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1 Department of Diagnostic and Surgical Sciences, University of Minnesota, Minneapolis, Minnesota 55455; 2 Department of Oral Science, University of Minnesota, Minneapolis, Minnesota 55455; 3 Department of Psychiatry, University of Minnesota, Minneapolis, Minnesota 55455
Submitted 2 April 2003; accepted in final form 7 May 2003
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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, 60 A
, and 18
C fibers). Thirty-nine percent of all fibers were excited by acetic acid, but
a greater percentage of A (52%) and C fibers (44%) were excited than
A fibers (20%). Evoked responses of fibers increased with increasingly
more acidic pH until the greatest responses were evoked by acetic acid at pH
2.59–2.41. Application of acetic acid at pHs <2.41 evoked less
excitation, suggesting that fibers became desensitized. Similar percentages of
nociceptors and low-threshold mechanoreceptors were excited by acetic acid.
Thus primary afferent fibers were excited by acetic acid at pHs that have been
shown to evoke the wiping response in our previous study. The results of the
present study suggest that the model of acetic acid-induced nociception in
frogs may be useful for studying the mechanisms by which tissue acidosis
produces pain. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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; Lindahl
1961; Reeh and Steen
1996; Revici et al.
1949). Mean pHs as low as 6.6 ± 0.2 (mean ± SE) have
been measured in inflamed tissues in humans
(Geborek et al. 1989;
Goldie and Nachemson 1970;
Harrison et al. 1986;
Jebens and Monk-Jones 1959;
Richman et al. 1981;
Treuhaft and McCarty 1971;
Ward and Steigbigel 1978) and
animals (Edlow and Sheldon
1971; Hutchins and Sheldon
1972; Kofoed 1986;
Punnia-Moorthy 1987). In
humans, application of acidic solutions to skin
(Keele and Armstrong 1964;
Lindahl 1961), muscle
(Issberner et al. 1996), and
veins (Klement and Arndt 1991)
evokes a sensation of pain. Continuous infusion of acidic buffer into skin
decreased tissue pH from 7.4 to 6.2 (Steen
et al. 1995a) and evoked a sensation of burning pain
(Steen and Reeh 1993;
Ugawa et al. 2002). The
intensity of pain increased as intradermal pH decreased
(Steen and Reeh 1993).
Moreover, a combination of inflammatory mediators (bradykinin, serotonin,
histamine, and prostaglandin E2, each at 10–7 M)
potentiated the algogenic effect of tissue acidosis
(Steen et al. 1996). Thus
tissue acidosis likely contributes to pain associated with inflammation.
Nociceptors are excited preferentially by stimuli that damage or
potentially damage tissue (Sherrington
1906). Acidic stimuli have been found to excite some nociceptors
(Belmonte et al. 1991;
Gallar et al. 1993). A
subpopulation of C polymodal nociceptors (
40%) in rats was excited by
acidified buffer (pH 7.4–4.3) applied to the skin in an in vitro
saphenous nerve-skin preparation (Steen et
al. 1992). These C polymodal nociceptors encoded the acidity of
the buffer down to pH 5.2. A combination of inflammatory mediators that
potentiated the algogenic effect of tissue acidosis in humans
(Steen et al. 1996) was found
to increase the excitation of C polymodal nociceptors evoked by the acidified
buffer (Steen et al. 1995b).
Moreover, experimentally induced inflammation increased expression of
acid-sensing ion channel (ASIC) isoforms in small dorsal root ganglion neurons
(Voilley et al. 2001). Thus
activation of C polymodal nociceptors by tissue acidosis can be potentiated by
inflammatory mediators, perhaps in part by increased expression of
acid-sensing ion channels, and likely contributes to pain associated with
inflammation.
A behavioral model using frogs may be useful for studying the role of
tissue acidosis in pain. Acetic acid applied topically to the hind limb
results in nocifensive behaviors, including a vigorous wiping of the exposed
skin, termed the wiping response (Pezalla
1983a). This model has been proposed as an alternative to
mammalian models of nociception (Stevens
1992) and has been used to study the analgesic actions of
pharmacological agents (e.g., opioids)
(Pezalla and Stevens 1984;
Stevens et al. 1994;
Willenbring and Stevens 1996)
and stress-induced analgesia (Pezalla
1983b; Pezalla and Dicig
1984). Using frogs as research subjects to study the contribution
of tissue acidosis to nociception is attractive because frog skin is permeable
to aqueous solutions (Boutilier et al.
1992). In contrast, mammalian skin is relatively impermeable to
aqueous solutions, such as acids (Flynn
1989; Smith 1990).
Thus acidosis in frog skin can be produced by applying acids topically
(Hamamoto et al. 2000), which
eliminates the need for an injection. An injection can mechanically injure the
skin and may sensitize nociceptors (Perl
1976; Reeh 1986).
Furthermore, frogs are not restrained during application of acetic acid.
Restraining animals can produce stress-induced analgesia
(Pezalla and Dicig 1984;
Stevens et al. 1995) and
confound the results of behavioral testing. Therefore this model of
aceticacid-induced nociception in frogs has advantages that make it attractive
for studying the mechanisms by which tissue acidosis produces pain.
Little is known about the primary afferent fibers in frogs that are excited
by tissue acidosis. Early electrophysiological studies in frogs reported that
noxious mechanical, thermal, and chemical stimuli (including acetic acid)
excited primary afferent fibers with slowly conducting axons (Adrian
1926,
1928;
Hogg 1935). However, the
percentage of fibers excited by acetic acid and their ability to encode the pH
of the acid were not reported. More recently, acidic pH has been shown to
induce inward currents in small- to medium-sized frog dorsal root ganglion
neurons in vitro (Kuffler et al.
2002; Philippi et al.
1995). Because acetic acid likely evokes the wiping response in
frogs by exciting nociceptors, further characterization of their response
properties is needed. Thus the aim of this study was to examine responses of
cutaneous primary afferent fibers evoked by topical application of acetic acid
and thereby determine which types of afferent fibers may contribute to the
wiping response in frogs.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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Northern grass frogs (Rana pipiens, 25–60 g, Sullivan,
Nashville, TN) were housed in large metal cages, fed live crickets three times
per week, and kept on a 12 h light/dark cycle. Room temperature was maintained
at 20°C. All experimental protocols were approved by the
Institutional Animal Care and Use Committee at the University of Minnesota and
conformed to the guidelines set forth by the International Association for the
Study of Pain (Zimmermann
1983).
Acids
A series of 13 solutions with differing pHs were made by serially diluting
glacial acetic acid (2 volumes of acid to 1 volume of distilled water)
(Pezalla 1983a;
Willenbring and Stevens 1996).
The two most concentrated solutions (17.4 and 11.6 M) were not used because in
previous experiments less-concentrated solutions always evoked the wiping
response (Hamamoto et al.
2000). Thus 11 solutions of acetic acid (0.13–7.75 M, pH
2.84–1.42) were used. The pH of each solution was measured before and
after each experiment and never drifted by >0.04 pH units.
Electrophysiological recording
Each frog was anesthetized with a subcutaneous injection of tricaine
methane sulfonate (MS 222, Sigma Chemical, St. Louis, MO) at 0.1 mg/g body wt
and then pithed. The frog was covered with wet gauze, and the skin was kept
moist with water. Action potentials were recorded extracellularly from the
sciatic nerve or the spinal nerves that join to form the sciatic nerve, using
conventional microdissection and recording techniques. An incision was made in
the skin overlying the nerve, and the skin was sewn to a plastic ring to form
a basin. The overlying muscle was removed to expose the nerve, which was then
separated from the surrounding connective tissue. The basin was filled with
mineral oil that was at room temperature (22°C). The nerve was placed
on a dissecting platform, and the epineural sheath was opened. Small fascicles
were cut and their proximal ends were placed on the platform for fine
dissection.
Fine filaments were teased from fascicles using sharpened jeweler's forceps and placed on a silver wire electrode. Neuronal activity was amplified, filtered, displayed on an oscilloscope, and audio-monitored. Only single units that could be easily discriminated were studied. Action potentials from the fiber of interest were discriminated from those of other fibers and from background noise using an amplitude window discriminator. Neuronal activity and discriminated pulses were recorded by a computer using a customized data-acquisition system (Labview, National Instruments, Austin, TX). In most experiments, recordings were obtained from only one afferent fiber.
Identification and classification of primary afferent fibers
MECHANICAL STIMULATION AND IDENTIFICATION OF RECEPTIVE FIELDS.
Receptive fields of primary afferent fibers were located using mechanical
stimulation of the skin with a wet cotton swab. Controlled mechanical
stimulation was delivered using von Frey monofilaments with bending forces
that ranged from 0.05 to 137 mN (0.005–14.0 g). The precise location of
the receptive field was determined using a suprathreshold von Frey
monofilament and mapped onto a drawing of the hind limb. Next, the mechanical
threshold of the fiber was ascertained by applying von Frey monofilaments with
increasing bending forces to the most sensitive area of the receptive field.
The threshold force was defined as the minimum force (mN) that evoked a
response in 
50% of the trials. The responses of each fiber to mechanical
stimulation was further studied by evaluating its response to gentle brushing
of the receptive field with a soft camel's hair brush and to gentle pinching
with a curved forceps. Care was taken not to injure the skin during the
pinching. Fibers that were excited by pinching but not brushing or that were
differentially excited by pinching were classified as nociceptors.
CONDUCTION VELOCITY. Conduction velocity (CV) for each fiber was
determined by electrically stimulating the receptive field with square-wave
pulses (0.5 Hz, 0.1–0.5 ms) of constant current delivered to the skin at
two times the current required to evoke an action potential in 50% of the
trials (0.09–3.8 mA). Copper surface electrodes were used to avoid
making holes in the skin that would allow the topically applied acid to
directly enter. Conduction velocity was calculated by dividing the conduction
distance (distance along the path of the nerve between the recording electrode
and the middle of the receptive field) by the conduction latency (latency from
the beginning of the stimulus artifact to the beginning of the action
potential). Fibers were classified by their CV as A fibers (CV 15.0
m/s), A fibers (2.0 < CV < 15.0 m/s), or C fibers (CV 
2.0
m/s). These classifications were based on analyses of compound action
potentials in preliminary studies and review of the literature
(Erlanger and Gasser 1930;
Erlanger et al. 1924; also see
DISCUSSION).
THERMAL STIMULATION. Thermal stimuli (heat and cold) were
delivered by a feedback controlled Peltier device (surface area = 1
cm2). Stimulus temperatures were recorded from a thermocouple
located at the interface between the Peltier device and the skin. Beginning
from an adapting temperature of 24°C, each thermal stimulus was applied
for 5 s. There was 1 min between trials. To quickly ascertain if a fiber was
excited by thermal stimuli, large increments in stimulus temperatures were
used initially. These temperatures were 27, 37, and 47°C (ramp rate =
20°C/s) for heat stimuli and 20, 10, 0, and –10°C (ramp rate =
5°C/s) for cold stimuli. In later trials, –4°C was used as the
coldest stimulus temperature because temperatures below –4°C
occasionally froze the skin as identified by the abrupt rise in temperature at
the surface of the skin produced by the exothermic crystallization of water in
the skin (Beise et al. 1998).
If a fiber was excited by one of these initial temperatures, then the initial
series of temperatures was interrupted and subsequent trials were performed to
determine the threshold temperature and to determine if the fiber increased
its response with increasing stimulus intensity. In these subsequent trials,
stimuli were increased (for heat) or decreased (for cold) by increments of
2°C from an adapting temperature of 24°C. In some cases, fibers were
excited only during cooling after a heat stimulus. These fibers were not
considered to be heat responsive, but because cooling during the cold stimuli
excited them, they were classified as cold responsive (see
Fig. 1 for an example).
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APPLICATION OF ACETIC ACID. Acetic acid was applied to the skin
in a manner analogous to that used in the nocifensive behavioral assay, the
Acetic Acid Test (Hamamoto et al.
2000; Pezalla
1983a). Five minutes after the end of the last cold stimulus,
fibers were tested for sensitivity to acetic acid. Neuronal activity was
recorded for a baseline period of 10 s. Next, a drop (30 µl) of one of the
acetic acid solutions was applied to the center of the receptive field using a
Pasteur pipette. After 5 s, the exposed skin was rinsed with distilled water
for an additional 5 s. The time of application of acetic acid and the period
during which the skin was rinsed were marked using a foot switch whose voltage
output was recorded by the computer. Neuronal activity was recorded for a
total of 60 s. Solutions of acetic acid were applied in order of decreasing
pH, from pH 2.84 to 1.42, with 2 min between applications. Because exposure of
the terminals of primary afferent fibers to noxious stimuli may alter fiber
response properties, fibers were studied only if their receptive fields were
2 cm away from those of previously studied fibers.
Statistical analyses
2 analyses were used to determine if the percentage of
fibers excited by each stimulus (pinch, heat, cold, or acetic acid) differed
between groups. Forces produced by the von Frey monofilaments are presented as
medians (mN) and ranges. Kruskal-Wallis nonparametric statistical analyses
were used to determine if mechanical thresholds differed among groups followed
by Mann-Whitney U tests to compare mechanical thresholds between
pairs of groups. Response thresholds for thermal stimuli (°C) are reported
as means ± SE and were compared between groups using one-way ANOVAs
followed by Duncan's multiple range test (Duncan's MRT) to compare mean
response thresholds between pairs of groups. Comparisons of response
thresholds for thermal stimuli and conduction velocities (m/s) between groups
were made using t-tests.
The least acidic solution of acetic acid that evoked impulses during the 5
s period after application of the acid was defined as the threshold solution.
The 5 s period was chosen because the nocifensive wiping response occurred
within 5 s after application of acetic acid
(Hamamoto et al. 2000;
Pezalla 1983a). The pH of the
threshold solution and the pH of the solution of acetic acid that evoked the
greatest number of impulses are presented as means ± SE. Comparisons of
pH of the threshold solutions (or the pH of the solutions that evoked the
greatest number of impulses) between fiber types were made using one-way
ANOVAs followed by Duncan's MRTs. When these comparisons were made between two
groups, t-tests were used.
The number of impulses evoked during the 5 s period after application of a solution of acetic acid is reported as mean ± SE. Some fibers with low mechanical thresholds were excited by application of a drop of acetic acid and responded with one to three impulses. These responses occurred within 0.5 s of application, and the number of impulses did not increase as more acidic solutions of acetic acid were applied. Thus these few impulses were likely evoked by the mechanical stimulation produced by the contact of the acetic acid. Fibers excited in this manner were not classified as being excited by acetic acid.
Stimulus-response functions comparing the number of impulses to pH of acetic acid were constructed by calculating the mean (±SE) number of impulses evoked within 5 s of application of acetic acid at each pH. Fibers were grouped based on their CV, functional subtype, and response to pH. A two-way repeated-measures ANOVA followed by Duncan's MRT was used to test for differences in number of impulses between pHs of acetic acid within a group of fibers (repeated measure) and between groups of fibers following application of acetic acid at each pH. For all analyses, P < 0.05 was considered statistically significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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GENERAL RESPONSE CHARACTERISTICS OF PRIMARY AFFERENTS FIBERS.
Recordings were made from 122 mechanosensitive primary afferent fibers
innervating skin on the hind limb of frogs. The majority of fibers (61%;
71/116) had their receptive field on the lateral surface of the lower leg
(between the ankle and knee; e.g., Figs.
1A,
2A, and
3A). This area of the
hind limb is where the acetic acid test is applied in behavioral studies
(Hamamoto et al. 2000). The
remaining fibers had receptive fields on the thigh (7%; n = 8), foot
(17%; n = 20), and toes (15%; n = 17).
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Fibers were classified according to their conduction velocities
(Table 1). A greater percentage
of C fibers were excited by pinching than were A or A fibers
(P < 0.01) and mechanical thresholds were higher for C fibers than
for A and A fibers (P < 0.01). The percentage of
fibers that were excited by heat stimuli was greater for C fibers than for
A and A fibers (P < 0.01). Although C fibers had
higher response thresholds to heat than did A fibers, this difference
was not statistically significant. The percentage of fibers excited by cold
stimuli did not differ between classes of fiber, but mean response threshold
temperature was lower for C fibers than for A or A fibers
(P < 0.01). Thus C fibers were more likely to be excited by
nociceptive stimuli (pinch and heat) and required more intense mechanical and
cold stimuli to evoke excitation than A and A fibers.
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RESPONSES OF PRIMARY AFFERENTS FIBERS EVOKED BY ACETIC ACID.
Figure 1 shows responses of a
single A fiber evoked by thermal stimuli and by solutions of acetic acid
at various pHs. The receptive field of this A fiber was located on the
lateral aspect of the lower leg (Fig.
1A). This fiber had a conduction velocity of 19.7 m/s
(Fig. 1B). The
mechanical threshold of this fiber was 0.5 mN, and it responded to
suprathreshold mechanical stimuli with a rapidly adapting burst of impulses at
the onset and offset of the stimulus (not shown). As illustrated in
Fig. 1, C and
D, this A fiber was excited by cooling in both heat
and cold trials. Thus this A fiber was classified functionally as a
cold-responsive low-threshold mechanoreceptor. The response of this A
fiber to the acetic acid is shown in Fig.
1E. Application of the least acidic solution of acetic
acid (pH 2.84) did not evoke any impulses. However, rinsing the skin produced
a brief discharge of impulses perhaps because of the low mechanical threshold
of this fiber. The least acidic solution of acetic acid that evoked discharges
before the skin was rinsed had a pH of 2.72. The number of impulses increased
following application of acetic acid at pH 2.59, which evoked the greatest
number of impulses (15) for this fiber. Application of acetic acid solutions
at more acidic pHs evoked fewer impulses; thus this A fiber desensitized
to application of acetic acid at pHs lower than pH 2.59. The mechanical
stimulation produced by rinsing the skin with water produced a brief discharge
in all of the acetic acid trials. However, acetic acid also evoked impulses
that continued during the rinsing after application of acetic acid at pH 2.72,
2.59, and 2.49; in the case of acetic acid at pH 2.72 and 2.49, the impulses
continued after rinsing had ended.
Responses of a single A fiber to the solutions of acetic acid are
shown in Fig. 2.
Figure 2A shows that
the receptive field was located on the lateral surface of the lower leg.
Figure 2B illustrates
the conduction latency (3.9 ms) of this fiber to electrical stimulation of its
receptive field; the conduction velocity was 11.0 m/s. This fiber had a very
low mechanical threshold (0.05 mN) and exhibited a rapidly adapting response
after suprathreshold mechanical stimuli. Heat stimuli 47°C and cold
stimuli down to –4°C did not excite this fiber. However, as
demonstrated in Fig.
2C, this fiber was excited by the least acidic solution
of acetic acid (pH 2.84) with 13 impulses. The number of impulses evoked by
acetic acid at pH 2.72, 2.59, and 2.49 ranged between 8 and 13 but increased
to 18 impulses after application of acetic acid at pH 2.41. In a previous
study, the median pH of acetic acid that evoked the nocifensive wiping
response in frogs was pH 2.41 (Hamamoto et
al. 2000). Hence, this fiber may be an example of the fibers
contributing to the wiping response in the acetic acid test. This fiber
exhibited impulses that continued for a few seconds after the rinsing of the
skin ended. Furthermore, subsequent application of acetic acid at more acidic
pHs (2.30–1.42) evoked fewer impulses suggesting that this fiber became
desensitized.
The C fiber illustrated in Fig. 3 had a small receptive field on the lateral surface of the lower leg (Fig. 3A) and was excited by pinching but not brushing. The conduction velocity of this fiber was 0.41 m/s (Fig. 3B). The mechanical threshold for this fiber was 8.05 mN, and this fiber exhibited a slowly adapting response to excitation with a suprathreshold von Frey monofilament. Responses to heat are shown in Fig. 3C. This fiber was unresponsive to cold stimuli down to –4°C. Responses of this fiber evoked by solutions of acetic acid are shown in Fig. 3D. Acetic acid at pH 2.06 evoked the greatest number of impulses and the impulses continued for 25 s. Application of acetic acid at pHs more acidic than 2.06 evoked only one impulse during the 5 s stimulus period. Thus this fiber became desensitized to more acidic solutions of acetic acid.
As illustrated in Fig.
4A, the percentage of fibers that were excited by acetic
acid was greater for A fibers (52%, 31/60) than for A fibers
(20%, 9/44; P < 0.01). Forty-four percent of C fibers (8/18) were
excited by acetic acid, but this was not significantly greater than the
percentage of A fibers excited by acetic acid. Mean pH of the threshold
solution of acetic acid was not different among A (pH 2.74 ±
0.06), A (pH 2.77 ± 0.02), and C (pH 2.60 ± 0.11) fibers.
In contrast, as shown in Fig.
4B, the mean pH of the acetic acid solution that evoked
the greatest number of impulses was significantly lower for C fibers (pH 2.00
± 0.15) than for A (pH 2.39 ± 0.10) and A (2.42
± 0.05) fibers (P < 0.01). However, the greatest number of
evoked impulses did not differ among A (18.4 ± 4.1), A
(13.5 ± 1.6), and C fibers (13.1 ± 3.4).
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The stimulus-response relationships for A
(Fig. 4C), A
(Fig. 4D), and C
fibers (Fig. 4E)
demonstrate that the responses of A and A fibers peaked at less
acidic pHs (2.42 for A fibers and 2.59 for A fibers) than did the
responses of C fibers (pH 1.71). Moreover, responses of A and A
fibers decreased to near zero as acetic acid at more acidic pHs were applied.
In contrast, the responses of C fibers did not decrease to zero even after
application of acetic acid at the most acidic pH (1.42).
Figure 4F demonstrates
that the number of impulses evoked by acetic acid was not significantly
different among fiber types at pH 2.84; this pH evoked the wiping response in
few (3%) frogs in our previous study
(Hamamoto et al. 2000). The
median pH of the solutions of acetic acid that evoked the wiping response was
pH 2.41, but the number of impulses evoked by this solution was not
significantly different between fiber types in the present study. In contrast,
acetic acid at pH 1.71 evoked a significantly greater number of impulses from
C fibers (8.6 ± 4.4 imp) than from A (0.9 ± 0.7 imp) and
A (0.4 ± 0.2 imp) fibers (P < 0.05).
Response characteristics of nociceptors
GENERAL RESPONSE CHARACTERISTICS OF NOCICEPTORS. Nociceptors were defined as those fibers that were differentially excited by pinching. As shown in Table 2, nociceptors had slower conduction velocities (P < 0.01) and higher thresholds to mechanical stimulation (P < 0.01) than did low-threshold mechanoreceptors. The percentage of fibers that were excited by heat was greater for nociceptors than for low-threshold mechanoreceptors (P < 0.01). Although the mean response threshold for heat was higher for nociceptors than for low-threshold mechanoreceptors, this difference was not statistically significant. Similar percentages of nociceptors and low-threshold mechanoreceptors were excited by cold stimuli, but the mean response threshold temperature for cold stimuli was significantly lower for nociceptors than for low-threshold mechanoreceptors (P < 0.01). Thus nociceptors required more intense mechanical and cold stimuli to excite them and were more likely to be excited by heat stimuli than were low threshold mechanoreceptors.
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RESPONSES OF NOCICEPTORS EVOKED BY ACETIC ACID. Similar percentages of nociceptors (46%, 16/35) and low-threshold mechanoreceptors (37%, 32/87) were activated by acetic acid, and there was no difference in the mean pH of the threshold solution of acetic acid between nociceptors (pH 2.68 ± 0.06) and low-threshold mechanoreceptors (pH 2.76 ± 0.03). Moreover, the greatest number of impulses evoked by acetic acid was not significantly different between nociceptors (13.1 ± 2.3 imp) and low-threshold mechanoreceptors (15.0 ± 1.8 impulses). However, the pH of the acetic acid that evoked the greatest number of impulses was lower for nociceptors (pH 2.19 ± 0.10) than for low-threshold mechanoreceptors (2.42 ± 0.06, P < 0.05).
Response characteristics of primary afferent fibers that were excited by acetic acid
To further elucidate the fibers that may contribute to the acetic
acid-induced wiping response, the characteristics of fibers that were excited
by acetic acid were compared with those of fibers that were not excited.
Stimulus-response relationships for fibers that were excited by acetic acid
and for fibers that did not are shown in
Fig. 5A. Fibers that
were excited by acetic acid exhibited an increasing number of impulses as
solutions of acetic acid at pH 2.84–2.59 were applied. The number of
impulses was greater when acetic acid at pH 2.59, 2.49, 2.41, and 2.30 were
applied than when acetic acid at pH 2.84 was applied (P < 0.05).
The number of impulses generally decreased as solutions of acetic acid at more
acidic pHs were applied. However, the response to acetic acid at pH 1.95 was
an exception to this observation. Thus fibers that responded to acetic acid
exhibited the greatest response to the solutions of acetic acid (pH
2.59–2.41) that evoked the wiping response in most frogs in our previous
study (Hamamoto et al.
2000).
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As shown in Fig.
5B, conduction velocity was slower for fibers that were
excited by acetic acid (9.0 ± 0.8 m/s) than for fibers that were not
excited (14.0 ± 1.2 m/s, P < 0.01). A greater percentage of
fibers that were excited by acetic acid also were excited by heat (22 vs. 4%,
P < 0.05); this is illustrated in
Fig. 5C. Although cold
stimuli excited a similar percentage of acetic acid sensitive (29%) and
insensitive (16%) fibers, Fig.
5D shows that fibers that were excited by acetic acid
responded to cold stimuli that were less cold (17.5 ± 1.6°C) than
fibers that were not excited by acetic acid (11.3 ± 1.3°C,
P < 0.05). Therefore fibers that were excited by acetic acid
exhibited their greatest responses at pHs that evoked the wiping response in a
majority of frogs (Hamamoto et al.
2000). These fibers differed from fibers that were not excited by
acetic acid by having slower conduction velocities, a greater likelihood of
being excited by heat, and being more sensitive to cold stimuli.
Previous studies have found that response characteristics of fibers differ
among classes of fibers (i.e., A vs. A vs. C fibers)
(Burgess and Perl 1979;
Raja et al. 1999). Thus
differences in the characteristics of fibers that were excited by acetic acid
compared with those that were not excited, as illustrated in
Fig. 5, could be due to
differences in the percentage of A and C fibers in each group (see
Fig. 4A). Hence, the
characteristics of fibers that were excited by acetic acid and fibers that
were not excited were compared within each class of fiber.
Figure 6 demonstrates that
A fibers that were excited by acetic acid differed from A fibers
that were not excited by acetic acid in several characteristics. Mean
conduction velocity for A fibers that were excited by acetic acid (16.5
± 0.6 m/s) was slower than that for fibers that were not excited (21.2
± 0.9 m/s, P < 0.05); this is illustrated in
Fig. 6A. Furthermore,
the percentages of fibers that were excited by pinch (11 vs. 0%,
Fig. 6B), heat (11 vs.
0%, Fig. 6C), and cold
(71 vs. 6%, Fig. 6D)
were greater for A fibers that were excited by acetic acid than A
fibers that were not (P < 0.05). Thus A fibers excited by
acetic acid were more likely to also be excited by other noxious stimuli such
as pinch, heat, and cold.
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Response characteristics of A fibers excited by acetic acid were
generally similar to the characteristics of A fibers that were not
excited. There were no significant differences in conduction velocities or
percentage of fibers that were excited by pinch, heat, or cold between these
two groups of fibers. However, A fibers that were excited by acetic
acid responded to cold stimuli that were less cold (19.0 ± 0.8°C)
than those that were not excited by acetic acid (11.1 ± 2.8°C,
P < 0.05).
As illustrated in Fig. 7A, C fibers that were excited by acetic acid had slower mean conduction velocities (0.55 ± 0.17 m/s) than C fibers that were not (1.26 ± 0.23 m/s, P < 0.05). A greater percentage of C fibers that were excited by acetic acid were also excited by pinch (100 vs. 60%, Fig. 7B) and heat (86 vs. 25%, Fig. 7C) than C fibers that were not excited by acetic acid (P < 0.05). Thus C fibers excited by acetic acid were more likely to be excited by other noxious stimuli such as pinch and heat and hence were most likely to have nociceptor function.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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Methodological considerations
There are few electrophysiological data collected from frogs that can be
used to select the conduction velocities by which to classify fibers into
A, A, and C fiber groups. In early studies, compound action
currents or compound action potentials were recorded from the sciatic nerve of
bullfrogs (Erlanger and Gasser
1930; Erlanger et al.
1924). From these studies, A fibers conducted at 14
m/s and C fibers conducted at velocities <1 m/s. In our own preliminary
electrophysiological studies, 28 compound action potentials were recorded from
the sciatic nerve of frogs (R. pipiens). The beginning of the
A wave had an average conduction velocity of 14.4 ± 1.0 m/s, and
the fastest C fibers averaged 2.2 ± 0.2 m/s. Additional information can
be obtained from studies of primary afferent fibers in frogs in which fibers
were classified based on their functional characteristics
(Adrian 1932;
Catton 1958;
Erlanger and Gasser 1930;
Hogg 1935;
Matthews 1929;
Spray 1974). In these studies,
the majority of the conduction velocities delineating A from A
fibers were 15 m/s (Adrian
1932; Catton 1958;
Matthews 1929;
Spray 1974). The average
conduction velocity used to delineate C from A fibers was 2 m/s
(Adrian 1932;
Catton 1958;
Erlanger and Gasser 1930;
Hogg 1935;
Spray 1974). Thus in the
present study, 15 m/s was selected as the conduction velocity cutoff by which
to classify fibers as either A or A fibers and 2 m/s was selected
as the conduction velocity cutoff by which to classify fibers as either
A or C fibers.
Primary afferent fibers were classified as nociceptors based on their
differential responses to innocuous (brush) and noxious (pinch) mechanical
stimuli. Responses to thermal stimuli were not used to classify fibers as
nociceptors because little is known about the stimulus temperatures that are
nociceptive for frogs. In a review paper, Stevens and Willenbring reported
that a thermal radiant heat source evoked the wiping response in frogs at a
threshold temperature of 33.1 ± 2.3°C (mean ± SD)
(Stevens and Willenbring
1997). In contrast, Kuffler and colleagues found that the
threshold for evoking the wiping response was 38 ± 0.5°C when the
feet of pithed frogs were placed in a water bath
(Kuffler et al. 2002). In the
present study, three fibers were excited by heat stimuli >33°C but were
not classified as nociceptors based on their responses to pinching. However,
reclassifying these fibers as nociceptors did not change the results from the
statistical analyses of their responses to acetic acid. Fifteen fibers were
excited by brushing and by cold stimuli. Relatively innocuous temperatures
ranging from 18 to 22°C excited 12 of these brush- and cold-responsive
fibers. The remaining three fibers had a slightly colder response threshold of
14°C. Holloway reported that "some" cutaneous nociceptors in
frogs were excited by ice placed on their receptive fields
(Holloway 1973). These
nociceptors required noxious pinch or pinprick to excite them and thus were
different from the brush- and cold-responsive fibers found in the present
study. In mammals, nociceptors that were excited by noxious thermal stimuli
were differentially excited by pinch
(Burgess and Perl 1979;
Leem et al. 1993; Simone and
Kajander 1996,
1997) or were insensitive to
mechanical stimulation (Handwerker et al.
1991; LaMotte and Thalhammer
1982). However, Leem and colleagues found that some low-threshold
C mechanoreceptors were excited by noxious cold stimuli, but they did not
classify these fibers as nociceptors (Leem
et al. 1993). Thus thermally responsive nociceptors in mammals
differ from brush- and cold-responsive fibers found in the present study
because the latter responded to innocuous mechanical stimuli. In the present
study, fibers that were excited by brushing and thermal stimuli but were not
differentially excited by pinching were classified as low-threshold
mechanoreceptors.
Responses of primary afferent fibers to application of acids
Few studies have examined responses of cutaneous primary afferent fibers in
frogs to application of acidic stimuli. Adrian
(1930) found that a 5% solution
of acetic acid applied to the skin evoked both "rapid and slow
impulses" in the dorsal cutaneous nerve of decerebrate frogs. Using an
in vitro preparation consisting of the dorsal skin and attached nerve from
frogs, Hogg (1935) found that
lower concentrations of acetic acid (i.e., 2%) only excited fibers with slow
conduction velocities (1.5–4.5 m/s). However, at higher concentrations
(unspecified by Hogg), fibers with fast conduction velocities (again
unspecified by Hogg) were excited. In toads, application of acetic acid
(5–10%) to an excised nerve-skin preparation excited fibers with
conduction velocities that ranged from 0.1 to 15 m/s
(Maruhashi et al. 1952). The
findings from the present study are in agreement with these previous
observations in that fibers excited by acetic acid had a relatively wide range
of conduction velocities (0.16–19.7 m/s). In contrast, fibers with
faster conduction velocities (A and A fibers) were not excited by
acidified buffers in a rat in vitro skin-nerve preparation
(Steen et al. 1992). Thus in
amphibians, acetic acid excites fibers with a wide range of conduction
velocities, whereas in mammals, acids excite only fibers with slower
conduction velocities.
In the present study, similar percentages of nociceptors (46%) and
low-threshold mechanoreceptors (37%) were excited by acetic acid. Previous
reports have also found that acetic acid excited "tactile" fibers
in frogs (Adrian 1930;
Hogg 1935). In contrast, all
fibers recorded from toads that were excited by acetic acid were also excited
by pinprick (Maruhashi et al.
1952). Similarly, in a rat in vitro skin-nerve preparation,
low-threshold mechanoreceptors were not excited by acidified buffers; only C
polymodal nociceptors showed stimulus-related responses that increased as the
pH of the buffer was decreased (Steen et
al. 1992). Thus primary afferent fibers in frogs appear to differ
from those in mammals in that acids excite both nociceptors and low-threshold
mechanoreceptors in frogs but only excite nociceptors in mammals.
Relationship between subepidermal pH and excitation of primary afferent fibers after application of solutions of acetic acid that evoke the wiping response in frogs
In the present study, the method of applying the solutions of acetic acid
was identical to that used in our previous study
(Hamamoto et al. 2000) so that
comparisons could be made between behavioral responses, subepidermal pH, and
electrophysiological responses of primary afferent fibers to the same
solutions of acetic acid. In our previous study, solutions of acetic acid at
pH 2.59–2.41 evoked the wiping response in the majority (58%) of frogs.
These same solutions of acetic acid evoked the greatest number of impulses
from primary afferent fibers, suggesting that excitation of these fibers
contributes to the acetic acid-induced wiping response in frogs. Application
of acetic acid at pH 2.41 decreased subepidermal pH to 6.69 ± 0.30.
This subepidermal pH is similar to the pHs that have been shown to excite
nociceptors (pH 6.9–6.1) (Steen et
al. 1992) and to produce hyperalgesia to mechanical stimulation
(pH 6.4–6.0) (Hamamoto et al.
1998) in rats. Moreover, subepidermal injection of acidic buffer
in humans decreased tissue pH down to 6.2 and produced pH-dependent pain
(Steen et al. 1995a). Thus in
the present study, primary afferent fibers were excited by solutions of acetic
acid that evoked the wiping response in frogs. These solutions of acetic acid
decreased subepidermal pH to levels that excited nociceptors and produced
hyperalgesia in rats and produced pain in humans. Hence, primary afferent
fibers excited by acetic acid likely contribute to the acetic acid-induced
wiping response in frogs.
The stimulus-response relationship of primary afferent fibers to acetic
acid in frogs was similar to that observed in C polymodal nociceptors in rats.
Using an in vitro skin-nerve preparation, Steen and colleagues found that C
polymodal nociceptors in rats had response thresholds to acidified buffers
ranging from pH 6.9 to 6.1 (Steen et al.
1992). The threshold solutions of acetic acid in the present study
produced an average subepidermal pH of 7.14 ± 0.06 in our previous
study (Hamamoto et al. 2000).
Hence, the response threshold for cutaneous afferent fibers in frogs was at a
slightly less acidic pH than that for C polymodal nociceptors in rats. For the
C polymodal nociceptors in rats and the primary afferent fibers in frogs,
responses increased as more acidic stimuli were applied until a peak response
was evoked and then responses decreased. In the rat preparation, buffer at pH
5.2 evoked the maximum discharge (Steen et
al. 1992). In contrast, the solutions of acetic acid that evoked
the greatest number of impulses in primary afferent fibers decreased
subepidermal pH to 6.30 ± 0.15
(Hamamoto et al. 2000).
Therefore acetic acid evoked the greatest excitation of primary afferent
fibers in frogs at a less-acidic pH than the pH that evokes the greatest
excitation in C polymodal nociceptors in rats.
Interestingly, when responses of only the C fibers in frogs were
considered, the stimulus-response relationship was similar to that of C
polymodal nociceptors in rats. The solutions of acetic acid that evoked the
threshold response from the C fibers in frogs produced a subepidermal pH of
6.88 ± 0.30 in our previous study
(Hamamoto et al. 2000).
Moreover, the solutions of acetic acid that evoked the greatest number of
impulses in C fibers in frogs were previously found to decrease subepidermal
pH to 5.45 ± 0.42 (Hamamoto et al.
2000). Thus the stimulus-response relationship for C fibers in
frogs was similar to that found in rats.
Conclusions
In summary, 39% of primary afferent fibers in frogs were excited by acetic acid. Primary afferent fibers were excited by solutions of acetic acid that evoked the wiping response in frogs and that decreased subepidermal pH to levels that have been found to excite nociceptors and produce hyperalgesia to mechanical stimuli in rats and to produce pain in humans. In rats, only nociceptors were excited by acidic stimuli, whereas similar proportions of nociceptors and low-threshold mechanoreceptors were excited in frogs. However, the stimulus-response relationship of C fibers in frogs was similar to that found in C polymodal nociceptors in rats. Thus the results of the present study suggest that the model of acetic acid-induced nociception in frogs may be useful for studying mechanisms by which tissue acidosis excites primary afferent fibers and produces pain. Further studies are needed to determine the relative roles of nociceptors and low-threshold mechanoreceptors in evoking the nocifensive wiping response in frogs.
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DISCLOSURES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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FOOTNOTES |
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Address for reprint requests: D. T. Hamamoto, Dept. of Diagnostic and Surgical Sciences, 7-536 Moos Tower, 515 Delaware St. SE, University of Minnesota, Minneapolis, MN 55455 (E-mail: hamam001{at}umn.edu).
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REFERENCES |
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