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Meeting Abstracts  |   November 2007
Persistent Low-frequency Spontaneous Discharge in A-fiber and C-fiber Primary Afferent Neurons during an Inflammatory Pain Condition
Author Affiliations & Notes
  • Wen-Hua Xiao, M.D.
    *
  • Gary J. Bennett, Ph.D.
  • * Senior Research Associate, Department of Anesthesia and Centre for Research on Pain, † Professor, Department of Anesthesia, Centre for Research on Pain, and Faculty of Dentistry, McGill University.
Article Information
Meeting Abstracts   |   November 2007
Persistent Low-frequency Spontaneous Discharge in A-fiber and C-fiber Primary Afferent Neurons during an Inflammatory Pain Condition
Anesthesiology 11 2007, Vol.107, 813-821. doi:10.1097/01.anes.0000286983.33184.9c
Anesthesiology 11 2007, Vol.107, 813-821. doi:10.1097/01.anes.0000286983.33184.9c
TISSUE injury generally evokes pain of nearly immediate onset. If the injury is severe, the pain will persist for minutes to hours. This persistent pain is said to be ongoing  , in the sense that it seems to be a continuation of the pain felt at the time of injury. If the injury evokes a significant inflammatory reaction, pain may persist for a long time. This pain is said to be spontaneous  , in the sense that it arises without an easily identifiable stimulus. The inflamed tissue, and the surrounding uninjured tissue, may also become the source of abnormal stimulus-evoked pains (allodynia and hyperalgesia). Most of our knowledge about postinjury pain comes from experimental studies that have examined the neuronal mechanisms underlying peripheral and central sensitization that occur in the first few minutes to an hour or two after injury. Within this early time frame, sensitized nociceptors are believed to be key generators of ongoing pain, allodynia, and hyperalgesia, with their ongoing discharge also initiating and maintaining central sensitization mechanisms.1–9 
It is becoming increasingly clear that the mechanisms that underlie acute pain are different than those that underlie inflammatory pain, and that acute inflammatory pain mechanisms may differ from chronic inflammatory pain mechanisms. Numerous changes in primary afferent nociceptors that begin after the onset of inflammation have been described. Recent examples include the up-regulation of cyclin-dependent kinases,10 increased expression of the transient receptor potential vanilloid-1 channel,11 and changes in the expression of sodium channel α-subunit isoforms.12 
We know little about the activity of primary afferent neurons during a persistent inflammatory pain condition. A few studies have examined primary afferent spontaneous discharge during the first few days of the inflammatory response,6,7,13,14 but none have examined spontaneous discharge beyond the fourth day of inflammation. We have surveyed spontaneous discharge in primary afferent A fibers and C fibers in a commonly used inflammatory pain model (subcutaneous injection of complete Freund adjuvant [CFA] in the rat's plantar hind paw) over a time course of 14 days. This time course spans the time of peak pain severity to the time of pain resolution as determined in parallel experiments using behavioral assays of inflammation-evoked mechanoallodynia, mechanohyperalgesia, and heat hyperalgesia.
Materials and Methods
These experiments conformed to the guidelines of the Guide for the Care and Use of Laboratory Animals  of the National Academy of Science, the ethical guidelines for pain research in animals of the International Association for the Study of Pain,15 and the regulations of the Canada Institutes of Health Research and the Canadian Council on Animal Care. The experimental protocol was approved by the Animal Care and Use Committee of the Faculty of Medicine, McGill University, Montreal, Quebec, Canada.
Animals
Adult (250–350 g) male Sprague-Dawley rats from the Frederick, Maryland, breeding colony of Harlan Inc. (Indianapolis, IN) were housed on sawdust bedding in plastic cages. Artificial lighting was provided on a fixed 12-h light–dark cycle, and food and water were available continuously.
Inflammation
Undiluted CFA (100 μl; Calbiochem-Novabiochem Corp., La Jolla, CA) was injected subcutaneously into the plantar hind paw while the animals were anesthetized with isoflurane. The injection was made via  a 27-gauge needle that was inserted between the tori at the bases of digits III and IV and directed beneath the skin until the tip was in the approximate center of the circle formed by the digital and lateral tori. Control animals received an identical injection of 100 μl sterile saline.
Edema
The severity of hind paw edema was assessed by measuring the thickness of the mid hind paw with calipers at 2 h, 6 h, 1 day, 2 days, 3 days, 7 days, and 14 days after injection of CFA or saline.
Behavioral Measures
Mechanosensitivity and heat sensitivity were assayed 2 h, 6 h, 1 day, 2 days, 3 days, 7 days, and 14 days after injection of CFA or saline. As described in detail elsewhere,16 responses to 4-g and 15-g von Frey hairs applied to the plantar hind paw were measured to assess mechanoallodynia (increased response frequency to the 4-g von Frey hair—a normally innocuous stimulus) and mechanohyperalgesia (increased response frequency to the 15-g von Frey hair—a normally painful stimulus). Heat sensitivity was assessed with the method of Hargreaves et al.  ,17 with three withdrawal latencies obtained from each hind paw.
Electrophysiologic Studies of Primary Afferent Axons
We examined axons in the sural nerve in animals 2 days (n = 9), 7 days (n = 7), and 14 days (n = 6) after CFA injection, in a control group of animals 7 days after an intraplantar injection of saline (n = 5), and in a naive control group (n = 6) that received neither an intraplantar injection nor behavioral pain tests. We chose the sural nerve because it contains few axons from motor neurons,18,19 thus assuring that most of the impulses that we recorded were from sensory fibers.
An endotracheal tube and a cannula in the jugular vein were installed while the animal was anesthetized with an intraperitoneal injection of sodium pentobarbital (55 mg/kg). The rat was subsequently placed on a respirator, and anesthesia was maintained with isoflurane in oxygen (1%; 200 ml/min). Areflexia was maintained with an intravenous infusion of pancuronium bromide (1.0 mg · kg−1· h−1). End-tidal carbon dioxide was monitored continuously and kept within physiologic limits by adjusting the rate and volume of respiration. The electrocardiogram was monitored continuously. Core temperature was monitored with a rectal thermode and maintained at 37°–38°C via  a feedback-controlled heating pad beneath the animal. Care was taken to position the heating pad around the hind paw. Keeping the paw warm avoids the ongoing discharge of cooling-specific afferents that might be mistaken for inflammation-evoked activity. An adequate depth of anesthesia was inferred from the absence of heart rate change after pinching the tail and by the absence of a pinch-evoked tail flick before the administration of neuromuscular blockade.
The sural nerve was exposed in the popliteal fossa, transected, and submerged in a pool of warm mineral oil. A pair of needle electrodes was inserted subcutaneously at the lateral surface of the ankle for stimulation of sural afferent axons innervating the hind paw. Nerve impulses were amplified, band-pass filtered, led to an audio amplifier and to a computer interface, displayed on a video monitor for on-line analysis, and recorded for off-line analysis (Spike2 software; Cambridge Electronic Design, Cambridge, United Kingdom).
We used the teased microfilament recording method as we have described previously.20,21 Microfilaments were dissected from the distal stump of the transected nerve and draped over a hook electrode made of silver wire that was referenced to a needle electrode inserted in adjacent muscle. The nerve was dissected in its entirety, yielding 7–11 (mean, 8.5) appropriately small microfilaments per nerve. Appropriate microfilaments were estimated to be 35–45 μm in diameter, as judged by comparison to the 0.001-inch (25-μm) diameter of the wire used to form the recording electrode.
The following procedure was used for each microfilament. First, the recording was monitored for 5 min. If fibers with spontaneous discharge were present, they were identified by their waveform, and the pattern and frequency of their discharge were recorded. We defined spontaneous discharge as all activity greater than 1 impulse/min.22–24 Next, shocks (first 0.1-ms pulses to activate large fibers, subsequently 1.0-ms pulses to activate small fibers) of gradually increasing intensity were delivered via  the subcutaneous needle electrodes. The response latency was determined for every individually identifiable fiber (invariant waveform, discrete all-or-nothing threshold, and fixed latency of response) that was recruited as the intensity of stimulation was gradually increased. Spontaneously active fibers that could not be unequivocally identified in the response to nerve shock were excluded from the analysis. For A fibers and C fibers separately, the percentage of spontaneously active fibers was then computed as the ratio of spontaneously active individually identifiable neurons whose waveforms could be identified in the response to nerve shock, relative to the total number of individually identifiable fibers that could be identified in the response to nerve shock.20,25,26 It is not possible to individually identify every A fiber that responds to nerve shock in the microfilament because as stimulation intensity increases to the level where all fibers are activated, their waveforms will superimpose to form a multiunit potential.25 This will not influence the ratio described above because only spontaneously active fibers that could be identified after nerve shock were included in the ratio. The ability to individually identify the waveform of any given fiber after the nerve shock is a random event, depending on the fiber's position within the microfilament and its proximity to the recording and stimulating electrodes. Therefore, the ratio's numerator and denominator are both unbiased samples. The procedure is straightforward as long as there are no more than six to eight spontaneously discharging A fibers recognizable in the microfilament,27 as was the case in the current study. C fibers present less difficulty because their slow conduction velocities result in less superimposition; however, their impulses are smaller than those of A fibers and more difficult to differentiate from background activity. C fibers were counted here only if their impulse amplitudes were unequivocally distinguishable from the background voltage in addition to meeting the other criteria (invariant waveform, discrete all-or-nothing threshold, and fixed latency of response).
Conduction velocities were computed from the latency of response and the distance between the recording and stimulating electrodes as determined by measuring the length of a thread placed along the course of the nerve. Fibers were classified according to their conduction velocity (CV) as myelinated A fibers if the CV was greater than 2.0 m/s or as unmyelinated C fibers if the CV was less than 2.0 m/s.28 We do not differentiate Aδ and Aβ fibers here. Although A-fiber nociceptors generally have Aδ conduction velocities, in the normal rat, a large percentage conduct in the Aβ range28; moreover, inflammation may increase the conduction velocity of Aδ nociceptors such that they fall into the Aβ range.13 
Characterization of the functional properties of primary afferent axons requires the repeated application of high-intensity heat or mechanical stimuli to identify nociceptors. Such stimulation can itself cause primary afferent sensitization and spontaneous discharge. Our goal was a survey of the spontaneous discharge that was a result of the CFA-evoked inflammation itself, and we wished to avoid the possibility of spontaneous discharge due to repeated testing. Therefore, we did not perform stimulus–response characterizations.
Statistical Analysis
Data were analyzed using GraphPad InStat (version 3.0; GraphPad, Inc., San Diego, CA). Data from the edema and behavioral measures were analyzed with repeated-measures analyses of variance followed by Dunnett t  tests for pairwise comparisons to the preinjection baseline values. Differences in the mean conduction velocities of C fibers with spontaneous discharge versus  those without spontaneous discharge in the three CFA-injected groups were evaluated by unpaired t  tests. Similar comparisons for A fibers used the nonparametric Mann–Whitney test because the CV distributions were significantly different from normal. Differences between the CFA-injected groups relative to the control group in the incidence of spontaneous discharge in A fibers and C fibers were evaluated by Fisher exact test.
Results
Edema
Statistically significant edema was present as early as 2 h after CFA injection, peaked at 1 day after injection, and slowly declined over the following 2 weeks (fig. 1A). Statistically significant edema was still present at 14 days. The intraplantar injection of saline did not produce edema.
Fig. 1. Complete Freund adjuvant (CFA)–evoked edema and stimulus-evoked pain responses. Data are mean ± SEM from the hind paws ipsilateral (ipsi.) and contralateral (contra.) to the injection of CFA or the saline control (cont.) injection. (  A  ) Edema assessed as dorsoventral paw thickness in millimeters.  Error bars  are smaller than the symbols. (  B  ) Heat hyperalgesia assessed as the latency to withdraw from a radiant heat stimulus. (  C  ) Mechanoallodynia assessed as the frequency of withdrawal from a normally innocuous stimulus (4-g von Frey hair). (  D  ) Mechanohyperalgesia assessed as the frequency of withdrawal from a normally noxious stimulus (15-g von Frey hair). n = 6/group. *  P  < 0.05, **  P  < 0.01  versus  preinjection baseline (B). 
Fig. 1. Complete Freund adjuvant (CFA)–evoked edema and stimulus-evoked pain responses. Data are mean ± SEM from the hind paws ipsilateral (ipsi.) and contralateral (contra.) to the injection of CFA or the saline control (cont.) injection. (  A  ) Edema assessed as dorsoventral paw thickness in millimeters.  Error bars  are smaller than the symbols. (  B  ) Heat hyperalgesia assessed as the latency to withdraw from a radiant heat stimulus. (  C  ) Mechanoallodynia assessed as the frequency of withdrawal from a normally innocuous stimulus (4-g von Frey hair). (  D  ) Mechanohyperalgesia assessed as the frequency of withdrawal from a normally noxious stimulus (15-g von Frey hair). n = 6/group. *  P  < 0.05, **  P  < 0.01  versus  preinjection baseline (B). 
Fig. 1. Complete Freund adjuvant (CFA)–evoked edema and stimulus-evoked pain responses. Data are mean ± SEM from the hind paws ipsilateral (ipsi.) and contralateral (contra.) to the injection of CFA or the saline control (cont.) injection. (  A  ) Edema assessed as dorsoventral paw thickness in millimeters.  Error bars  are smaller than the symbols. (  B  ) Heat hyperalgesia assessed as the latency to withdraw from a radiant heat stimulus. (  C  ) Mechanoallodynia assessed as the frequency of withdrawal from a normally innocuous stimulus (4-g von Frey hair). (  D  ) Mechanohyperalgesia assessed as the frequency of withdrawal from a normally noxious stimulus (15-g von Frey hair). n = 6/group. *  P  < 0.05, **  P  < 0.01  versus  preinjection baseline (B). 
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Time Course of Heat Hyperalgesia
Statistically significant hypersensitivity to heat began within 2 h after CFA injection (fig. 1B). Heat hyperalgesia reached its maximum severity at 1 day, gradually declined during the following days, and resolved by 14 days. No change in heat sensitivity was seen in the hind paw contralateral to the CFA injection. There were no significant ipsilateral versus  contralateral differences in the saline-injected control group.
Time Course of Mechanoallodynia
Mechanoallodynia (increased response frequency to the 4-g von Frey hair stimulus) was first detected 2 h after CFA injection, gradually increased until it peaked at 7 days, and was reduced but still present at 14 days (fig. 1C). Relatively small changes were seen in the sensitivity of the hind paw contralateral to the CFA injection on day 2–7. Saline injection produced no change ipsilaterally or contralaterally.
Time Course of Mechanohyperalgesia
Statistically significant mechanohyperalgesia was present by 2 h after CFA injection, peaked at 6 h, remained at maximal intensity until at least 7 days, and was reduced but still present at 14 days (fig. 1D). Relatively small changes were seen contralateral to the CFA injection on days 2–7. There were no significant differences in the saline-injected control group.
Spontaneous Discharge in A Fibers and C Fibers in Control Rats
In the naive control group, we recorded 153 A fibers and 49 C fibers from 42 microfilaments. Few fibers had spontaneous discharge: 1.3% (2 of 153) of the A fibers and 0% (0 of 49) of the C fibers. In the control group that was examined 7 days after an intraplantar injection of saline, we recorded 145 A fibers and 46 C fibers from 39 microfilaments. Again, few fibers had spontaneous discharge: 0.7% (1 of 145) of the A fibers and 0% (0 of 46) of the C fibers. The results from the two control groups were nearly identical; combining their data to form a single control group yielded an incidence of spontaneous discharge of 1.0% (3 of 298) in A fibers and 0% (0 of 95) in C fibers.
The CVs for A fibers in the combined control group (fig. 2) varied widely and were not distributed normally. The median CV was 18.5 m/s. The CVs of the C fibers in the combined control group (fig. 3) had a normal distribution with a mean (±SD) of 0.9 ± 0.3 m/s.
Fig. 2. A-fiber conduction velocities (CVs) in the combined control group and 2, 7, and 14 days after complete Freund adjuvant (CFA) injection.  Blackened segments  of the bars represent fibers with spontaneous discharge. 
Fig. 2. A-fiber conduction velocities (CVs) in the combined control group and 2, 7, and 14 days after complete Freund adjuvant (CFA) injection.  Blackened segments  of the bars represent fibers with spontaneous discharge. 
Fig. 2. A-fiber conduction velocities (CVs) in the combined control group and 2, 7, and 14 days after complete Freund adjuvant (CFA) injection.  Blackened segments  of the bars represent fibers with spontaneous discharge. 
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Fig. 3. C-fiber conduction velocities (CVs) in the combined control group and 2, 7, and 14 days after complete Freund adjuvant (CFA) injection.  Blackened segments  of the bars represent fibers with spontaneous discharge. 
Fig. 3. C-fiber conduction velocities (CVs) in the combined control group and 2, 7, and 14 days after complete Freund adjuvant (CFA) injection.  Blackened segments  of the bars represent fibers with spontaneous discharge. 
Fig. 3. C-fiber conduction velocities (CVs) in the combined control group and 2, 7, and 14 days after complete Freund adjuvant (CFA) injection.  Blackened segments  of the bars represent fibers with spontaneous discharge. 
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Incidence of Spontaneous Discharge in A Fibers and C Fibers after CFA Injection
For the A fibers (fig. 4), statistically significant increases in the incidence of spontaneous discharge were present at 2 days (23% [71 of 315]; 95% confidence interval, 18–28%), at 7 days (9% [32 of 348]; 95% confidence interval, 6–13%), and at 14 days (7% [22 of 305]; 95% confidence interval, 5–11%). The increase seen at 2 days is significantly greater than those seen at 7 days and 14 days (P  < 0.01).
Fig. 4. Percentage of A fibers and C fibers with spontaneous discharge in the combined control group and 2, 7, and 14 days after complete Freund adjuvant (CFA) injection. ***  P  < 0.001  versus  control group. 
Fig. 4. Percentage of A fibers and C fibers with spontaneous discharge in the combined control group and 2, 7, and 14 days after complete Freund adjuvant (CFA) injection. ***  P  < 0.001  versus  control group. 
Fig. 4. Percentage of A fibers and C fibers with spontaneous discharge in the combined control group and 2, 7, and 14 days after complete Freund adjuvant (CFA) injection. ***  P  < 0.001  versus  control group. 
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For the C fibers (fig. 4), statistically significant increases in the incidence of spontaneous discharge were present at 2 days (24% [21 of 88]; 95% confidence interval, 15–34%) and at 7 days (24% [26 of 110]; 95% confidence interval, 16–33%). There was a dramatic decrease to 4% (4 of 107) in the incidence of spontaneous discharge at 14 days; although greater than the control value (0%), this difference was not statistically significant.
Conduction Velocities of A Fibers and C Fibers with Spontaneous Discharge after CFA Injection
The CV distributions of fibers with and without spontaneous discharge are shown in figures 2 and 3. For both A fibers and C fibers, the CV distributions of fibers without spontaneous discharge were similar to the CV distributions found in the control group. However, A fibers with spontaneous discharge had a median CV that was significantly slower than the median CV of A fibers without spontaneous discharge at 2 days (15.0 vs.  19.0 m/s; P  < 0.05) and 7 days (13.5 vs.  18.5 m/s; P  < 0.05), but the median CVs at 14 days did not differ (19.0 vs.  18.0 m/s). There were no statistically significant differences between the mean CVs of C fibers with and without spontaneous discharge (range, 0.9–1.2 m/s), and their CV distributions were also similar.
Frequency and Pattern of A-fiber and C-fiber Spontaneous Discharge after CFA Injection
At 2 days, 7 days, and 14 days, the frequency of A-fiber spontaneous discharge was highly variable (range, 0.03–30 Hz), but the mean rates were low (fig. 5). A large majority of the fibers (79–95%) discharged at 1.0 Hz or less and a small majority (58–68%) discharged at 0.3 Hz or less. There was no statistically significant change in the mean frequency over time (from 2.4 ± 4.7 Hz at 2 days to 0.9 ± 1.1 Hz at 14 days). In every case, the A-fiber discharge was irregular (i.e.  , highly variable interspike intervals). No fibers discharged in distinct bursts. Specimen recordings are shown in figure 6.
Fig. 5. Distribution of discharge frequencies in spontaneously discharging A fibers and C fibers 2, 7, and 14 days after complete Freund adjuvant (CFA) injection. Only one C fiber discharged at greater than 2.0 Hz; its data (2.2 Hz) are excluded from the distribution. The  x-axis labels  give the upper boundary of each bin relative to the preceding bin. Note that the intervals between bins are not equal. 
Fig. 5. Distribution of discharge frequencies in spontaneously discharging A fibers and C fibers 2, 7, and 14 days after complete Freund adjuvant (CFA) injection. Only one C fiber discharged at greater than 2.0 Hz; its data (2.2 Hz) are excluded from the distribution. The  x-axis labels  give the upper boundary of each bin relative to the preceding bin. Note that the intervals between bins are not equal. 
Fig. 5. Distribution of discharge frequencies in spontaneously discharging A fibers and C fibers 2, 7, and 14 days after complete Freund adjuvant (CFA) injection. Only one C fiber discharged at greater than 2.0 Hz; its data (2.2 Hz) are excluded from the distribution. The  x-axis labels  give the upper boundary of each bin relative to the preceding bin. Note that the intervals between bins are not equal. 
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Fig. 6. Specimen records of spontaneously discharging fibers after complete Freund adjuvant (CFA) injection. (  Top  ) Rapidly conducting A fiber. (  Middle  ) Slowly conducting A fiber. (  Bottom  ) C fiber. The C fiber's waveform is shown  below the trace  . Note the highly irregular interspike intervals. CV = conduction velocity. 
Fig. 6. Specimen records of spontaneously discharging fibers after complete Freund adjuvant (CFA) injection. (  Top  ) Rapidly conducting A fiber. (  Middle  ) Slowly conducting A fiber. (  Bottom  ) C fiber. The C fiber's waveform is shown  below the trace  . Note the highly irregular interspike intervals. CV = conduction velocity. 
Fig. 6. Specimen records of spontaneously discharging fibers after complete Freund adjuvant (CFA) injection. (  Top  ) Rapidly conducting A fiber. (  Middle  ) Slowly conducting A fiber. (  Bottom  ) C fiber. The C fiber's waveform is shown  below the trace  . Note the highly irregular interspike intervals. CV = conduction velocity. 
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The frequency of C-fiber spontaneous discharge was also variable (range, 0.05–2.2 Hz) at 2 days, 7 days, and 14 days (fig. 6). The majority of fibers (59–75%) discharged at 1.0 Hz or less, and many (23–24%) discharged at 0.3 Hz or less. There was no change in the C fibers' mean discharge frequency over time. All C-fiber discharge was irregular and without bursting (fig. 6).
Discussion
We find that there is a persistent discharge of very low frequency from 4% to 24% of primary afferent A fibers and C fibers during the course of a 14-day inflammatory pain condition. The incidence, but not the frequency, of this discharge evolves over time.
As we recorded from the distal stump of the transected nerve, we can be certain that this discharge originates distal to the recording site. Its site of origin is likely to be in the afferents' sensory terminals, but we cannot exclude the possibility of an ectopic origin in the length of axon lying between the terminals and the recording site. We did not examine the possibility that inflammation evokes spontaneous discharge in the afferents' cell bodies in the dorsal root ganglion.
The low incidence of spontaneous discharge in the control animals is consistent with our procedures. Keeping the hind paw warm will prevent discharge in cooling-specific afferents (mostly small A fibers). Warming the paw will activate some warm-specific afferents (C fibers), but these fibers either are not numerous or have especially small action potentials that make them difficult to detect.29 As is usual with the teased fiber recording method, the relative number of individually identifiable A-fiber potentials in each microfilament was larger than the number of individually identifiable C-fiber potentials, despite the much larger number of C fibers in the nerve. This is simply because the action potentials of C fibers are smaller than those of A fibers and consequently more difficult to isolate from the background activity.
Our preparations were anesthetized with isoflurane, and there is evidence that isoflurane may directly excite C fibers and A fibers innervating the cornea and respiratory track.30,31 However, we found a nearly total absence of spontaneous activity in the fibers innervating the hind paw in the combined control group, suggesting that these fibers are not excited by isoflurane. This is consistent with the reported absence of an isoflurane effect on the nociceptor innervation of the tail.32 
The A-fiber Response to Inflammation
We found that 23% of A fibers had spontaneous discharge after 2 days of inflammation; this declined markedly to 9% by 7 days and persisted at the same low level until at least 14 days. In the rat, others have reported a marked decline (from 51% to 2%) in the incidence of spontaneously discharging A fibers between 1 day and 4 days of inflammation produced by an atypical protocol in the rat (two subcutaneous injections of 100 μl CFA, one intraplantar and the other at the level of the knee).14 Pogatzki et al.  7 found an incidence of spontaneous discharge of 38% of A-delta fibers 1 day after an incision in the plantar hind paw. Hamalainen et al.  6 found no A-fiber spontaneous discharge at 45 min after hind paw incision. Therefore, it seems that inflammation evokes a high incidence of A-fiber spontaneous discharge that begins at 1 day or earlier, persists until at least 2 days, and thereafter declines sharply over the period of 4–14 days after injury. Our results indicate that throughout the duration of the inflammatory pain condition, this discharge is slow, usually less than 1.0 Hz and often less than 1 impulse every 3 s.
The C-fiber Response to Inflammation
There was a high incidence (24%) of spontaneously discharging C fibers that lasted until at least 7 days, with a rapid decline to 4% by 14 days. In rats, Djouhri et al.  14 found an increased incidence of spontaneous discharge of 31% of C-fiber nociceptors 1 day after the onset of inflammation and of 18% at 4 days. In the plantar incision model, 40% of C fibers had spontaneous discharge at 1 day.7 Therefore, it seems that inflammation evokes spontaneous discharge in a large percentage of C fibers within 1 day of the onset of inflammation and that this persists for at least 7 days but subsequently declines to a low incidence by 14 days. At all times examined, this C-fiber discharge is also slow, with most firing at 0.3–1.0 Hz. Similar low-frequency (0.2- to 0.6-Hz) C-nociceptor spontaneous discharge has been noted 1 day and 4 days after inflammation.14 
Pattern of Spontaneous Discharge
Both A fibers and C fibers discharged continuously in a clearly irregular pattern. The spontaneous discharge seen after inflammation is thus distinctly different than the ectopic spontaneous discharge seen in axotomized A-fiber and C-fiber neurons, where the A fibers have a distinctly higher frequency (approximately 30 Hz) and both A fibers and C fibers generally have a continuous or bursting discharge with very regular interspike intervals.33 
Identity of Spontaneously Discharging Fibers
At 2 and 7 days after inflammation, A fibers with spontaneous discharge had significantly slower CVs than the A fibers that were not discharging. This is consistent with most of the spontaneously active A fibers being nociceptors.28 In previous work, nearly all of the spontaneously active A fibers and C fibers seen 1 day and 4 days after CFA-evoked inflammation were nociceptors.14 It is thus probable that most of the spontaneously active fibers described here were nociceptors. However, it is possible that other afferent types were also present in our sample, e.g.  , edema might have activated A-fiber stretch receptors. Nonnociceptive spontaneous discharge might underlie the dysesthetic sensations that sometimes accompany inflammatory pain.
Relation of Spontaneous Discharge to Spontaneous Pain
There is no validated way to measure spontaneous pain in an animal.34,35 Attempts to use “spontaneous” behaviors such as hind paw guarding, lifting, and shaking as an index of spontaneous pain14,36 have not controlled for the possibility that the behavior may be evoked by incident pain (e.g.  , weight bearing). It has been argued that holding the paw in an elevated position that guards it from incident stimulation is indicative of spontaneous pain,36,37 but this is probably better thought of as avoidance behavior. Nevertheless, our finding of persistent low-frequency discharge throughout the period of inflammation raises the question of whether this might underlie spontaneous pain sensations.
The C-fiber spontaneous discharge frequency that we found (most fibers firing at 0.3–1.0 Hz) is comparable to the “ongoing” C-nociceptor discharge of roughly 0.5–2.0 Hz that is seen during the first few tens of seconds to minutes after the immediate high-frequency response evoked by injury. However, the A-fiber discharge frequency that we found (nearly all firing at 1.0 Hz or less, and a majority at 0.3 Hz or less) is far lower than the ongoing discharge frequency of 2.0–20.0 Hz seen in A-fiber nociceptors immediately after injury.5,38 It is possible that the perceptual consequences of even very low-frequency spontaneous nociceptor discharge may be amplified after central sensitization has been established.
Relation of Spontaneous Discharge to Allodynia, and Hyperalgesia
It is well established that in the normal case, a small proportion of A-fiber and C-fiber nociceptors have a low-frequency discharge when activated by a stimulus intensity that is distinctly below the perceptual threshold for pain.39,40 Experimental evidence in human subjects suggests that this relatively minor amount of C-nociceptor discharge is capable of initiating central sensitization if the discharge persists for tens of minutes.41 For neuropathic pain, there is evidence that C-fiber nociceptor spontaneous discharge of even exceedingly low frequency (approximately 1 impulse/min; 0.02 Hz) may have a role in the initiation or maintenance of central sensitization.22–24 Therefore, it is possible that even a low incidence of low-frequency C-nociceptor discharge is sufficient to maintain central sensitization, especially if the discharge persists for many days, as we show here. In the normal case, the initiation of central sensitization is due to C-fiber nociceptor input, but not A-fiber nociceptor input.4 However, in inflamed tissue, input from both C fibers and A fibers may be capable of maintaining the sensitized state.42 Therefore, both the A-fiber and the C-fiber persistent spontaneous discharge that we found may be involved in the maintenance of central sensitization.
The relation between spontaneous nociceptor discharge and allodynia and hyperalgesia is likely to be different for hypersensitivity to mechanical and thermal modalities.8,9 This may be related to our demonstration that there are different time courses for the severity of inflammation-evoked heat hyperalgesia, mechanoallodynia, and mechanohyperalgesia (fig. 1).
We found that the incidence of spontaneous discharge in A fibers was clearly increased at 2 days, when heat hyperalgesia, mechanoallodynia, and mechanohyperalgesia were all present at near peak severity. However, the marked decrease in the incidence of A-fiber spontaneous discharge that occurred from 2 to 7 days (from 23% to 9%) was not associated with any decrease in the severity of mechanoallodynia or mechanohyperalgesia. There was a decrease in the severity of heat hyperalgesia between 2 and 7 days, but this was gradual compared with the sharp decline in the incidence of A-fiber spontaneous discharge. On the other hand, the incidence of C-fiber discharge matched the behavioral time courses fairly well. In particular, there was a marked decrease in the incidence of spontaneously discharging C fibers (from 24% to 4%) from 7 to 14 days, and this coincided with clear decreases in the severity of mechanoallodynia and mechanohyperalgesia, but not for heat hyperalgesia.
At 14 days, the incidence of A-fiber, but not C-fiber, spontaneous discharge was still significantly greater than normal. Mechanoallodynia and mechanohyperalgesia were clearly waning but still present at this time, but significant heat hyperalgesia was no longer present. It is probable that an ongoing inflammatory process was still present at 14 days because edema was still very pronounced and CFA is a long-lasting oil-based depot for antigenic stimuli.
Conclusions
It is probable that most of the inflammation-evoked spontaneous activity shown here is from sensitized nociceptors, perhaps including activated “silent” nociceptors.43 Therefore, our results suggest that in the context of an inflammatory pain condition, a very low-frequency nociceptor discharge persists for days and is one of the mechanisms underlying chronic pain, allodynia, and hyperalgesia. It is possible that the persistent low-frequency spontaneous discharge is sufficient to initiate and maintain central sensitization. If so, chronic inflammatory pain is likely to be due to both peripheral and central mechanisms.
The authors thank Lina Naso (Laboratory Technician, Department of Anesthesiology, McGill University, Montreal, Quebec, Canada) for technical support.
References
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Fig. 1. Complete Freund adjuvant (CFA)–evoked edema and stimulus-evoked pain responses. Data are mean ± SEM from the hind paws ipsilateral (ipsi.) and contralateral (contra.) to the injection of CFA or the saline control (cont.) injection. (  A  ) Edema assessed as dorsoventral paw thickness in millimeters.  Error bars  are smaller than the symbols. (  B  ) Heat hyperalgesia assessed as the latency to withdraw from a radiant heat stimulus. (  C  ) Mechanoallodynia assessed as the frequency of withdrawal from a normally innocuous stimulus (4-g von Frey hair). (  D  ) Mechanohyperalgesia assessed as the frequency of withdrawal from a normally noxious stimulus (15-g von Frey hair). n = 6/group. *  P  < 0.05, **  P  < 0.01  versus  preinjection baseline (B). 
Fig. 1. Complete Freund adjuvant (CFA)–evoked edema and stimulus-evoked pain responses. Data are mean ± SEM from the hind paws ipsilateral (ipsi.) and contralateral (contra.) to the injection of CFA or the saline control (cont.) injection. (  A  ) Edema assessed as dorsoventral paw thickness in millimeters.  Error bars  are smaller than the symbols. (  B  ) Heat hyperalgesia assessed as the latency to withdraw from a radiant heat stimulus. (  C  ) Mechanoallodynia assessed as the frequency of withdrawal from a normally innocuous stimulus (4-g von Frey hair). (  D  ) Mechanohyperalgesia assessed as the frequency of withdrawal from a normally noxious stimulus (15-g von Frey hair). n = 6/group. *  P  < 0.05, **  P  < 0.01  versus  preinjection baseline (B). 
Fig. 1. Complete Freund adjuvant (CFA)–evoked edema and stimulus-evoked pain responses. Data are mean ± SEM from the hind paws ipsilateral (ipsi.) and contralateral (contra.) to the injection of CFA or the saline control (cont.) injection. (  A  ) Edema assessed as dorsoventral paw thickness in millimeters.  Error bars  are smaller than the symbols. (  B  ) Heat hyperalgesia assessed as the latency to withdraw from a radiant heat stimulus. (  C  ) Mechanoallodynia assessed as the frequency of withdrawal from a normally innocuous stimulus (4-g von Frey hair). (  D  ) Mechanohyperalgesia assessed as the frequency of withdrawal from a normally noxious stimulus (15-g von Frey hair). n = 6/group. *  P  < 0.05, **  P  < 0.01  versus  preinjection baseline (B). 
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Fig. 2. A-fiber conduction velocities (CVs) in the combined control group and 2, 7, and 14 days after complete Freund adjuvant (CFA) injection.  Blackened segments  of the bars represent fibers with spontaneous discharge. 
Fig. 2. A-fiber conduction velocities (CVs) in the combined control group and 2, 7, and 14 days after complete Freund adjuvant (CFA) injection.  Blackened segments  of the bars represent fibers with spontaneous discharge. 
Fig. 2. A-fiber conduction velocities (CVs) in the combined control group and 2, 7, and 14 days after complete Freund adjuvant (CFA) injection.  Blackened segments  of the bars represent fibers with spontaneous discharge. 
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Fig. 3. C-fiber conduction velocities (CVs) in the combined control group and 2, 7, and 14 days after complete Freund adjuvant (CFA) injection.  Blackened segments  of the bars represent fibers with spontaneous discharge. 
Fig. 3. C-fiber conduction velocities (CVs) in the combined control group and 2, 7, and 14 days after complete Freund adjuvant (CFA) injection.  Blackened segments  of the bars represent fibers with spontaneous discharge. 
Fig. 3. C-fiber conduction velocities (CVs) in the combined control group and 2, 7, and 14 days after complete Freund adjuvant (CFA) injection.  Blackened segments  of the bars represent fibers with spontaneous discharge. 
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Fig. 4. Percentage of A fibers and C fibers with spontaneous discharge in the combined control group and 2, 7, and 14 days after complete Freund adjuvant (CFA) injection. ***  P  < 0.001  versus  control group. 
Fig. 4. Percentage of A fibers and C fibers with spontaneous discharge in the combined control group and 2, 7, and 14 days after complete Freund adjuvant (CFA) injection. ***  P  < 0.001  versus  control group. 
Fig. 4. Percentage of A fibers and C fibers with spontaneous discharge in the combined control group and 2, 7, and 14 days after complete Freund adjuvant (CFA) injection. ***  P  < 0.001  versus  control group. 
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Fig. 5. Distribution of discharge frequencies in spontaneously discharging A fibers and C fibers 2, 7, and 14 days after complete Freund adjuvant (CFA) injection. Only one C fiber discharged at greater than 2.0 Hz; its data (2.2 Hz) are excluded from the distribution. The  x-axis labels  give the upper boundary of each bin relative to the preceding bin. Note that the intervals between bins are not equal. 
Fig. 5. Distribution of discharge frequencies in spontaneously discharging A fibers and C fibers 2, 7, and 14 days after complete Freund adjuvant (CFA) injection. Only one C fiber discharged at greater than 2.0 Hz; its data (2.2 Hz) are excluded from the distribution. The  x-axis labels  give the upper boundary of each bin relative to the preceding bin. Note that the intervals between bins are not equal. 
Fig. 5. Distribution of discharge frequencies in spontaneously discharging A fibers and C fibers 2, 7, and 14 days after complete Freund adjuvant (CFA) injection. Only one C fiber discharged at greater than 2.0 Hz; its data (2.2 Hz) are excluded from the distribution. The  x-axis labels  give the upper boundary of each bin relative to the preceding bin. Note that the intervals between bins are not equal. 
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Fig. 6. Specimen records of spontaneously discharging fibers after complete Freund adjuvant (CFA) injection. (  Top  ) Rapidly conducting A fiber. (  Middle  ) Slowly conducting A fiber. (  Bottom  ) C fiber. The C fiber's waveform is shown  below the trace  . Note the highly irregular interspike intervals. CV = conduction velocity. 
Fig. 6. Specimen records of spontaneously discharging fibers after complete Freund adjuvant (CFA) injection. (  Top  ) Rapidly conducting A fiber. (  Middle  ) Slowly conducting A fiber. (  Bottom  ) C fiber. The C fiber's waveform is shown  below the trace  . Note the highly irregular interspike intervals. CV = conduction velocity. 
Fig. 6. Specimen records of spontaneously discharging fibers after complete Freund adjuvant (CFA) injection. (  Top  ) Rapidly conducting A fiber. (  Middle  ) Slowly conducting A fiber. (  Bottom  ) C fiber. The C fiber's waveform is shown  below the trace  . Note the highly irregular interspike intervals. CV = conduction velocity. 
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