Department of Oral and Craniofacial Biological Sciences, University of Maryland Dental School, Baltimore, Maryland 21201
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Andrew, David and Joel D. Greenspan. Mechanical and Heat Sensitization of Cutaneous Nociceptors After Peripheral Inflammation in The Rat. J. Neurophysiol. 82: 2649-2656, 1999. Tissue injuries commonly cause an increase in pain sensitivity, so that normally painful stimuli become more painful (hyperalgesia), and those usually associated with nonnoxious sensations evoke pain (allodynia). The neural bases for these sensory phenomena have been explored most extensively using heat injuries and experimental arthritis as models. Heat sensitization of cutaneous nociceptors is observed after burns, and sensitization of articular afferents to limb movements occurs after knee joint inflammation. These are likely to be peripheral mechanisms of hyperalgesia. Others, using different models of peripheral inflammation, have only rarely found mechanical sensitization of cutaneous nociceptors. In general these studies have failed to evaluate suprathreshold mechanical sensitivity, which has led to the concept of enhanced spinal cord processing ("central sensitization") serving as the neural substrate for mechanical hyperalgesia. In the current experiments, the mechanical and heat responses of cutaneous nociceptors supplying the glabrous skin of the rat hindpaw were studied 16-24 h after induction of acute inflammation with complete Freund's adjuvant. Single-fiber recordings were made from nociceptors in the sciatic nerve of barbiturate-anesthetized animals, and their responses compared with those obtained from nociceptors tested identically in normal animals. Nociceptors were characterized by the following: 1) graded mechanical stimuli (5-90 g) delivered with probes of tip area of 1 and 0.1 mm2, 2) their adaptive responses to 2-min mechanical stimuli at three intensities, and 3) their responses to graded heat stimuli (40-50°C). Forty-three nociceptors were studied in the inflamed state; 20 were A fibers, and the remainder were C fibers. Mechanical thresholds, determined with calibrated monofilaments, were not significantly different from controls. Sensitization to suprathreshold mechanical stimuli was observed for both A- and C-fiber nociceptors, although it was greater for the A fibers. Similarly, sensitization during testing of adaptive properties of A- and C-fiber nociceptors was seen, although it was limited to the dynamic (initial) and not the static (plateau) phase of the response. Heat sensitization was observed in 25% of A-fiber nociceptors, but the responses of C fibers to heat were depressed. Other indicators of neuronal sensitization, such as spontaneous activity and expanded receptive fields, were also observed. It was concluded that the mechanical hyperalgesia caused by peripheral inflammation could be explained by nociceptor sensitization. Central mechanisms cannot be completely ruled out as contributing to such hyperalgesia, although their role may be much smaller than previously envisaged.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The effects of tissue damage and inflammation are
characterized by increased pain sensitivity, so that the curve that
describes the relationship between stimulus level pain intensity is
shifted leftward. If the shift is sufficiently large, there are two
behavioral consequences: 1) painful stimuli become more
painful (i.e., hyperalgesia), and 2) previously
nonpainful stimuli such as light touch become painful (i.e.,
allodynia). Both central and peripheral sensitization in
nociceptive pathways have been implicated in the generation of enhanced
pain sensitivity (for review see Dubner and Ruda 1992). Recent studies of the effects of injectable or topical agents that
evoke an inflammatory response (e.g., carrageenan, complete Freund's
adjuvant, turpentine), have produced apparently conflicting data on the
contributions of peripheral and central sensitization after injury.
There is no doubt that physical injuries such as burns cause threshold
reductions and increases in suprathreshold discharge of heat-sensitive
cutaneous nociceptors (LaMotte et al. 1982
; Meyer
and Campbell 1981
). However, until recently, only afferents
supplying deep tissues (muscle: Berberich et al. 1988
; joints: Schaible and Schmidt 1985
, 1988
) have been shown
to respond to inflammatory agents with increases in mechanosensitivity.
In contrast, several studies of cutaneous afferents have reported heat,
but not mechanical, sensitization of C-fiber nociceptors (Kocher
et al. 1987
; Reeh et al. 1986
) or that cutaneous
nociceptors innervating inflamed tissue were indistinguishable from
controls supplying uninflamed skin (Hylden et al. 1989
;
Woolf and MacMahon 1985
). Inflamed cutaneous nociceptors
that were spontaneously active in the absence of stimulation have been
reported by Kocher et al. (1987)
and by Hylden et
al. (1989)
. All of these studies have used threshold reductions
to indicate sensitization, but reliance on threshold data alone to
predict mechanical sensitization of nociceptors is prone to false
negatives (Cooper et al. 1991
). Therefore, failure to
observe nociceptor sensitization to mechanical stimulation may be
because suprathreshold sensitivity was not studied.
We have therefore reinvestigated the effects of inflammation on
cutaneous nociceptor sensitivity, by using sufficiently sophisticated stimuli to identify sensitization. The inflammatory model we have used
is unilateral injection of complete Freund's adjuvant (CFA) into the
plantar surface of the rat's hindpaw. This model has been subject to
extensive behavioral characterization (Hylden et al.
1989, Iadarola et al. 1988a
, 1988b
) and causes
intense edema and hyperalgesia in the injected paw within a matter of hours. Animals treated this way maintain their weight, display normal
grooming behavior, and provided they can guard the affected limb, do
not appear to be in inescapable pain (Iadarola et al. 1988a
,b
). Statistical comparisons were made with a similar
sized population of nociceptors supplying the uninflamed rat hindpaw (Andrew and Greenspan, 1999
).
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Experiments were performed on 18 male Sprague-Dawley rats (300-400g; Harlan, Indianapolis, IN) under institutionally approved protocols.
Induction of inflammation
Subcutaneous injection of CFA (Sigma) was used to induce acute
inflammation of the right hindpaw. The animals were anesthetized with
5% Isoflurane in O2, and 150 µl of a 1-mg/ml
solution of CFA was injected into the paw over 30 s. The animals
were returned to their cages and allowed to regain consciousness. To
limit discomfort of the animals after the injection, the ethical
guidelines of the International Association for the Study of Pain were
followed (Zimmerman 1983). The animals were housed
singly in cages, the floors of which were covered with soft bedding,
and the survival period was kept as short as possible.
Animal preparation and nociceptor identification
Sixteen to 24 h after CFA injection, the animals were
prepared for a terminal experiment. At this time heat and mechanical hyperalgesia are near maximal (Hylden et al. 1989;
Iadorola et al. 1988b
). Each rat was anesthetized with
an intraperitoneal injection of sodium pentobarbital (50 mg/kg;
Nembutal, Abbott, North Chicago, IL) and surgically prepared for
recording the activity of single hindpaw nociceptors with glabrous skin
receptive fields, as described in the companion article (Andrew
and Greenspan, 1999
). Nociceptors were identified in fine
filaments of sciatic nerve by their response to noxious (squeezing),
but not nonnoxious (brushing, gentle pressure), mechanical stimuli. By
using these criteria, it is possible that nociceptors with very low
mechanical thresholds were discarded. Nociceptor-receptive fields,
conduction velocity, mechanical threshold, and tissue compliance were
determined, as described in the companion article (Andrew and
Greenspan, 1999
).
Evaluation of nociceptor mechanical and heat sensitivity
Nociceptor responses to short-duration graded mechanical stimuli
applied with probes of contact areas of 1 and 0.1 mm2 were recorded as described in the companion
article (Andrew and Greenspan, 1999), as were adaptive
responses to long-duration (2 min) mechanical stimuli. Heat sensitivity
was investigated by applying discrete stimuli with a contact thermal
stimulator (probe tip area 1.1 cm2; Taylor
et al. 1993
). Ramp-and-hold (rise time 2.0 s, hold time 5.0 s, interstimulus interval 25 s) stimuli in the range of
40-50°C were delivered in 2°C steps, from an adapting skin
temperature of 35°C. Heat thresholds were defined as the temperature
that evoked a single impulse from a fiber. When a stimulus evoked more than 1 impulse and was preceded by a subthreshold stimulus, threshold was defined as the mean of the sub- and suprathreshold temperatures. For spontaneously active units, thresholds were defined as the stimulus
intensity (force or temperature) that increased the firing rate of a
unit by greater than twice the standard deviation of its ongoing
discharge frequency.
Data analysis
Electrophysiological data were sampled and digitized as
described in the companion article (Andrew and Greenspan,
1999). Responses of spontaneously active units were corrected
for background activity. Responses of inflamed nociceptors to graded
mechanical stimuli were compared with those of controls using
three-factor repeated-measures ANOVA (RM ANOVA), with the three factors
being unit type (control or inflamed), stimulus intensity, and probe
size and the repeated measures being stimulus intensity and probe size.
Tissue compliance and responses to heat were compared across fiber
types using two-factor RM ANOVA. Tukey's test was used to compare
groups with one another if ANOVA revealed a significant-factor effect.
Statistical differences between the adaptation of nociceptors supplying
normal and inflamed skin were determined by resolving the time course
of individual responses into two constants, a and
b, using the two-parameter single-exponential-decay
relationship y = aebx
that relates firing rate (y) to time
(x). With this function, a is proportional to the
peak firing rate (the "dynamic" phase of the fiber's response),
and b is proportional to the rate of response decline (the
"static" phase of the response). For each stimulus intensity,
comparisons between these constants for control and inflamed fibers
were made with the Mann-Whitney rank sum test.
For all statistical tests, P < 0.05 was considered significant.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
General properties of inflamed nociceptors
Recordings were made from 43 nociceptors with receptive fields on the glabrous skin of the hindpaw; 20 were A fibers, and the remainder were C fibers. The conduction velocities of the A fibers ranged from 5.3-34.8 m/s (17.1 ± 9.2 m/s, mean ± SD), those of the C fibers were between 0.7 and 1.2 m/s (1.0 ± 0.1 m/s). Mean monofilament threshold of the A-fiber nociceptors was 756 kPa (764 ± 278 kPa, median ± SD; range 520-1639), and that of the C fibers was 933 kPa (930 ± 308, range 520-1639). The median threshold values of nociceptors supplying inflamed tissue were not significantly different from those of controls (P > 0.1 for both A- and C-fiber nociceptors, Mann-Whitney rank sum test). Although the areas of receptive fields were not precisely measured by planimetry, eight of the units in inflamed paws had receptive fields that were much larger than those that could be accounted for solely by the increase in paw volume that inflammation causes (Fig. 1). Fourteen of the units were spontaneously active (9 C fibers and 5 A fibers), their rates of ongoing activity were between 0.1 and 1.3 imp/s (0.7 ± 0.3 imp/s, mean ± SD).
|
Responses to mechanical stimulation
Although there were no significant differences between the monofilament thresholds of units supplying normal or inflamed skin, there was significant sensitization to mechanical stimuli (Fig. 2A and C). Although inflamed A-fiber nociceptors were considerably more variable in their responses than their control counterparts, they were significantly more responsive to suprathreshold mechanical stimuli than were controls for both the 1 (P < 0.003, 3-factor RM ANOVA) and 0.1 mm2 (P < 0.04, 3-factor RM ANOVA) probes. There was also a significant interaction between fiber type (control vs. inflamed) and stimulus intensity (P < 0.008, 3-factor RM ANOVA) and also between fiber type and stimulus intensity and probe size (P < 0.005, 3-factor RM ANOVA). The C-fiber nociceptors were significantly more responsive than controls for the 0.1 mm2 probe (P < 0.05, 3-factor RM ANOVA), but not for the 1 mm2 probe (P > 0.14, 3-factor RM ANOVA). There were no significant interactions among factors for the C-fiber nociceptors.
|
Adaptation of nociceptor discharge
Stimuli of intensity 25, 50, and 100 g delivered for 2 min
with a probe of tip area 0.1 mm2 were used to
investigate nociceptor mechanical adaptation. Single exponential decays
were fitted to individual responses and the peak firing rate and decay
rate computed. The R2 values
(mean ± SD) for these fits for control and inflamed A fibers,
respectively, were 25 g: 0.35 ± 0.16, 0.58 ± 0.25;
50 g: 0.40 ± 0.17, 0.67 ± 0.17; and 100 g:
0.47 ± 0.19, 0.66 ± 0.14. Those for the C fibers were
25 g: 0.31 ± 0.18, 0.38 ± 0.19; 50 g: 0.56 ± 0.15, 0.48 ± 0.26; 100 g: 0.47 ± 0.19, 0.66 ± 0.16. The adaptational properties of inflamed A- and C-fiber
nociceptors are shown in Fig. 3, and the
best-fit exponential function parameters are shown in Fig.
4. For the A-fiber nociceptors,
sensitization was observed in the peak of the dynamic phase of
responses (P < 0.05, Mann-Whitney rank sum test), but
not during the response decay (P > 0.3, Mann-Whitney
rank sum test) for all three stimulus intensities. The proportion of
A-fiber nociceptors that exhibited rapidly adapting
(A-HT[RA]) responses to tonic mechanical stimuli declined from 50% in controls to 30% after inflammation (P < 0.04, 2 test). The
C-fiber nociceptors were also sensitized during the dynamic phase of
the response, but only at the highest intensity tested (100 g;
P < 0.05, Mann-Whitney rank sum test). There were no
significant differences between the normal and inflamed units in their
rates of adaptation at any of the stimulus intensities tested
(P > 0.4, Mann-Whitney rank sum test).
|
|
In addition to nociceptor sensitization during the dynamic phase of
responses to long-duration mechanical stimuli, 15 units (12 C fibers, 3 A fibers) displayed bursting activity during stimulation. This was
observed only rarely in control units and seldom exceeded more than two
or three pairs or triplets of action potentials during a stimulus (see
Fig. 2 in Andrew and Greenspan, 1999). An example is
shown in Fig. 5. Despite the observation
that nociceptor bursting was dramatically increased during
inflammation, there was no significant relationship between stimulus
intensity and any of the following burst characteristics that were
measured: number of bursts per stimulus (P > 0.1, ANOVA), latency to first burst (P > 0.3, ANOVA),
interburst interval (P > 0.8, ANOVA), peak firing rate
within a burst (P > 0.9, ANOVA), or the number of
impulses in a burst (P > 0.5, ANOVA).
|
Inflammatory effects on skin compliance
Skin compliance was determined by measuring probe displacement
during testing a unit with graded mechanical stimuli, to investigate whether changes in skin stiffness could account for the changes in
nociceptor mechanosensitivity after inflammation. There was no
significant difference between the compliance of control and inflamed
A-fiber-receptive fields (P > 0.2, 2-factor RM ANOVA; Fig. 6). The receptive fields of inflamed
C fibers were significantly more compliant than were controls
(P < 0.05, 2-factor RM ANOVA; Fig. 6), and there was
also an interaction between stimulus intensity and unit type
(P < 0.001, 2-factor RM ANOVA). There is no obvious explanation for this differential effect on A- versus C-fibers. It may
reflect a difference in proportion of receptive fields in the center
versus the edges and digits of the paw (Fig. 1). Nevertheless, the fact
that both A- and C-fibers demonstrated significant mechanical
hyperresponsiveness, yet only the C-fiber-receptive fields showed a
compliance increase after inflammation, implies that the changes in
mechanosensitivity in C-fiber nociceptors were due to receptor
sensitization and not to better stimulus-receptor coupling produced by
edema (Cooper 1993).
|
Heat-evoked responses
Of 20 control C-fiber nociceptors, 16 responded to heating in the
range tested. Threshold was 42.5 ± 1.9°C (mean ± SD;
range 40-45 °C,), and the units monotonically increased their firing rate as stimulus intensity increased (Fig.
7A). Of 20 control A-fiber
nociceptors only 1 responded to heat, and it did so with a single
impulse at the highest temperature tested (50°C). This negligible
sensitivity of rat A-fiber nociceptors to noxious heat has been
reported previously (Lynn and Shakhanbeh 1988).
Inflammation increased the proportion of A fibers that were heat
sensitive (5 of 20 tested), but not significantly so (P > 0.1, Fisher exact test). Their thresholds were in the range
43-48°C (45.2 ± 1.9°C), and they were significantly more
sensitive than controls (P < 0.04, Mann-Whitney rank
sum test on responses to 50°C; Fig. 7B). Of 23 C-fiber
nociceptors tested after CFA-induced inflammation 19 responded; the
proportion of heat-sensitive C-fibers was not significantly different
between groups (P = 1, Fisher Exact test). Mean heat
threshold of the C-fibers supplying inflamed skin was 42.1°C (range
40-45 ± 1.9°C), which was not significantly different from
that of controls (P > 0.6, unpaired
t-test). After inflammation, the heat-evoked responses of
C-fibers were significantly reduced (Fig. 7A;
P < 0.05, 2-factor RM ANOVA). None appeared to be
sensitized to heat, and a group of fibers with very weak heat responses
seemed to cause the population response to be depressed (Fig. 7,
C and D).
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In the present experiments we have described mechanical and
heat sensitization of glabrous skin nociceptors in the rat's hindpaw after acute inflammation. Mechanical sensitization of nociceptors by
agents evoking an inflammatory response has previously been demonstrated only for deep tissue afferents (Berberich et al. 1988; Schaible and Schmidt 1985
, 1988
), and for
goat palate mechanonociceptors (Cooper et al. 1991
). The
failure of other studies (Hylden et al. 1989
;
Kocher et al. 1987
; Reeh et al. 1986
;
Woolf and MacMahon 1985
) to show mechanical
sensitization of cutaneous nociceptors could be used to infer that
mechanical sensitization is peculiar to mechanonociceptors supplying
specialized tissues. However, these studies relied solely on mechanical
thresholds as a measure of mechanical sensitization, and as reported by
Cooper et al. (1991)
, mechanical sensitization is not
necessarily associated with significant reductions in nociceptor
thresholds. Our experiments show first, in agreement with Cooper
et al. (1991)
, that mechanical thresholds are inadequate
predictors of sensitization and second, mechanical sensitization was
observed for cutaneous nociceptors with both A- and C-fiber conduction
velocities, including mechanoheat nociceptors.
Heat sensitization was evident for A-fiber but not for C-fiber
nociceptors that showed significantly depressed responses. This
reduction in mean responsiveness per fiber was unexpected because
inflammation-induced heat sensitization of rat hairy skin nociceptors
has been described previously (Kocher et al. 1987). However, our experiments were performed on units supplying glabrous skin, where burn injuries in monkey have been shown to sensitize A-fiber nociceptors but to desensitize C-fiber nociceptors to heat
(Campbell and Meyer 1983
; Campbell et al.
1979
; Meyer and Campbell 1981
). Because our
study was performed several hours after inflammation induction, it is
not possible to state with certainty that the reduced responsiveness of
C-fibers to heat was caused by receptor desensitization, as shown by
Campbell and Meyer's (1983)
experiments. An alternative
interpretation of these results is that weak heat sensitivity developed
in previously heat-insensitive receptors. Therefore, the total
nociceptor input to the spinal cord would be increased, despite a
reduction in the average firing rate per nociceptor. A similar finding
has been made in man by Schmidt et al. (1995)
after
topical application of mustard oil or capsaicin to the receptive fields
of nociceptors that were initially heat insensitive.
The enhancement of A-fiber nociceptor heat sensitivity after peripheral
inflammation is similar to that reported in monkey after burns
(Meyer and Campbell 1981). Given that the rat A-fiber nociceptors show almost no heat sensitivity, even after repeated stimulation (Lynn and Shakhanbeh 1988
), the development
of heat sensitivity implies upregulation of heat-transducing
elements within the cell membrane. Whether some of the other
observations on sensitized nociceptors are due to changes in ion
channel expression is unclear. Mechanical sensitization of
nociceptors in hairy skin is an effect of several lipoxygenase
products, such as (8R, 15S)-dihydroxyicosa(5E-9, 11, 13Z)tetraenoic acid and leukotriene B4
(Martin et al. 1988
; White et al. 1990
;
see also Kress and Reeh 1996
; Levine and Tiawo 1994
for recent reviews), and could be due to chemical
modulation of receptor sensitivity. Similarly, the bursting behavior
seen during long-duration mechanical stimuli could either be due to chemomodulation, or the incorporation of new ion channels into the
receptor membrane.
Evidence was also obtained that under control conditions parts of the
terminal arborization of nociceptors were unresponsive but became
responsive after inflammation. This conclusion was based on the
observation that fibers supplying inflamed skin had larger receptive
fields than that of the largest control unit encountered. It is
unlikely that these larger fields are false positives due to the
increase in size of the inflamed paw, because many of the units (35/43)
were of a size similar to control nociceptors. Nociceptor-receptive
field expansion has been described after burns in monkeys
(Thalhammer and LaMotte 1982); after sustained, strong,
blunt pressure in rats (Reeh et al. 1987
); and after
topical mustard oil or capsaicin application in humans (Schmelz
et al. 1994
). The short time course of receptive field
expansion in monkeys (22 min) suggests some sort of local sensitizing
phenomenon, perhaps due to inflammatory mediators, to improved
stimulus-receptor coupling because of edema (Cooper
1993
) or even to strengthening of ephaptic junctions between
coupled nociceptors (Schmelz et al. 1994
).
We have obtained electrophysiological evidence of a peripheral basis
for mechanical hyperalgesia after inflammation: The stimulus-response functions of nociceptors supplying inflamed skin were significantly steeper than those of controls. Also, the proportion of A-fiber rapidly
adapting nociceptors was reduced after inflammation. Coupled with the
development of nociceptor bursting to tonic stimuli, the afferent
inflow into the spinal cord is greatly increased, and as such these
peripheral changes are likely to be mechanisms of mechanical
hyperalgesia. Thus, the idea that mechanical hyperalgesia is solely
mediated at the spinal level seems no longer tenable. In contrast,
mechanical allodynia is likely to be mediated through sensitization of
spinal cord neurons receiving convergent nociceptive and
mechanoreceptive inputs (Torebjörk et al. 1991).
However, it is still possible that some inflamed nociceptors may have
very low mechanical thresholds and would not have been evaluated in the
present study. Heat sensitization was observed for a small proportion
of A-fiber nociceptors, and the population response to heat of
heat-sensitive C-fiber nociceptors was significantly reduced. This
reduced C-fiber response could be interpreted as meaning such fibers
have little role in heat hyperalgesia, despite its being prominent
behaviorally in this model (Hylden et al. 1989
;
Iadorola et al. 1988a
). However, the total input to the spinal cord would be increased if, as we suspect, some previously heat-insensitive nociceptors display weak heat sensitivity after inflammation. Other possible explanations for heat hyperalgesia include
the following: 1) enhanced sensitivity of heat-specific nociceptors, which we would have been unable to detect with our unitary
identification criteria and that have not previously been described in
rat glabrous skin. They are present in monkeys (Baumann et al.
1991
) and pigs (Lynn et al. 1995
) in which they
make up to 7-19% of unmyelinated units; 2) enhanced
central processing in the spinal cord, possibly due to central
sensitization (Dubner and Ruda 1992
) or ongoing activity
in unmyelinated nociceptors producing "wind-up" (Mendell and
Wall 1965
). The latter is supported by our observation that
spontaneously active units are dominated by unmyelinated fibers (64%),
and their discharge frequencies seem to be sufficient to produce a
sustained depolarization of central neurons (Mendell and Wall
1965
).
![]() |
ACKNOWLEDGMENTS |
---|
We thank Dr. J. C. Gunsolley of the Department of Periodontics for statistical advice.
This work was supported by Grant IBN-9696127 from the National Science Foundation.
Current address of D. Andrew: Division of Neurosurgery, Barrow Neurological Institute, 350 W. Thomas Rd., Phoenix, AZ 85013.
![]() |
FOOTNOTES |
---|
Address reprint requests to J. D. Greenspan, Dept. OCBS, Rm. 5-A-12, University of Maryland Dental School, 666 W. Baltimore St., Baltimore MD 21201.
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 26 August 1998; accepted in final form 16 July 1999.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|