Department of Anatomy and Neuroscience, Marine Biomedical Institute, The University of Texas Medical Branch, Galveston, Texas 77555-1069
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Lin, Qing,
Jing Wu, and
William D. Willis.
Dorsal Root Reflexes and Cutaneous Neurogenic Inflammation After
Intradermal Injection of Capsaicin in Rats.
J. Neurophysiol. 82: 2602-2611, 1999.
The role of dorsal
root reflexes (DRRs) in acute cutaneous neurogenic inflammation induced
by intradermal injection of capsaicin (CAP) was examined in
anesthetized rats. Changes in cutaneous blood flow (flare) on the
plantar surface of the foot were measured using a laser Doppler
flowmeter, and neurogenic edema was examined by measurements of paw
thickness. To implicate DRRs in neurogenic inflammation after CAP
injection, the ipsilateral sciatic and femoral nerves were sectioned,
dorsal rhizotomies were performed at L3--S1,
and antagonists of GABA or excitatory amino acid receptors were
administered intrathecally. Intradermal injection of CAP evoked a flare
response that was largest at 15-20 mm from the injection site and that
spread >30 mm. Acute transection of the sciatic and femoral nerves or
dorsal rhizotomies nearly completely abolished the blood flow changes
15-20 mm from the CAP injection site, although there was only a
minimal effect on blood flow near the injection site. These procedures
also significantly reduced neurogenic edema. Intrathecal bicuculline,
6-cyano-7-nitroquinoxaline-2,3-dione, (CNQX) or
D()-2-amino-7-phosphonoheptanoic acid (AP7), but not phaclofen, also reduced dramatically the increases in blood flow 15-20
mm from the CAP injection site, but had only a minimal effect on blood
flow near the injection site. Neurogenic edema was reduced by the same
agents that reduced blood flow. Multiunit DRRs recorded from the
central stumps of cut dorsal rootlets in the lumbar spinal cord were
enhanced after CAP injection. This enhanced DRR activity could be
reduced significantly by posttreatment of the spinal cord with
bicuculline, CNQX or AP7, but not phaclofen. It is concluded that
peripheral cutaneous inflammation induced by intradermal injection of
CAP involves central nervous mechanisms. DRRs play a major role in the
development of neurogenic cutaneous inflammation, although a direct
action of CAP on peripheral nerve terminals or the generation of axon
reflexes also may contribute to changes in the skin near the injection site.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Neurogenic inflammation forms part of the
peripheral tissue response to injury. This inflammatory response is
generally attributed to an effector function of primary afferent
terminals (Jancsó et al. 1967, 1968
; Lewis
1927
) and is initiated by the release of vasoactive peptides,
such as substance P (SP) and calcitonin gene-related peptide (CGRP)
(Brain and Williams 1985
; Ferrell and Russell
1986
). The primary afferents involved are thought to be mainly
C fibers, although A
-fibers also play a role (Colpaert et al.
1983
; Lam and Ferrell 1993
; Lewin et al.
1992
).
Although the effector action of primary afferent fibers is often
attributed to axon reflexes (Barron and Matthews 1938;
Carpenter and Lynn 1981
; Lewis 1927
), it
also can be initiated in the spinal cord by primary afferent
depolarization (PAD) large enough to trigger dorsal root reflexes
(DRRs) (Eccles et al. 1961
, 1962
; Koketsu
1956
; Willis 1999
). Previous experiments by our
group have shown that neural processing in the spinal cord dorsal horn contributes to the development of knee joint inflammation by the generation of DRRs (Rees et al. 1994
, 1996
). These nerve
impulses are conducted antidromically in large and small myelinated and in unmyelinated axons, as shown by recordings of compound action potentials in primary afferent fibers from the central stumps of cut
dorsal root filaments or the cut medial articular nerve after induction
of acute arthritis (Sluka et al. 1995a
). This antidromic
activity could, in turn, result in the release of inflammatory mediators in the knee joint (Ferrell and Russell 1986
;
Yaksh 1988
), setting up a positive feedback loop between
the dorsal horn and periphery that tends to prolong the inflammatory
state (Sluka et al. 1995b
; Willis 1999
;
Willis et al. 1998
).
The experiments described in this paper examined the effector role of
primary cutaneous afferents in acute cutaneous inflammation and
determined the possible involvement of DRRs in this process. Intradermal CAP injection is a simple means to evoke an acute neurogenic inflammation in rats (Jancsó et al. 1967,
1968
). When applied to the skin, CAP causes vasodilation if the
sensory innervation is intact (Bernstein et al. 1981
;
Carpenter and Lynn 1981
). To evoke an acute cutaneous
inflammation, CAP was injected intradermally into the plantar skin of
the foot. The flare (a major characteristic of inflammation) was
evaluated by measuring changes in cutaneous blood flow in the foot.
Neurogenic edema was estimated by measurements of paw thickness. DRRs
were evaluated by recording antidromic discharges from the central ends
of cut dorsal rootlets in the lumbar spinal cord. DRRs also could be
demonstrated in cutaneous afferents supplying the foot.
Some of the present results have been reported in abstract form
(Lin et al. 1997b, 1999
).
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
A total of 116 male Sprague-Dawley rats weighing 250-350 g were used for this study. All experimental protocols were approved by the Animal Care and Use Committee and were in accordance with the guidelines of the National Institutes of Health and the International Association for the Study of Pain.
Animals were initially anesthetized by pentobarbital sodium (50 mg/kg
ip). The external jugular vein was cannulated, and anesthesia was then
maintained by infusion of sodium pentobarbital (5-8 mg · kg1 · h
1 ) in a
saline solution. The level of anesthesia was monitored by frequent
examination of pupillary size and responses to stimulation, absence of
a flexion reflex, and stability of the level of end-tidal CO2. Once a stable level of surgical anesthesia
was reached, the animals were paralyzed with pancuronium (0.3-0.4
mg/hr iv) and ventilated artificially. End-tidal
CO2 was kept between 3.5 and 4.5% by adjusting
the respiratory parameters. Rectal temperature was maintained near
37°C by a servo-controlled heating blanket.
Cutaneous blood flow measurement
Changes in cutaneous blood flow in the plantar skin of the foot
were measured to reflect the local vasodilation (flare) that followed
intradermal injection of CAP. The measurements were done by attaching
laser Doppler flowmeter probes (Moor Instruments, UK) to the skin of
the foot with adhesive tape. Blood flow, detected as blood cell flux by
the flowmeter (laser wavelength 633 nm), was processed by a computer
analysis system (CED1401 plus, with Spike2 software), and
blood flow was shown as a voltage level. It has been reported that the
depth of laser penetration can be 500-700 µm below the surface
where the probe is placed (Silverman et al. 1994
).
Therefore the laser Doppler flow probe presumably picked up the blood
flow signal mainly from the microvasculature in the dermis. A volume of
25 µl of CAP, dissolved in Tween 80 (7%) and saline (93%) to a
concentration of 1%, was injected into the plantar surface of the
foot. For control purposes, the vehicle for dissolving CAP was injected
intradermally in the same volume as the CAP solution.
Experiments first were made to map the distribution of flare on the foot skin ipsilateral to CAP injection by placing three probes at sites ~5, 15-20, and 30 mm, respectively, from the spot where CAP was injected intradermally (Fig. 1A). Figure 1, B and C, shows that the maximal flare reaction was recorded from a site (Probe III) ~15-20 mm away from the CAP injection spot and that the blood flow changes were much less at a site (Probe II) ~30 mm away from the injection spot. Therefore blood flow responses at ipsilateral sites >20 mm from the CAP injection site were not studied further.
|
To determine if elimination of presumed DRRs would result in a reduction in the flare produced by CAP injection, the femoral and sciatic nerves were sectioned ipsilaterally or dorsal roots L3-S1 were cut on the day when experiments were performed. CAP was then injected to determine if the flare reaction could be evoked. Before peripheral nerves were sectioned, 2% lidocaine was applied to them at sites where these nerves were to be cut to minimize injury discharges. For control purposes, sham surgery was done in other animals.
In another series of animals, the tip of a catheter
(PE10) was implanted into the spinal subarachnoid
space at the T12-L1 vertebral level for intrathecal administration of drugs. The spinal cord was pretreated with a GABAA receptor
antagonist (bicuculline, 5 µg), a GABAB
receptor antagonist (phaclofen, 15 µg), a
non-N-methyl-D-aspartate (non-NMDA) receptor
antagonist [6-cyano-7-nitroquinoxaline-2,3-dione, (CNQX) 0.1 µg] or
an NMDA receptor antagonist
[D()-2-amino-7-phosphonoheptanoic acid (AP7), 4 µg],
to determine if blockade of spinal GABA or excitatory amino acid (EAA)
receptors could reduce the flare. Drugs were dissolved in artificial
cerebrospinal fluid (ACSF) in a volume of 15 µl and were given
intrathecally 20 min before CAP injection. The same volume of ACSF was
used in other animals for control purposes.
In four rats, the spinal cord was transected at the
T12-L1 level 1 h before
the blood flow measurements were performed. The effects of CAP
injection on blood flow were evaluated in these spinalized rats.
The degree of cutaneous inflammation due to CAP injection was also assessed by paw-thickness measurements. This was done with a digital caliper, which was placed near the site where Probe 2 was placed. Care was taken to assure that the caliper was placed at the same site on the paw for each measurement. Paw thickness was measured before and at 2 h after CAP injection. Paw-thickness changes due to CAP injection also were observed in experiments in which the sciatic and femoral nerves were sectioned or dorsal roots were cut or in which the spinal cord was pretreated with GABA or EAA receptor antagonists. Additionally, paw thickness was measured after the same volume of vehicle was injected intradermally in some rats as controls.
DRR recordings
The lumbosacral spinal cord was exposed by laminectomy to
provide access to dorsal roots for recordings of DRRs. The spinal cord
was protected from drying and cooling by formation of a mineral oil
pool between skin flaps. Heated water was circulated through metal
tubes placed in the pool. A dorsal rootlet from the
L4-L6 level was teased
into several filaments and cut distally. Multiunit DRRs were recorded
by placing the proximal stump of one dorsal root filament on a platinum
unipolar hook electrode. DRRs were amplified and observed on analogue
and digital storage oscilloscopes and discriminated from noise using a
window discriminator. Digitized signals were processed by an interface
(CED 1401) connected to a Pentium PC to construct peristimulus rate
histograms for counting the firing rates. To assure that nerve
impulses, instead of noise, were recorded throughout the experiment,
analogue signals also were processed simultaneously with the use of
Spike-2 wavemark software, which captured the original spikes after
subtracting the noise level (see Fig. 5C). Data storage
space is conserved by saving only the spikes and not the baseline.
Different spikes can be distinguished on the basis of their waveforms.
Spontaneous DRRs always were recorded, but these often had a low
discharge rate. Additional DRR activity was evoked by applying a series of calibrated von Frey filaments having graded bending forces to a
certain area on the foot. The sites from which DRRs could be evoked
were considered the "receptive field" for the DRRs. Because the
threshold for evoking DRRs by mechanically stimulating peripheral
afferent terminals varied with each experiment, an appropriate set of
von Frey filaments was chosen for each animal. The forces applied to
the receptive fields to evoke DRRs were between 20 and 1,274 mN, which
are in the noxious range of stimuli (Leem et al. 1993).
After control spontaneous antidromic activity and evoked DRRs were recorded, CAP was injected intradermally into the foot in the way described in the preceding section. DRRs then were recorded at 15 min after CAP injection. To verify that these antidromic discharges after CAP injection were influenced by the same drugs that reduced flare, the spinal dorsal horn was posttreated with the same GABA, non-NMDA or NMDA receptor antagonists. A catheter (PE10) was placed near the surface of the dorsal horn at the point where the dorsal root from which DRRs were recorded entered the spinal cord. Bicuculline, phaclofen, CNQX or AP7 was dissolved in 15 µl of artificial cerebrospinal fluid (ACSF) at the same concentrations as used for reducing flare. The drugs were administered topically onto the spinal cord at 30 min after CAP injection. For control purposes, the same volume of ACSF was administered onto the spinal cord. Each animal was posttreated with only one drug, and so the animals were grouped according to drug treatment. Recordings of DRRs were made 30 (immediately after drug administration), 60, 90, and 120 min after CAP injection.
In some animals separate from the preceding section, the vehicle (7% Tween 80 and 93% saline) that was used for dissolving CAP was injected into the foot in the same volume while DRRs were recorded.
In two animals, recordings of DRRs were made from the central end of a
cut sural nerve. The effects of CAP on DRRs recorded from ipsilateral
sural nerve were observed when CAP was injected intradermally into
the area that was innervated by the tibial or saphenous nerves
(Takahashi et al. 1994).
Data analysis
Baseline blood flow level was expressed as 100% and percentage changes after CAP injection were compared for different groups of animals. Recorded DRR activity was analyzed off-line from peristimulus time histograms. Spontaneous antidromic discharges were quantitated in terms of multiunit discharge rates. Evoked DRRs were evaluated by subtracting spontaneous discharges from all evoked responses and by adding all responses evoked by stimulation using five graded von Frey filaments to produce a total response value. The responses to CAP injection were expressed as a percentage of baseline, with baseline set at 100%. Statistical significance was tested using ANOVA with repeated measures and differences across time were assessed with post hoc paired t-tests. A grouped t-test was used to compare the difference in responses between groups having different treatments. A P < 0.05 was taken as significant. Values are expressed as means ± SE.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Changes in cutaneous blood flow recorded from different areas of the ipsilateral foot after capsaicin injection and effects of nerve sectioning
A laser probe was placed on a spot near the CAP injection
site (site of Probe I, Fig. 1A) and another 15-20 mm
distally (site of Probe II, Fig. 1A) to observe
simultaneously blood flow changes both near and distal to the CAP
injection site. As shown in Fig. 1B, baseline cutaneous
blood flow was recorded continuously for ~30-40 min before CAP was
injected intradermally into one foot. The CAP effect then was observed
for 2 h.
Experiments were initially done on rats with sham surgery without cutting the sciatic and femoral nerves (n = 7). An elevated blood flow was seen at both sites after CAP injection, but the enhanced responses recorded from Probe I (Fig. 2A) were less than the responses measured from the distal site (Probe II, Fig. 2B). Peak increases were 294 ± 53% (P = 0.01, compared with baseline level) recorded from Probe I and 643 ± 131% (P = 0.0067) recorded from Probe II. After the sciatic and femoral nerves were cut (in separate experiments, n = 6), the enhanced blood flow recorded from both sites (Fig. 2, A and B) was less than in rats with sham surgery, but the magnitude of the reduction in the response at the site distal to CAP injection site was much larger than that at the site near the CAP injection spot (Fig. 2, B vs. A). Peak increases were 137 ± 9% (P = 0.027) recorded from the Probe II and 185 ± 26% (P = 0.027) recorded from Probe I. The peak increase recorded from Probe II in the nerve-cut group became much smaller than that in the sham-operated group (P = 0.002, Table 1). However, there was no statistical difference in the peak increases measured by the Probe I between the nerve-cut and the sham-operated groups (P = 0.11, Table 1).
|
|
In six rats, a dorsal rhizotomy was performed at the L3-S1 levels before blood flow was measured. A result consistent with the nerve-cut study was obtained in animals that underwent dorsal rhizotomy (Fig. 2). The peak increases in blood flow after CAP injection were reduced to 295 ± 72% (P = 0.045) at Probe I (Fig. 2A) and 198 ± 24% (P = 0.009) at Probe II (Fig. 2B). In sham-surgery animals (n = 7), the peak increases were 383 ± 34% (P = 0.005) at Probe I (Fig. 2A) and 589 ± 42% (P = 0.0006) at Probe II (Fig. 2B). There was no statistically significant difference in the peak increases at Probe I between these two groups of animals (P = 0.97, Table 1). However, a significant attenuation of enhanced blood flow responses was seen when the peak increase from Probe II in dorsal rhizotomized rats was compared with that in sham-operated rats (P = 0.0001, Table 1).
Control experiments were done in six rats separate from those used for
CAP injection. The same volume of vehicle (Tween 80 and saline) that
did not contain CAP was injected intradermally into the foot. Blood
flow was recorded from the site of Probe II and observations were made
40 min after vehicle injection. A slight increase in blood flow was
seen (peak value 114 ± 5%) after vehicle injection, but this was
without statistical significance.
To examine if supraspinal modulation affected the neurogenic inflammation after CAP injection, additional observations were made on the effects of CAP injection on blood flow in four spinalized rats. Peak increases recorded from Probes I and II were 148 ± 24% and 491 ± 39%, respectively. These values were slightly less than the changes seen in intact animals. However, the differences did not reach statistical significance (P = 0.16 and P = 0.41, respectively).
Effects of blockade of spinal GABA and excitatory amino acid receptors on blood flow responses after capsaicin injection
Pretreatment of the spinal cord by intrathecal infusion of the GABAA receptor antagonist, bicuculline, or the non-NMDA receptor antagonist, CNQX, reduced profoundly the CAP-induced blood flow responses at the site distal to CAP injection spot (Probe II, Fig. 3B). Peak increases were 222 ± 42% (P = 0.032) and 255 ± 53% (P = 0.018), respectively. These values were much smaller when compared with the peak increases in rats pretreated with ACSF (P = 0.0004 and P = 0.009, respectively, Table 2), in which CAP injection resulted in a 563 ± 78% peak increase (P = 0.002) at Probe II. The responses at the site (Probe I) near the CAP injection spot were also reduced slightly (Fig. 3A), but the changes did not reach statistical significance when compared with the peak increases in rats pretreated with ACSF (P = 0.22 and P = 0.11, respectively, Table 2).
|
|
Interestingly, blood flow responses induced by CAP injection also were reduced by pretreatment with an NMDA receptor antagonist, AP7 (Fig. 3, A and B). The peak increases recorded from Probes I and II were 200 ± 27% (P = 0.031) and 257 ± 52% (P = 0.03), respectively. Compared with peak increases in rats pretreated with ACSF, the peak increase at Probe II was reduced significantly (P = 0.015, Table 2) with AP7 pretreatment. However, the reduction in the response recorded from Probe I was not significant when compared with the response at Probe I of ACSF-pretreated group (P = 0.15, Table 2).
In summary, the amplitudes of the blood flow responses at the site distal to CAP injection were reduced significantly compared with those of the blood flow responses at the site near the CAP injection site when the spinal cord was pretreated with GABAA, non-NMDA or NMDA receptor antagonists.
In contrast, a GABAB receptor antagonist, phaclofen, did not significantly affect blood flow responses (P = 0.83 for Probe I and P = 1.0 for Probe II compared with the responses of the ACSF-pretreated group, Table 2). Peak increases were 319 ± 63% (P = 0.016) at Probe I and 583 ± 125% (P = 0.007) at Probe II.
Changes in paw thickness after capsaicin injection and effects of nerve sectioning and blockade of spinal GABA and excitatory amino acid receptors
Both in sham-operated rats (n = 14) and in rats in which the sciatic and femoral nerves were cut (n = 6) or in which dorsal rhizotomies were performed (n = 6), the development of edema in the hindpaw on the side of CAP injection was observed. Within 2 h after CAP injection, paw thickness increased significantly in sham-operated rats (Fig. 4A). However, the increase in paw thickness was reduced significantly after section of sciatic and femoral nerves or after dorsal rhizotomy. (Fig. 4A).
|
In four rats in which the same volume of vehicle was injected intradermally, the paw thickness was 5.4 ± 0.05 mm before vehicle injection. There was slight increase in paw thickness (5.5 ± 0.06 mm) after vehicle injection, but this was without statistical significance (P = 0.09).
Pretreatment of the spinal cord with ACSF did not obviously affect the increases in paw thickness (n = 6). The increase in paw thickness was similar to that in sham-nerve cut or sham-dorsal rhizotomized rats (Fig. 4B). However, when the spinal cord was pretreated with bicuculline (n = 6), the increase in paw thickness due to CAP injection was almost completely blocked (Fig. 4B). Pretreatment of the spinal cord with either CNQX (n = 6) or AP7 (n = 6) also prevented most of the increase in paw thickness due to CAP injection (Fig. 4B). However, blockade of spinal GABAB receptors with phaclofen did not affect significantly the enhanced paw thickness (n = 6).
Dorsal root reflexes recorded from the central ends of cut dorsal root filaments and changes after intradermal injection of capsaicin
Baseline multiunit spontaneous antidromic discharges were recorded
from 42 animals. The discharges were irregular and had rates that
varied over a wide range of frequencies (0.03-5.9 Hz). Usually, the
activity had a very low rate. In most recorded units, additional DRR
activity could be evoked by mechanically applying a series of von Frey
hairs of increasing bending force to the skin of the foot. Figures
5, A and B, and
6 show examples of spontaneous and
evoked DRRs recorded from four rats, respectively. Intradermal injection of CAP caused an increase in spontaneous and/or evoked activity (2nd rows of Figs. 5A and
6A), although enhanced responses could be predominantly in
either spontaneous or in evoked activity in particular cases (see
2nd row of Fig. 6A). The enhancement of the
responses lasted for 2 h after CAP injection.
|
|
In contrast, no significant change was observed in spontaneous antidromic activity and evoked DRRs after the same volume of vehicle was injected intradermally into the foot in six rats. Spontaneous activity and evoked DRRs 15 min after vehicle injection increased slightly to 120 ± 24% and 102 ± 11%, respectively. However, the changes were not statistically significant when compared with the baseline (P = 0.15 and P = 0.11, respectively).
Enhanced DRRs could also be recorded from the cut central end of a peripheral sensory nerve after CAP injection. Experiments were done in two animals, and Fig. 5C shows the results in one rat. Recordings were made from the central end of the cut ipsilateral sural nerve. CAP injection into the area of the foot that is innervated by the tibial nerve resulted in an increase in spontaneous antidromic discharges. The activity recovered within 90 min after CAP injection.
Effects of posttreatment of the spinal cord with GABA, non-NMDA and NMDA receptor antagonists on enhanced DRRs after capsaicin injection
CAP injection produced a long-lasting increase both in spontaneous
antidromic discharges and evoked DRRs. Posttreatment of the spinal cord
with ACSF at 30 min (3rd rows of Figs. 5A and 6A) after CAP injection did not interfere with this enhanced
activity. There was a significant increase in DRRs for 90 min after
CAP injection with ACSF posttreatment (n = 8, P = 0.04, Fig. 7,
A and B). However, increased responses were
reversed nearly completely after the spinal cord was posttreated with
bicuculline (3rd rows of Figs. 5B and
6B). The responses of eight animals with bicuculline posttreatment are summarized in Fig. 7, A and B,
and Table 2. The CAP-induced responses were significantly reduced after
posttreatment with bicuculline when compared with the values at the
same time points after CAP injection in the ACSF group
(P = 0.028 and P = 0.0009, respectively).
|
To establish if the enhanced DRR responses after CAP injection were selectively reversed by the GABAA receptor antagonist, bicuculline, similar experiments (n = 7) were done using the GABAB antagonist, phaclofen. Posttreatment with phaclofen failed to reverse the increase in DRRs after CAP injection (P = 0.463 and P = 0.301, respectively, Fig. 7, A and B, Table 2).
A reversal of the enhanced DRRs was seen when the non-NMDA (n = 7) or NMDA (n = 8) receptor antagonists, CNQX or AP7, were applied to the spinal dorsal horn, using the same procedure as for bicuculline administration. The reversal of the enhanced DRRs is shown in Fig. 7, A and B, and Table 2.
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The main finding of this study was that intradermal injection of
CAP into the rat foot evoked vasodilation in that foot. This response
occurred both near the CAP injection site and in a surrounding area
that extended >30 mm away from the injection site. Flare is defined as
a cutaneous vasodilation surrounding a site of damage (see Lewis
1927). Because the CAP-evoked spread of vasodilation found in
this work is similar to the flare seen in human skin after CAP
injection (LaMotte et al. 1991
), we refer to it as
flare. Flare was accompanied in these experiments by the development of
edema as shown by an increase in paw thickness. Several lines of
experimental evidence, including the data from DRR recordings provided
by this study, indicate that much of the vasodilation and edema after
intradermal injection of CAP is mediated by way of the dorsal horn of
the spinal cord via an effector action of primary afferent fibers. We
propose that the mechanism of these components of neurogenic cutaneous
inflammation depend on antidromic discharges traveling from the dorsal
horn along primary afferent fibers, that is on DRRs (Fig.
8).
|
It has been thought that inflammation-induced vasodilation around a
cutaneous injury is initiated by axon reflexes (Barron and
Matthews 1938; Carpenter and Lynn 1981
;
Lewis 1927
). This term denotes a reflex that takes place
entirely within a single neuron (Lewis 1927
). It was
proposed that sensory nerve fibers have branches that end in adjacent
areas of skin. One branch forms a sensory ending for reception of the
irritant stimulus, whereas another supplies a blood vessel. When the
sensory ending is activated by inflammatory mediators released by
tissue injury, such as after an intradermal injection of CAP, nerve
impulses travel not only centrally but at the branch point also pass
antidromically to the vicinity of the blood vessel to cause
vasodilation by releasing vasoactive substances. This hypothesis is
based on two lines of experimental evidence: 1) reflex flare
depends on intact sensory innervation because it does not take place in
denervated skin (Bernstein et al. 1981
; Carpenter
and Lynn 1981
; Jancsó et al. 1968
;
Lewis 1927
); and 2) the spread of flare is
prevented by administration of a local anesthetic (Dux et al.
1996
; Jancsó et al. 1968
; Lewis
1927
). Additionally, there is morphological evidence for an
axon reflex arrangement because there are extensive arborizations of
sensory nerve processes and some branches have been described near
blood vessels (Chapman and Goodell 1964
; Helme and McKernan 1985
).
It recently was demonstrated in rats that antidromic stimulation of
identified cutaneous C fibers produces an increase in blood flow in a
localized area, the size of which coincides well with the afferent
receptive field. (Gee et al. 1997). Therefore it is
difficult to explain the flare that we observed simply by a local axon
reflex because the area of flare extends greatly beyond the receptive
fields of rat nociceptive primary afferent neurons. From our
observations, an enhanced blood flow occurs both near the CAP injection
site and up to
30 mm away. Although the part of the vasodilation near
the injection site could be mediated by local axon reflexes or by a
direct action of CAP, it seems unlikely that this mechanism could
account for increased blood flow more than a few millimeters from the
CAP injection site because nociceptors in the rat foot have receptive
fields <6 mm2 in area (Bharali and Lisney
1992
). Other arguments from our study that are also against the
view that neurogenic inflammation is mediated only by a local axon
reflex include 1) the blood flow that could be measured
15-20 mm from the CAP injection spot was increased more than that
recorded at a site within 5 mm of the CAP injection spot; 2)
proximal sectioning of the sciatic and femoral nerves (any sensory
nerve bifurcations in the periphery remained intact) or dorsal
rhizotomy abolished nearly completely the CAP-evoked increase in blood
flow at the distal site, but the change in blood flow near the
injection site was only slightly affected; and 3) more
importantly, blockade of spinal GABAA, non-NMDA or NMDA receptors reduced dramatically the CAP-evoked increase in blood
flow at the distal site, although the enhanced response near the
injection site was only slightly decreased. The vasodilation near the
site of injection could result mainly from local axon reflexes or from
a direct action of CAP on sensory terminals (Szolcsányi 1996
). The fact that there was a smaller vasodilation at a
distance of 5 mm than at 15-20 mm from the CAP injection site could be the result of a blockade by CAP of nerve conduction in CAP-sensitive afferent nerve fibers and thus of DRRs conducted in these fibers. The
widespread vasodilation or flare in the skin distal to the injection
site appears to involve a spinally mediated mechanism (PAD that
triggers DRRs) with an action on large numbers of primary afferent
fibers (Willis 1999
; Willis et al. 1998
).
However, one possible concern is that sympathetic efferents might
be involved in the mediation of the acute peripheral inflammatory response by interaction with primary afferent terminals
(Jänig et al. 1996). The effects of sectioning the
sciatic and femoral nerves and dorsal rhizotomy on the CAP-evoked
changes in blood flow may indicate a central control of neurogenic
inflammation by efferent activity of sensory neurons, but cannot rule
out the possible involvement of sympathetic nerves. The role of
sympathetic nerves in neurogenic peripheral inflammation still remains
obscure. It has been shown that the development of peripheral
inflammation depends on sympathetic postganglionic neurons
(Green et al. 1993
, 1997
; Miao et al.
1996
). However, observations in previous studies by our group
and others showed that acute inflammation of the knee joint was
unaffected by chemical or surgical sympathectomy (Lam and
Ferrell 1991
; Rees et al. 1994
; Sluka et
al. 1994b
). In the present study, blockade of spinal
GABAA, non-NMDA or NMDA receptors reduced
profoundly the enhanced blood flow response after CAP injection. These
observations provide evidence consistent with the idea that spinal cord
modulation of neurogenic inflammation involves DRRs.
To obtain direct evidence that DRRs are involved in cutaneous
neurogenic inflammation, we have used the same model as was used in
experiments for blood flow measurements to determine if DRRs recorded
from the cut central end of dorsal roots at the L4-6 levels become enhanced during acute
cutaneous inflammation of the foot after CAP injection. Our results
reveal that DRRs originating from the spinal cord are enhanced after
intradermal injection of CAP. These antidromic impulses were blocked
pharmalogically by posttreatment of the spinal cord with GABA and EAA
receptor antagonists. The results are consistent with the data on
neurogenic flare and edema. It thus is suggested that DRRs are likely
to play a significant role in cutaneous neurogenic inflammation. The
main reason why we recorded DRRs chiefly from dorsal root filaments
instead of from peripheral cutaneous sensory nerves was based on the
concern that the peripheral nerves contain sympathetic fibers, which
needed to be excluded carefully in this experiment. The role of
sympathetic nerves in neurogenic peripheral inflammation still needs to
be considered (Green et al. 1993, 1997
; Miao et al. 1996
). DRRs also were recorded from the cut central ends of the sural nerve in separate experiments, and DRRs in this peripheral nerve responded to CAP injection in a manner similar to DRRs recorded from dorsal root filaments, indicating that DRRs generated in the
spinal cord do travel along cutaneous afferents to the periphery where
some presumably release inflammatory agents.
Therefore we conclude that the DRRs recorded from either dorsal root filaments or peripheral nerves are enhanced by activation of neighboring intact afferent fibers after CAP injection via central circuits within the spinal dorsal horn. This is supported by the observation that DRRs often could be evoked by mechanical stimulation applied to peripheral receptive fields.
There are several mechanisms by which inflammation might increase the
likelihood for DRRs to be triggered by peripheral stimulation. Inflammation causes an upregulation of GABAergic mechanisms in the
dorsal horn (Castro-Lopes et al. 1994a,b
; Nahin
and Hylden 1991
). An increased release of GABA from GABAergic
interneurons of the dorsal horn could result in more PAD and
secondarily trigger DRRs (Willis 1999
). PAD also is
found to be mediated by spinal non-NMDA receptors (Evans and
Long 1989
; Hackam and Davidoff 1991
). The most
likely explanation of this is that primary afferent fibers release
EAAs, which then activate non-NMDA receptors on GABAergic interneurons,
causing them to release GABA at axoaxonic or dendroaxonic synapses on
primary afferent terminals (Carlton and Hayes 1990
; Sluka et al. 1995b
). A significant alleviation of
inflammatory responses, such as swelling, an increase in the
temperature of the knee joint and hyperalgesia, was accompanied by an
inhibition of DRRs when spinal GABAA or non-NMDA
receptors were blocked (Sluka and Westlund 1993
;
Sluka et al. 1993
, 1994a
). Another mechanism could be an
increased activity of the
Na+-K+-Cl
cotransporter in primary afferent neurons (Willis 1999
).
This cotransporter is regulated by second-messenger systems that are affected by a variety of neurotransmitters, hormones, and growth factors (Alvarez-Leefmans et al. 1998
). Second-messenger
systems may control the cotransporter by phosphorylation and
dephosphorylation. Central sensitization of spinothalamic tract neurons
after intradermal injection of CAP is associated with the activation of
protein kinase pathways (Lin et al. 1996
, 1997a
). Thus
it is plausible to suggest that the
Na+-K+-Cl
cotransporter could be phosphorylated by protein kinases that are
activated after CAP injection.
The results of this and our preceding paper support previous studies
done in models of arthritis (Rees et al. 1994-1996
;
Sluka et al. 1993
, 1994a
, 1995a
) and suggest that
excessive PAD leading to the generation of DRRs may be an important
factor in acute cutaneous neurogenic inflammation. The neurotransmitter
receptors that mediate DRR activity in arthritis include
GABAA and non-NMDA glutamate receptors. However,
the present study indicates that spinal NMDA receptors also are
involved in the generation of DRRs in an inflammatory model involving
the skin, suggesting that the mechanisms by which DRRs are generated in
the development of cutaneous inflammation differ somewhat from those in
joint inflammation. Thus the dorsal horn circuits that produce DRRs in
cutaneous nerve fibers presumably involve GABAA,
non-NMDA and NMDA receptors (Fig. 8). The primary afferent fibers
activate GABAergic interneurons by release of EAAs (Evans and
Long 1989
), and PAD then is initiated by the action of GABA on
GABAA receptors on primary afferent terminals (Curtis and Lodge 1982
; Eccles et al.
1963
). The PAD may become excessive and trigger DRRs, which in
turn propagate peripherally and release inflammatory agents in
peripheral tissue, including the skin (Fig. 8). The result is the
induction of the initial stages of neurogenic inflammation.
![]() |
ACKNOWLEDGMENTS |
---|
The authors thank G. Gonzales for assistance with the illustrations.
This work was supported by Recruitment Grant 2517-98 from the Sealy Memorial Endowment Fund for Biomedical Research and National Institute of Neurological Disorders and Stroke Grant NS-09743.
![]() |
FOOTNOTES |
---|
Address for reprint requests: W. D. Willis, Dept. of Anatomy and Neuroscience, Marine Biomedical Institute, The University of Texas Medical Branch, 301 University Blvd., Galveston, TX 77555-1069.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 5 March 1999; accepted in final form 2 August 1999.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|