E.M. (note that some error bars are hidden behind the symbols).
Postoperative measurements were taken 1 month following CCI to the
left sciatic nerve. An asterisk indicates significant difference from
preoperative values (paired t-test), a cross indicates significant difference between left (solid symbols) and right (open symbols) sides
(paired t-test), and an x indicates significant difference between young
(group 3/0 or 3/1) and aging (group 16/0 or 16/1) animals (t-test).
In this study we have shown that 1) sympathetic innervation of rat DRG increases normally with age; 2) innervation density in response to CCI is also significantly greater
in aged rats compared with younger rats; 3) aged, uninjured animals exhibit subtle but statistically significant
increases in nocifensive behavior in response to some
thermal and mechanical stimulation; and 4) aged, nerveinjured animals show increased duration of response to
thermal stimulation and an increased frequency of withdrawal from nociceptive mechanical stimulation in response to a CCI. However, aged animals do not show a
significantly decreased thermal nociceptive threshold following injury.
An important technical consideration in the present
experiment was whether or not the extent of the lesion was
similar in the young and old rats. In rats with polyethylene
cuffs of varying sizes placed around the sciatic nerve, the
resulting behavior and nerve damage depended on the cuff
diameter (Mosconi and Kruger, 1996). Similarly, the extent
of sympathetic sprouting in DRG varies with the extent of
sciatic nerve injury: In the first 2 weeks following CCI,
sprout density is increased compared with rats that have
completely transected sciatic nerves (Ramer and Bisby,
1997). The CCI is a relatively subjective lesion and varies
between laboratories. Although a detailed histological
analysis of the injured nerves was not carried out, we feel
confident that in the present experiment, the extent of the
lesion in young and old animals was similar for a number
of reasons. First, the criterion for determining the tightness of the ligatures was met for all animals (the ligatures
44
were tightened slowly until a leg twitch was observed), and
all lesions were performed by the same individual (M.S.R.).
In this way, the induction of the lesion could be controlled.
Second, the extent of the motor deficits resulting from the
injury was identical for both groups. Third, the gross
appearance of the injured nerve at the time of killing was
similar. Finally, the persistence of an afferent connection
to the sciatic territory (apparent during both early intermittent tests as well as at the end of the experiment) showed
that the injury did not result in complete axotomy for
either group. These factors are strongly suggestive that
the injuries are indeed comparable.
Sympathetic sprouting and neuropathic pain
Although sympathetic-sensory coupling has been documented in human neuromas following nerve injury (Chabal et al., 1992), there are now several reports that show a
connection between sympathetic invasion of the DRG and
behavior associated with neuropathic pain following nerve
injury in rats (Chung et al., 1993, 1996; Davis et al., 1994;
Kim et al., 1996). Whether this association can be linked to
the genesis and/or the maintenance of painful behavior is
currently a matter of controversy, but previous work from
our laboratory shows good temporal correlation between
CCI-induced allodynia and hyperalgesia and sympathetic
sprouting that accompanies the injury (Ramer and Bisby,
1997). In addition, the more rapid sprouting and sympathetic basket formation that occurs following a spinal
nerve ligation (Chung et al., 1993, 1996; Kim et al., 1996)
is accompanied by equally rapid establishment of mechanical and cold allodynia in that model of neuropathic pain
compared with more distal nerve injuries (Kim et al.,
1996). The present study clearly shows that there is a
relationship between the extent of sympathetic innervation of the DRG and some types of abnormal painful
behavior: Specifically, there seems to be a tight relationship between thermal hyperalgesia and mechanoallodynia
(which develop in both young and old animals) and sympathetic sprouting, whereas thermal allodynia and sympathetic sprouting do not always coexist (for example, in old,
nerve-injured animals).
These findings have obvious implications in terms of the
dependence of neuropathic pain on the sympathetic nervous system. However, different models of neuropathic
pain resulting from partial nerve injuries are relieved to
varying extents on surgical or chemical sympathectomy
(Neil et al., 1991; Seltzer and Shir, 1991; Kim et al., 1993;
Kinnman and Levine, 1995; Chung et al., 1996). Results
from separate experiments employing distal partial sciatic
nerve injuries (Seltzer and Shir, 1991; Wakisaka et al.,
1991), proximal injuries (Chung et al., 1996), or both (Kim
and Chung, 1992; Kim et al., 1997) indicate that the more
proximal the partial nerve injury is to the DRG, the more
effective sympathectomy is at relieving the pain. Not
surprisingly, then, the dependence of distal CCI-induced
pain on the sympathetic nervous system has also been
debated (Wakisaka et al., 1991; Perrot et al., 1993; Desmeules et al., 1995). This inconsistency of the effects of
sympathectomy after CCI may be due to the absence of a
sympathetic component to the pain, but it is more likely
due to the involvement of mechanisms in addition to but
separate from the sympathetic nervous system in the
maintenance of the pain. Not only does the ability of
sympathectomy to relieve pain vary with distance of the
lesion from the DRG, but so does the degree to which
M.S. RAMER AND M.A. BISBY
sympathetic axons invade the DRG: Distal lesions have
been shown to have less pronounced effects in terms of
sympathetic sprouting in the DRG than proximal lesions
(Kim et al., 1996).
Age effects
Previous studies on the effect of age on partial nerve
injury-induced neuropathic pain have yielded contradictory results: L5 spinal nerve ligation causes more pronounced symptoms in young rats (Chung et al., 1995),
whereas ligation of one of the spinal nerves of the rat tail
has a greater behavioral effect in aged rats (Kim et al.,
1995). One report on the effect of age on behavior following
chronic constriction of the sciatic nerve showed increased
thresholds to thermal and mechanical stimulation in
107-day-old rats compared with 54- and 71-day-old rats
(Tanck et al., 1992). However, the oldest rats in that study
were the same age as the youngest rats in the present
study (90–100 days). We report here for the first time the
exacerbating effect of advanced age on CCI-induced pain.
We also report that thermal hyperalgesia (but not thermal
allodynia) and mechanoallodynia develop with age (albeit
slightly), even in the absence of injury. This may well be
analogous to the finding that older humans show decreased pain sensitivity (that is, no allodynia) to contact
thermal stimulation but increased severity of thermal
pain (hyperalgesia; Harkins et al., 1986). The failure of the
aged rats to exhibit a decreased threshold for thermal
stimulation following nerve injury may be related to
changes in the thermal properties of the skin or to
alterations in sensory fiber function with age, because
antidromic electrical stimulation of the sciatic nerve elicits
a decreased substance P-mediated, cutaneous, vascular
response compared with young rats (Khalil et al., 1994),
and substance P levels in DRG are significantly reduced
with age (unpublished observation cited in Khalil et al.,
1994).
The finding that sympathetic fibers invade the DRG
with age, even in the absence of injury, raises the question
of the stimulus for sprouting following injury or during
normal aging. In both cases, sympathetic axons arborize
almost exclusively around large-diameter DRG neurons,
and, in both cases, sympathetic axons invade the DRG
from the vasculature and from the ganglion perimeter
(McLachlan et al., 1993; present results), suggesting a
common mechanism.
What is the sprouting stimulus?
The sprouting of sympathetic fibers from perivascular
nerves and the DRG capsule may be due to nerve growth
factor (NGF). When a peripheral nerve is injured, the
distal stump undergoes Wallerian degeneration, which is
characterized by Schwann cell proliferation, macrophage
invasion, and the production of NGF. NGF is also elevated
in denervated target tissues (Mearow et al., 1993), and
NGF mRNA is elevated in the DRG following injury
(Sebert and Shooter, 1993, Wells et al., 1994). We have
previously proposed that a mechanism for sympathetic
sprouting in the DRG relies on retrograde transport to the
DRG of NGF (or some other product of Wallerian degeneration) produced in the distal stump of a partially injured
nerve via axons spared by the injury. Retrograde-transported NGF would then effect perivascular sympathetic
axon sprouting (Ramer and Bisby, 1997; Ramer et al.,
1997). Others have also suggested an involvement of NGF
ADRENERGIC SPROUTING IN DRG IN AGING
(Davis et al., 1994; Zhou et al., 1996). The NGF-specific
receptor trkA, which is present on sympathetic axons (for
review, see Barbacid, 1994), very likely plays a role in the
sprouting response of sympathetic fibers. The retrograde
transport of NGF in DRG neurons also requires trkA, and,
although the receptor is expressed mainly in small primary afferents, it is possible that the sympathetic baskets
encapsulate a subpopulation of large-diameter neurons,
which also express trkA (Wright and Snider, 1995). Whatever stimulatory agent is involved, it seems that the
requirement is some product of the degenerating nerve,
because behavioral manifestations of a CCI are suppressed in C57Bl/Wld (Wld) mice, a strain that is deficient
in Wallerian degeneration (Myers et al., 1996) and in
which sympathetic invasion of the DRG is also delayed
(Ramer et al., 1997).
The mechanism underlying the increased sympathetic
innervation of the DRG in aged rats is unknown. Sympathetic sprouting and pain behavior in the absence of injury
may be related to degenerative changes that are known to
occur in aging rat DRG, such as sensory neuron involution
(for review, see Devor, 1991). Age-related changes in
sympathetic innervation seem to be tissue specific: Innervation of the cerebral vasculature decreases, whereas that
of the tail artery increases (Andrews and Cowen, 1994). If
this is true of the peripheral vasculature generally, then a
side effect may be increased sprouting into the DRG. The
increased innervation in the aged DRG following nerve
injury may again be ascribed to NGF. First, aged sympathetic fibers have a slightly enhanced outgrowth ability
when they are provided with a peripheral target (Gavazzi,
1995), and they respond to applied NGF with more widespread growth (Andrews and Cowen, 1994). Second, degenerating nerves of old rats have elevated NGF levels
compared with young rats (Date et al., 1994). This may
explain why sympathetic innervation of the DRG after CCI
is more pronounced in the aged rats, but a more trivial
explanation is simply that there are more sympathetic
fibers resident within the DRG when the nerve is injured,
providing a richer source of sprouts.
Contralateral effects
The mechanisms for the contralateral effects of CCI are
unclear. In the present experiment, we found no significant contralateral changes in thermal nociceptive thresholds, nocifensive response duration, but the withdrawal
frequency from mechanical stimulation was elevated
slightly but significantly on the contralateral side of aging
nerve-injured rats. Kupers et al. (1992) also noted saphenous-mediated bilateral changes in nocifensive behavior
following a sciatic nerve injury. In addition, chronic constriction of the rat infraorbital nerves (IoN) results in increased
sensitivity in the territory of both the injured and the
contralateral IoN (Vos et al., 1994). Human cases have
been reported in which allodynia is present bilaterally
after a unilateral mechanical insult (Baron and Maier,
1995). Like the work of McLachlan et al. (1993) and our
own previous work (Ramer and Bisby, 1997), we found that
ganglia opposite the injury had been invaded by sympathetic axons. Other bilateral neuroanatomical changes
have been observed in response to unilateral experimental
manipulations: Bilateral changes in neuropeptide expression are seen after a unilateral intraplantar injection of
NGF (Amann et al., 1996) and after unilateral adjuvantinduced arthritis (Donaldson et al., 1995). In addition, the
45
expression of the low-affinity NGF receptor (p75) increases
bilaterally in DRG satellite cells after a sciatic nerve
injury (Zhou et al., 1996). There remains no satisfactory
explanation for these bilateral changes, but elevated targetderived factors (for example, from the skin; Mearow et al.,
1993) or trophic factors from the degenerating peripheral
nerve may be acting on DRG neurons and sympathetic
axons in the contralateral ganglion via the circulation.
Sympathetic-sensory coupling
The mechanism by which sympathetic fibers in the DRG
might increase pain sensitivity and/or pain severity is also
unknown, but the ability of DRG neurons to express
adrenergic receptors has been investigated (Gold et al.,
1997), and it has been shown that some small-diameter
DRG neurons up-regulate a receptors following injury
(Sato and Perl, 1991; Nishiyama et al., 1993). Adrenergic
a-2 receptors have been shown to be coupled to N-type
calcium channels in cultures of previously axotomized rat
sensory neurons, and application of norepinephrine had
the greatest effects on small cells (possibly nociceptive)
and on DRG neurons cultured from autotomizing rats
(Abdulla and Smith, 1997). Alternatively, as suggested by
Levine and colleagues (1986), increased levels of released
norepinephrine may be acting on the sympathetic fibers
themselves, causing the release of prostaglandins PGE2
and PGI2 in the vicinity of DRG axons (or cell bodies),
resulting in increased afferent sensitivity. At any rate,
there is an accumulating body of evidence that shows
increased DRG neuron activity in response to sympathetic
stimulation after nerve injury (McLachlan et al., 1993;
Devor et al., 1994; Michaelis et al., 1996).
The specific submodalities of pain influenced by the
sympathetic nervous system remain unknown. The baskets of sympathetic fibers eventually form preferentially
around large-diameter (low-threshold mechanosensory or
possibly proprioceptive) DRG neurons, although some
small neurons are initially surrounded (Chung et al.,
1996). Injury induces neurochemical changes in the phenotype of large-diameter DRG neurons: Preprotachykinin
(the precursor to the pain-transmitting peptide substance
P) mRNA can be found in large DRG neurons following
injury, but not in uninjured animals (Noguchi et al., 1994).
In addition, large DRG neurons undergo a change in
connectivity in the spinal cord following injury, sprouting
into the more superficial laminae of the dorsal horn (which
normally receives much of the input from small diameter
nociceptive afferents; Woolf et al., 1992; Shortland and
Woolf, 1993; Doubell et al., 1997). Increased responses of
large DRG neurons to normally innocuous mechanical
stimuli through sympathetic coupling, combined with phenotype changes and sprouting into the dorsal horn, could
underlie some abnormal behaviors that are characteristic
of neuropathic pain.
CONCLUSIONS
In conclusion, we show that sympathetic fibers accumulate in the DRG of uninjured animals as a function of age
and that the CCI-induced sympathetic innervation of DRG
of older animals is greater than that of younger animals.
This work provides further evidence of a link between
sympathetic sprouts in the sensory ganglia and behavioral
anomalies associated with partial nerve injury. The importance of these phenomena, particularly with respect to
46
M.S. RAMER AND M.A. BISBY
aging, is that they may provide insight into neuropathic
pain in the elderly, who are especially afflicted by this
debilitating clinical condition.
ACKNOWLEDGMENTS
We thank Serap Erdebil and Kevan McRae for fine
technical assistance. M.S.R. receives an MRC studentship.
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