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2Neuropathic Pain Unit, Hospital General de Catalunya, 08190 Barcelona, Spain; 3Departamento de Ciencias Neurológicas, Universidad de Chile, Santiago 10D, Chile; and 4Sobell Department, Institute of Neurology, University College London, Queen Square, London WC1N 3BG, United Kingdom; and 1Oregon Nerve Center, Good Samaritan Hospital and Medical Center, Oregon Health Sciences University, Portland, Oregon 97210.
Submitted 12 June 2003; accepted in final form 12 January 2004
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONCONCLUSIONACKNOWLEDGMENTSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONCONCLUSIONACKNOWLEDGMENTSREFERENCES |
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; Kilo et al. 1994; LaMotte et al. 1991; Simone et al. 1989). However, the spatial extent and underlying mechanisms of the flare, the psychophysical profile of the hyperalgesias, and the neural mechanisms responsible for them remain disputed (Ali et al. 1996; Serra et al. 1998).
The primary hyperalgesia at the site of capsaicin application features increased sensory responses to both mechanical and heat stimuli, attributed to sensitization of cutaneous nociceptor terminals, both of the common polymodal type (Campbell and Meyer 1983; LaMotte et al. 1982, 1983, 1992; Torebjörk et al. 1984) and of the more recently described mechano-insensitive type of C nociceptor (Davis et al. 1993; Meyer et al. 1991; Schmelz et al. 2000b; Schmidt et al. 1995). The neural mechanisms underlying the secondary hyperalgesia in surrounding skin remain unclear. Common polymodal C nociceptors have been reported not to become sensitized in the area of experimental secondary hyperalgesia in both animals and humans (Baumann et al. 1991; Campbell and Meyer 1983; LaMotte et al. 1992; Torebjörk et al. 1992). Schmelz et al. (2000b) found that application of capsaicin to one branch of a mechano-insensitive C nociceptor did not sensitize other branches not exposed to capsaicin. These findings were consistent with previous reports on the absence of spread of sensitization to neighboring branches of a stimulated C-nociceptor (Baumann et al. 1991; LaMotte et al. 1992; Schmelz et al. 1996; Thalhammer et al. 1982). However, possible sensitization of remote mechano-insensitive C nociceptors by capsaicin was not specifically tested in those studies.
Partly because of the lack of evidence of receptor sensitization, it has been proposed that capsaicin-induced secondary hyperalgesia is mediated by dorsal horn neurons, sensitized by a primary C-nociceptor barrage (Koltzenburg et al. 1994; LaMotte et al. 1991; Sang et al. 1996; Simone et al. 1991; Torebjörk et al. 1992; Woolf and Mannion 1999). More specifically, Magerl et al. (2001), using combinations of cutaneous desensitization and pressure nerve blocks, found evidence that secondary hyperalgesia to pinprick is mediated by capsaicin-insensitive A
afferents, centrally facilitated by capsaicin-sensitive C fiber input. However, evidence has also been reported of a likely contribution of mechano-insensitive or "silent" C nociceptors to the pathophysiology of secondary hyperalgesia (Serra et al. 1993, 1994, 1995, 1998). With the aid of improved methods to track the behavior of multiple identified C fibers, we have now confirmed that these units are activated and can become sensitized in the area of secondary hyperalgesia.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONCONCLUSIONACKNOWLEDGMENTSREFERENCES |
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Eleven healthy adult volunteers participated in a total of 25 microneurographic recordings. There were seven males and four females, with ages ranging from 18 to 40 yr (mean, 35 yr). The study was approved by the local ethics committee and conformed to the Declaration of Helsinki. All subjects gave their informed written consent.
Microneurographic recordings
Microneurography was used to record action potentials of human C fibers from cutaneous nerve fascicles of the superficial peroneal nerve at the ankle (10 subjects) or the superficial radial nerve at the wrist (1 subject). The subjects sat relaxed, with the leg or arm firmly supported in a padded platform. The general technique of microneurography has been described in detail by Vallbo and Hagbarth (1968). Intraneural recordings were obtained using a 0.2-mm-diam lacquer-insulated tungsten microelectrode (MNG active/1 M
. FHC, Bowdoinham, ME), which was inserted percutaneously into a sensory nerve. A subcutaneous reference electrode was inserted 1–2 cm outside the nerve trunk. The neural signals were amplified by a commercial differential amplifier (3+ Cell Isolated Microamplifier, FHC) and filtered with an adjustable analogue filter (band-pass 100–2,000 Hz). Line interference was removed with an on-line noise eliminator (Hum Bug, Quest Scientific, North Vancouver, Canada). Signals were displayed on a Tektronix 5113a oscilloscope and digitized by a personal computer with a Data Translation DT2812 A/D board at a sampling rate of 10 kHz. Digitized signals were stored on the hard drive of the personal computer as raw data for off-line analysis. Digital filtering (band-pass 300–2,000 Hz) and clamping of the baseline were performed both on-line and during off-line analysis for a better visualization of the action potentials. Temperature of the skin was measured with a thermocouple placed on the skin adjacent to the receptive fields of the units under study. Skin temperature was maintained above 30°C with an infrared lamp.
Protocol of electrical stimulation of the cutaneous receptive fields
Search for the electrical receptive fields of C fibers was conducted by stimulating electrically with a pair of needle electrodes in areas of skin to which painful sensations were referred during intraneural electrical microstimulation at near threshold levels (Torebjörk and Ochoa 1990). Stimulation was performed using rectangular pulses of 0.25- to 0.3-ms duration (Grass S48, stimulus isolation unit SIU 5) at a rate of 0.25 Hz. Only fibers with latencies compatible with conduction velocities in the C fiber range (<2 ms-1) were studied. When time-locked responses with such latencies were recorded at 0.25-Hz baseline stimulation, a sequence of a 3-min pause followed by a 6-min baseline and a 3-min 2-Hz train was given (see Fig. 1). Stimulation at low rates following a pause has been reported to differentiate mechano-sensitive from mechano-insensitive C nociceptors (i.e., CM from CMi units) in the human skin (Weidner et al. 1999), while stimulation at 2 Hz for 3 min differentiates patterns of slowing among functional types of C fibers (Serra et al. 1999). For convenience and because no axonal property has yet been shown to correlate with heat sensitivity, we will here use the abbreviations CM and CMi for mechano-sensitive and mechano-insensitive nociceptors, respectively, without regard for heat sensitivity. CM therefore includes common polymodal CMH units, and CMi embraces both CH and CMiHi units.
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Responses were recorded and analyzed with QTRAC software (Institute of Neurology, London, UK), specially modified to track peak latencies and display them as a latency "profile" or as a raster plot. In the latency raster plots, each peak in the filtered voltage signal that exceeded a specified level is represented by a dot on a plot with latency as the ordinate and elapsed time as the abscissa (see Serra et al. 1999). Depending on the level chosen, the dots could represent action potentials or noise (Fig. 1A). For the raster figures in this paper, latencies of selected units with adequate signal-to-noise were remeasured from the raw data, so that each dot represents an identified single unit. These remeasured figures are referred to as modified raster plots (Fig. 1B). A full description of the method is provided in Serra et al. 1999.
Measurements of conduction velocity changes and assignment of axonal type
Conduction velocity was estimated by dividing the conduction distance by the baseline latency at the stimulation rate of 0.25 Hz. Slowing after the 3-min pause was measured as the percentage increase in latency from the first spike after the pause to the baseline latency. Percentages of conduction slowing relative to the baseline latency were also determined at 1 and 3 min after the onset of the 2-Hz stimulus train. As shown in a previous report (Serra et al. 1999), when stimulation frequency is increased from 0.25 to 2 Hz, different patterns of slowing can be observed among human C fibers: "type 1" fibers slowed progressively (average latency increase at 3 min = 28.3%), whereas "type 2" fibers slowed to reach a plateau (average latency increase = 5.2%) within 1 min. The former were identified as nociceptors, whereas the latter have been recently identified as specific cold receptors (Campero et al. 2001). We quickly found that the 3-min pause enabled the type 1 fibers to be divided into two groups: those that were essentially unaffected by the pause and those that showed an appreciable reduction in latency at the end of the pause. We designated the former type 1A and the latter type 1B. According to this classification, the five units in Fig. 1B can be assigned (in order of increasing latencies) to types 2, 1A, 1A, 2, and 1B. Only type 1A and 1B units, putative nociceptors, are considered further in this study.
Measurement of recovery
The time course of recovery of conduction velocity after cessation of stimulation at 2 Hz was also measured. Following Thalhammer et al. (1994), we used two indices of the recovery rate: 1) the time necessary to reverse 50% of the activity-induced latency change and 2) the percentage of recovery at 30 s after the end of the stimulus train.
Functional characterization of the units
Identification of the receptor properties of the units under study was determined from raster plots while the unit was being electrically stimulated regularly at 0.25 Hz, using the "marking technique" as described previously (Hallin and Torebjörk 1974; Schmelz et al. 1995). In short, natural stimuli that activate a unit induce an abrupt increase in conduction latency, due to activity-dependent slowing (Fig. 2). This has been shown to be a reliable method to follow units, both at rest and during natural activation, for long periods of time. This method is able to discriminate, among several units, which ones have been activated with natural stimulation.
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45°C). To exclude that the units under study might have been sympathetic efferents, maneuvers known to activate sympathetic fibers, i.e., the Valsalva maneuver, startle by an unexpected shout, and stress caused by mental arithmetic (Delius et al. 1972; Hallin and Torebjörk 1974) were performed, and sudden latency shifts, indicative of activation of that unit, were tracked. After some experiments had been performed, it became evident that the protocol combining the pause and 2-Hz stimulation allowed unambiguous identification of the unit under study as either a CM or CMi unit (see Fig. 3). Subsequently, some of the units were never tested for mechanical or heat sensitivity before being challenged with capsaicin. Therefore possible sensitization of the units with repeated testing before capsaicin injection was completely avoided.
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Following the 2-Hz, 3-min period, an ensuing 6-min (or longer if required) stimulation period at 0.25 Hz was allowed until the unit had recovered to its previous stable latency. Flare and hyperalgesia were induced by injecting an aqueous solution containing 1% capsaicin dissolved in 7.5% Tween 80. A volume of 10 µl, containing 100 µg of capsaicin, was injected intradermally in the skin of the dorsum of the foot or the hand at a distance of 
1 cm from the stimulating electrodes, always within the innervation territory of the implicated nerve, using a 28-gauge hypodermic needle. To test for effects of capsaicin injection lasting longer than the duration of a microneurographic recording, on two occasions, capsaicin was injected 30 min before the start of the microneurography. At the end of the experiment, short trains of electrical stimuli (0.25-ms pulses at 20 Hz) at the maximum intensity tolerated by the subject (50 mA) were applied with the aid of a rounded tip stimulator (as described by Meyer et al. 1991) to search for possible branches of the recorded units in the area of capsaicin injection.
Monitoring of responses following remote capsaicin injection
Stimulation at the usual baseline 0.25-Hz frequency was continued during and after the capsaicin injection. This enabled any spontaneous activity of the units to be revealed by sudden increases in their otherwise stable latencies on the raster plot. Subsequently, the units under study were again tested to check for possible development of mechanical or heat sensitivity.
Determination of the area of visual flare and mechanical hyperalgesia
The extent of the early visual flare that appeared immediately after capsaicin injection was outlined on the skin with a soft pen, and a picture was taken. As previously reported (Serra et al. 1998), the extent of this early visual flare is much larger and shorter lived than the flare that remains afterwards for several hours surrounding the injection site. Also, the area of mechanical hyperalgesia was determined using a von Frey hair exerting a force of 2.02 N (1.02 mm diam, pressure 24.75 bars). In normal skin this stimulus is clearly suprathreshold for mechanical pain in all subjects. The filament was applied at right angles for 5 s to each of a series of points 0.5 cm apart along radial paths converging at the injection site. Care was taken to avoid stretching the skin laterally, which might activate CMi units (Schmelz et al. 2000b). Stimulation started at points well beyond the area where hyperalgesia was typically detected. The area of hyperalgesia was outlined with a soft pen on the skin, and a picture was taken.
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RESULTS |
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A total of 29 units classified as axonal type 1B were recorded with adequate signal-to-noise ratio to be included in the study. An additional 29 units classified as axonal type 1A with good signal-to-noise ratio were also extracted from the same raster plots for comparison.
The results, like the experiments, fall into two parts. In the first part of an experiment, as shown in Fig. 2, the units were tested with the pause and 2-Hz train to define the axonal type, and in most cases, the units were also tested with mechanical and heat stimuli to characterize their responsiveness to natural stimuli. These results confirmed that axonal behavior could be used to determine receptor type without ambiguity, so that mechanical testing (which carries a slight risk of sensitizing CMi units) could be avoided. In the second part of an experiment (shown in Fig. 7, which is a continuation of Fig. 2), capsaicin was injected at a distance, and any spontaneous activity or changes in sensitivity was noted.
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LATENCY CHANGES INDUCED BY THE PAUSE AND RECEPTOR RESPONSIVENESS. Percentages of slowing of conduction velocity following the 3-min pause are shown in Fig. 3, together with the classification of the units into types 1A and 1B. Seventeen of the 29 type 1B units were tested for mechanical responsiveness using a von Frey hair exerting a bending force of 722 mN. None of the units tested in such a way responded to mechanical stimulation and they are indicated by I (insensitive) in Fig. 3. All 29 type 1A units were tested mechanically, and all responded vigorously to von Frey hairs ranging from 52 to 722 mN (mean, 129.3 ± 150.8 mN). Therefore type 1A units were identified as CM, while type 1B were identified as CMi, in confirmation of the report by Weidner et al. (1999). Heat responses were not tested systematically because of the difficulty of applying the thermode without disturbing the stimulating electrodes.
FURTHER DIFFERENCES IN CONDUCTION PROPERTIES BETWEEN TYPE 1A AND 1B UNITS. Weidner et al. (1999) found that all CMi units had lower velocities than CM units. We also found that the type 1B (CMi) units conducted significantly more slowly than the type 1A (CM) units, but there was considerable overlap (see Fig. 4A). Although both velocity and slowing after the pause were related to axonal type, there was no correlation between these variables within each type of fiber.
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Apart from the effect of the pause, the parameter that most clearly differentiated the type 1A and 1B units was the time to 50% recovery of latency after the end of the 2-Hz train. These two parameters are compared in Fig. 4D, which shows that, although these parameters were correlated among the type 1 population as a whole, there was no correlation within either the type 1A or type 1B subgroup. Figure 4, A and D, indicates that the type 1A and type 1B units form two distinct classes, which differ in multiple axonal properties as well as in their receptor properties.
Part 2: effects of capsaicin injection
SPONTANEOUS ACTIVITY INDUCED BY CAPSAICIN INJECTION. Capsaicin was injected in 10 experiments while recording from 29 type 1A and 22 type 1B units. Distances from injection site to electrical receptive fields of the units ranged from 10 to 50 mm (median, 30 mm). In agreement with previous reports (Baumann et al. 1991; LaMotte et al. 1992), none of the 29 Type 1A (CM) fibers were affected by the remote injection of capsaicin. Examples are shown in Figs. 5 (unit 1) and 7 (units 1–3). In contrast, capsaicin induced spontaneous bursts of activity in 11 of the 15 type 1B (CMi) units for which responses were recorded, after a delay that ranged from 0.5 to 18 min (median, 2.7 min). There was no correlation between time to onset of the burst and distance from injection site. Some units bursted for long periods of time, up to almost 30 min in some cases (Fig. 6, units 1 and 2). These bursts of activity could be readily identified in the raster plots as sudden shifts in the stable latencies to the continuous stimulation at 0.25 Hz. Examples of this are shown in Figs. 5 (unit 2 only), 6 (units 1–3), and 7 (unit 4). On some occasions, a single burst of activity appeared (Fig. 7), while on other occasions, there was a succession of bursts, sometimes one on top of the other (Figs. 5 and 6).
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). The maximal area of this initial flush was similar to the area that subsequently became hyperalgesic (e.g., Fig. 10).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONCONCLUSIONACKNOWLEDGMENTSREFERENCES |
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Classification of C units by electrical and natural stimulation
Weidner et al. (1999) reported that CMi units in human skin could be recognized by the electrical properties of their axons: compared with CM units, they conducted more slowly and showed more prominent activity-dependent slowing at very slow stimulation rates, so that after at least 2 min without stimulation, they slowed appreciably on stimulation at only 0.125 or 0.25 Hz. Our results with a 3-min pause in baseline stimulation at 0.25 Hz (Fig. 3) are in excellent agreement and leave no doubt that our type 1A fibers correspond to their mechano-sensitive units and our type 1B fibers to their mechano-insensitive units. The slight difference in the average degree of slowing at 0.25 Hz after a pause for the CMi units, 6.75% in Weidner et al. (2000) and 4.5% in the present study, is probably due to the different recording distances of the two studies. The combination of pause and a 2-Hz train provides further differentiation of the nociceptor fibers, which all slow progressively for 3 min, from nonnociceptive cold fibers, which reach a latency plateau after slowing by about 5% (type 2, see Fig. 1 and Campero et al. 2001), and a rarer, unidentified third type of afferent that hardly slows at all (type 3, Serra et al. 1999). The great advantage of the protocol in this study was that it enabled mechano-sensitive and mechano-insensitive nociceptors to be identified unambiguously without extensive mechanical probing, which could have carried the risk of sensitizing units before the capsaicin was applied.
Delayed activation of CMi units by capsaicin in the area of secondary hyperalgesia
Previous studies have shown that both CM and CMi units are activated within a few seconds by local application of capsaicin but that CMi units give more prolonged discharges, corresponding better with the duration of burning pain (Schmeltz et al. 2000b). We have now found that, when capsaicin is injected at a distance of 10–50 mm, so that the recording site becomes engulfed in the area of secondary hyperalgesia, CM units are not activated, whereas CMi units often are activated but with appreciable delays. The mechanism of this delayed activation is uncertain. The possibility that it is caused by diffusion of capsaicin itself can be discounted: in other studies, with the same volume and concentration of capsaicin, it has been concluded that the drug does not spread more than a few millimeters from the site of injection (monkey: Baumann et al. 1991; LaMotte et al. 1991). Also, in this study, the capsaicin-sensitive CM units were unaffected, and among the CMi units, there was no correlation between activation delay and distance. Another possible explanation of the delayed activation could be that the activation is indirect, via a cascade system (cf. Lembeck and Gamse 1982; Lynn and Cotsell 1991).
The distances over which the CMi units were activated in this study are actually modest compared with their extensive arborizations in the skin described by Schmidt et al. (2002): CMi innervation territories are made up of multiple subfields, extending 
72 mm (mean, 45 mm). Although we were not able to excite branches of the CMi units recorded extending into the area of capsaicin injection, we cannot exclude the possibility that there were deep branches, which the capsaicin may have taken several minutes to reach. However, the time courses of the delayed responses were hardly consistent with a slow penetration of the algogen to the receptor membrane: the most delayed responses (e.g., unit 3 in Fig. 9) had a conspicuously abrupt onset, suggesting that they may have been mediated by another cell type, which could have integrated a weak capsaicin signal over several minutes, before releasing a packet of algogen close to the CMi unit.
A further possibility is that the delayed activity recorded in CMi units was caused by dorsal root reflexes set up by nociceptive inputs into the dorsal horn and recorded antidromically. Lin et al. 1999 have recorded such activity in rats and shown that it is an important contributor to the development of flare, 15–20 mm from the site of injection of capsaicin. However, the observation that flare can still be evoked in acutely denervated human skin until axonal degeneration has completely occurred (Lewis 1935) speaks against the possibility that dorsal root reflexes might have been the cause of the spontaneous activity recorded in our experiments.
Altered responsiveness of CMi units in the area of secondary hyperalgesia
Whereas all the type 1B units tested were insensitive to mechanical stimulation prior to remote capsaicin injection, a high proportion (11/17) became responsive afterward. These mechanically evoked discharges were usually very different in time course from those of the CM units. They were characteristically delayed and prolonged, and in some cases, resembled a resumption of spontaneous activity. Similar delayed responses were recorded by Schmelz et al. (2000b) from CMi units sensitized by local application of capsaicin to their electrical receptive field. The fact that the responses to mechanical stimuli are delayed and build up for a few seconds suggests an indirect mechanism in the mechanical response, most probably mediation by an algogenic chemical. The newly acquired mechanical responsiveness may be due to a heightened sensitivity to algogenic chemicals released from the skin by the noxious mechanical stimuli, and/or to an enhanced release of such substances from damaged skin rather than to acquired mechanosensitivity of the receptor membranes. However, the nature and origin of the hypothetical chemical mediator(s) are unknown.
We reported previously that some units fulfilling the definition of CMi units were activated spontaneously and became sensitized in areas of secondary hyperalgesia (Serra et al. 1995), but encountered the criticism that the activated or sensitized units might have been different from the ones recorded before the capsaicin injection. The use in this study of raster plots to follow the behavior of multiple single units for long periods avoids this potential criticism. On the other hand, the continuous stimulation of the CMi units incurs a potential risk that they may become sensitized by the electrical stimulation. Our recordings provided no evidence that this occurred: neither spontaneous discharges nor mechanically evoked responses were seen in any of the CMi units prior to capsaicin injection, even though we had in some cases already been stimulating electrically for over 1 h.
The behavior of CMi units to distant capsaicin injection was previously studied by Schmelz et al. (2000b), who also monitored the units by continuous stimulation at 0.25 Hz. They injected capsaicin into one part of the electrical receptive field and explored the behavior of the other parts of the receptive field that were not challenged directly by capsaicin. They found evidence of sensitization of the part of the unit where capsaicin had been injected, but no sensitization of adjacent parts of the electrical receptive field to which capsaicin had not spread. Our results appear to be in conflict, since Schmelz et al. (2000b) were testing CMi branches at distances from the capsaicin injection site well within the range where we have found frequent sensitization. The discrepancy could be due to their use of a lower dose of capsaicin (8–20 µg compare with our 100 µg). The lower dose was appropriate for limiting the number of units activated directly, whereas our higher dose is consistent with the amounts used in most psychophysical experiments on secondary hyperalgesia. Both studies are in agreement in showing that sensitization of CMi units in the area of secondary hyperalgesia is not due to impulses carried by axon reflexes in branches of the same units: Schmelz et al. (2000b) specifically activated distant branches of the same units and saw no sensitization, whereas we saw a discrepancy between activation and sensitization in four units. We infer that the sensitization is due to changes in the skin caused mainly by activity in CMi axons other than the one being stimulated electrically.
What is the role of CMi units in secondary hyperalgesia?
CMi UNITS AND THE SPREAD OF FLARE AND HYPERALGESIA. Several studies (LaMotte et al. 1991; Lewis 1935; Serra et al. 1998) have demonstrated that anesthetizing a narrow strip of skin can block the spread of hyperalgesia from the site of injury or capsaicin injection. Lewis (1935) postulated that a system of widely arborizing "nocifensor" fibers (probably without sensory function but with cell bodies in the dorsal root ganglion) mediated the spread by releasing chemicals in the skin. After a detailed psychophysical reinvestigation of capsaicin-induced secondary hyperalgesia, LaMotte et al. (1991) concluded that, although the spread of hyperalgesia is mediated by chemosensitive C afferents with long arborizing branches (or with functional coupling between pairs of fibers), the long-lasting facilitation must occur in the spinal cord.
Our new data, coupled with other recent studies of CMi units, show that they are excellent candidates to fulfill the role of Lewis's hypothetical nocifensor fibers or LaMotte et al.'s hypothetical chemo-sensitive fibers, even though they probably have a sensory function as well. First, CMi units are chemosensitive, activated directly by capsaicin (Schmelz et al. 2000b), and they arborize widely (Schmidt et al. 1998), so that they are well placed to take the capsaicin signal into the region of secondary hyperalgesia. Second, we have recorded from CMi units in the area of secondary hyperalgesia and found that they are activated, and the delays of 0.5–18 min are commensurate with the recorded delays in the spread of secondary hyperalgesia. Third, it is likely that CMi units are responsible for mediating flare (Schmelz et al. 2000a), and there is often a remarkably detailed correspondence between the area of flare (as described by Lewis (1935) or measured thermographically; Serra et al. 1998)) and the area of secondary hyperalgesia.
HOW DOES CMi UNIT ACTIVITY CONTRIBUTE TO SECONDARY HYPERALGESIA? The most conspicuous change in sensation in the area of secondary hyperalgesia is an increase in pricking pain to punctate stimuli, mediated by myelinated afferents (Ziegler et al. 1999). According to the models of LaMotte et al. (1991) and Ziegler et al. (1999), the role of CMi units in secondary hyperalgesia should be restricted to triggering or maintaining a central facilitation of A pathways. One reason why LaMotte et al. (1991) discounted a role of sensitized primary afferents in the secondary hyperalgesia was because no type of afferent with the required properties had been found (e.g., Baumann et al. 1991). Our demonstration that CMi units change their responsiveness in the area of secondary hyperalgesia reopens the controversial question of the extent to which peripheral mechanisms contribute to this phenomenon (Coderre and Katz 1997). Nevertheless, the demonstration of LaMotte et al. (1991), that punctate hyperalgesia is abolished if the capsaicin is applied during a proximal nerve block (even though the secondary hyperalgesia normally lasts much longer than the nerve block), and the evidence of Ziegler et al. (1999) from pressure blocks that this type of hyperalgesia depends on myelinated afferents, appear to exclude a significant role for CMi sensitization in the punctate secondary hyperalgesia.
What then are the likely sensory consequences of the changes in CMi responsiveness that we recorded? Lewis (1935) reported that, in the area of secondary hyperalgesia, needle pricks "give unusually intense, diffuse, and long-lasting pain." The newly acquired responses of CMi units to mechanical stimulation were mainly delayed and prolonged, and it seems likely that they contribute to the long-lasting component of the altered sensation, as previously argued for altered sensations at the site of capsaicin injection (Schmelz et al. 2000b). Also, since the CMi units arborize very widely, it is logical that they should only contribute to diffuse sensations. Sensitization of CMi units may also contribute to pathological hyperalgesia: a recent microneurographic study of patients with pain and hyperalgesia found C afferent fibers displaying activity-dependent slowing, similar to our type 1B units that were mechanically sensitive, and this responsiveness was regarded as evidence of sensitization of normally silent nociceptors (Orstavik et al. 2003).
The altered responsiveness of the CMi units observed in the area of secondary hyperalgesia implies that there was either an altered resting chemical environment in the skin or altered release of chemicals in response to noxious stimulation. The chemical(s) responsible are not known, but they appear not to affect the neighboring CM units (e.g., Fig. 10), in line with several reports that CM units respond normally in the area of secondary hyperalgesia (Baumann et al. 1991; Schmelz et al. 1996). However, it is not unlikely that some A nociceptors should have a similar chemical susceptibility to the CMi units and be able to contribute to the hyperalgesia. This possibility will be difficult to test, however, since A fibers are considerably more difficult to record by microneurography than C fibers, and their activity cannot be monitored so conveniently by activity-dependent slowing. This raises the possibility that there may be a secondary hyperalgesia to first pain (mediated by A fibers) and a secondary hyperalgesia to second pain (mediated by C fibers, in this case, mechano-insensitive C nociceptors). As far as we are aware, nobody has studied the differential behavior of first and second pain in the area of secondary hyperalgesia.
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CONCLUSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONCONCLUSIONACKNOWLEDGMENTSREFERENCES |
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), whose activation in the area of secondary hyperalgesia was proposed to lead to facilitation of A nociceptive pathways in the spinal cord. However, we also found that many of these units become responsive to noxious mechanical stimuli. Since CMi units are thought to have a nociceptive function (Schmelz et al. 2000b; Schmidt et al. 2000), this sensitization should contribute a direct, C fiber component to the secondary mechanical hyperalgesia. |
ACKNOWLEDGMENTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONCONCLUSIONACKNOWLEDGMENTSREFERENCES |
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This work was supported by National Institute of Neurological Disorders and Stroke Grant RO1 NS-39761.
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FOOTNOTES |
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Address for reprint requests and other correspondence: J. Serra, Unitat Dolor Neuropàtic, Hospital General de Catalunya, c. Josep Trueta s/n, 08190 Sant Cugat del Vallès, Barcelona, Spain (E-mail: jserrac{at}meditex.es).
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONCONCLUSIONACKNOWLEDGMENTSREFERENCES |
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, type 1A units; 
, type 1B units. Ellipses represent 95% confidence limits for a member of each group. B: effect of duration of repetitive stimulation on slowing in 30 type 1A and 26 type 1B fibers. Slowing (i.e., increase in latency, expressed as percentage of control) measured at 1 and 3 min after start of 2-Hz repetitive stimulation. Symbols connected by lines denote same unit. 
