Peripheral Coding of Tonic Mechanical Cutaneous Pain: Comparison of Nociceptor Activity in Rat and Human Psychophysics

David Andrew and Joel D. Greenspan

Department of Oral and Craniofacial Biological Sciences, University of Maryland Dental School, Baltimore, Maryland 21201


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Andrew, David and Joel D. Greenspan. Peripheral Coding of Tonic Mechanical Cutaneous Pain: Comparison of Nociceptor Activity in Rat and Human Psychophysics. J. Neurophysiol. 82: 2641-2648, 1999. These experiments investigated temporal summation mechanisms of tonic cutaneous mechanical pain. Human volunteers provided psychophysical estimates of pain intensity, which were compared with discharge patterns of rat cutaneous nociceptors tested with identical stimulus protocols. Human subjects made either intermittent or continuous ratings of pain intensity during stimulation of the skin between the thumb and first finger. Stimulus intensities of 25, 50, and 100 g were applied with a probe of contact area of 0.1 mm2 for 2 min. Pain perception significantly increased during stimulation (temporal summation) for the 50- and 100-g stimulus intensities. Sequential conduction block of the myelinated fibers supplying the stimulated skin was used to investigate the role of A-fiber mechanoreceptors and nociceptors in this temporal summation. Conduction block of the Abeta fibers resulted in an increase in mechanically evoked pain estimates and an increase in temporal summation, consistent with loss of Abeta -mediated inhibition. When only conduction in the unmyelinated fibers remained, pain estimates were reduced to the preblock levels, but temporal summation was still present. Electrophysiological recordings were made from filaments of the sciatic nerve supplying receptors in the plantar skin of barbiturate-anesthetized rats. Forty units fulfilled the identification criteria for nociceptors: 20 A-fiber and 20 C-fiber nociceptors. Each unit was characterized by recording its responses to graded mechanical and heat stimuli. Nociceptors were also tested with stimuli identical to those applied to the human subjects. The responses of all units to sustained mechanical stimuli were adaptive---that is, they exhibited a gradual decline in response with time. However, the time course of adaptation varied among units. All the C-fiber nociceptors and one-half of the A-fiber nociceptors had rapidly adapting responses. The remainder of the A-fibers displayed slowly adapting responses. One-third of all units also showed short-duration increases in firing rate during stimulation. The latency after stimulus onset of this rate acceleration was inversely related to stimulus intensity. Despite the apparent disparity between perceptual temporal summation and nociceptor adaptation, central and peripheral mechanisms are proposed that can reconcile the relationship between nociceptor activity and pain perception.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Adriansen et al. (1984) have described a novel sensory paradox: Long-duration noxious mechanical stimulation of human skin at constant intensity evokes a gradually increasing perception of pain despite prominent adaptation of cutaneous C-fiber nociceptors. Previous experiments on rat tail nociceptors have demonstrated that A-fiber nociceptors adapt more slowly than C-fiber nociceptors to such stimuli (Handwerker et al. 1987). However, the observation that some rat dorsal horn neurons maintain or even increase their firing rate during comparable stimulation (Cervero et al. 1988) is discordant with the responses of single nociceptors. The different time courses of C-fiber nociceptor and large-diameter mechanoreceptor adaptation led Adriansen et al. (1984) to propose a pattern theory of central integration of nociceptor and mechanoreceptor inputs, to explain the increase in perceived pain. An alternative central mechanism could be activity-dependent plasticity, involving summation of slow postsynaptic potentials from unmyelinated fibers ("wind-up"; Mendell and Wall 1965). However, recent experiments (Garell et al. 1996) have shown prominent differences in the mechanical encoding properties of A- and C-fiber nociceptors in the hairy skin of cat. In addition, the A fibers have heterogeneous mechanical response properties, suggesting that different functional groups of fibers may subserve separate roles for perception.

With this in mind, we have revisited the psychophysical and electrophysiological experiments of Adriansen et al. (1984) and Handwerker et al. (1987). In particular, we have addressed the following issues. 1) During long-duration mechanical stimulation of human skin does pain intensity increase significantly over time? 2) Can these perceptual changes be prevented by blocking conduction in one or more groups of myelinated afferent fibers? 3) Is there a population of A-fiber nociceptors that have mechanical adaptation properties more consistent with evoked pain intensity?


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Experiments were performed on 31 male Sprague-Dawley rats (300-400 g; Harlan, Indianapolis, IN) and on 18 human subjects (10 males, 8 females; aged 25-54). The experimental protocols for the animal studies were approved by the University of Maryland Dental School Animal Care and Use Committee. These experiments were performed on cutaneous nociceptors supplying the plantar (glabrous) skin of the rat hindpaw. This site was chosen for reasons of mechanical stability, because it could be rigidly fixed and was a relatively flat surface. The experiments involving human subjects complied with the Declaration of Helsinki and were approved by the Institutional Review Board of the University of Maryland, Baltimore. Each subject gave informed consent and was free to withdraw from the experiment at any time. Subjects were paid $15 (testing only) or $25 (testing with nerve block) per experimental session.

Anesthesia and preparation of animals

Rats were initially anesthetized with sodium pentobarbital (Nembutal, 50 mg/kg ip; Abbott, North Chicago, IL). Cannulae were inserted into the right jugular vein and carotid artery and into the trachea. Anesthesia was maintained with additional doses of pentobarbital (15-20 mg·kg-1 · h-1) administered intravenously. Anesthetic depth was maintained so that the animals were areflexic to pinching a paw. Blood pressure was recorded with a pressure transducer connected to the arterial cannula. Body temperature was maintained at 38.0 ± 0.5°C with an electric blanket controlled from a rectal thermistor. The animals were positioned prone and the hair over the dorsal surface of the right hindlimb clipped short. The foot was securely held, plantar surface uppermost, by fixing it with cyanoacrylate adhesive to a preformed matrix of thermoplastic dental impression compound (Impression Compound type 1; Kerr Manufacturing, Romulus, MI). The tibial and sciatic nerves were exposed by an incision extending from just proximal to the ankle to midthigh. To limit ongoing activity from other tissues the sural, gastrocnemius, and posterior biceps femoris and semitendinosis nerves were sectioned. The sciatic nerve was then cut as far centrally as possible and its proximal end placed on a plastic dissecting platform. A skin pool was formed from the surrounding tissues and filled with warm (38°C) mineral oil. The tibial nerve was freed from the surrounding tissues and placed on stimulating electrodes. The connective tissue surrounding the cut end of the sciatic nerve was slit with a chip of razor blade and fine filaments dissected from it with sharpened forceps. Pairs of filaments were placed on a pair of platinum wire electrodes (0.125-mm-diam), and differential recordings made from them.

Identification and characterization of nociceptors

Filaments were repeatedly subdivided until ideally only a single functional unit remained. More commonly, 2-4 units remained, and recordings were accepted only if the action potentials of the units were sufficiently different (e.g., size, polarity) to discriminate. Units were tested to determine their receptor type by stroking the skin with a camel's hair brush and by gentle squeezing with a pair of blunt forceps. A unit was classified as a nociceptor if it fulfilled the following criteria: 1) a sustained response to pinching a skin fold and 2) no response to brushing or gentle mechanical pressure with a blunt probe. Once a nociceptor was isolated, its latency at threshold and twice threshold was determined by constant current stimulation of the tibial nerve (0.1- or 0.5-ms stimulus duration, up to 10-mA intensity). The extent of a unit's receptive field was determined by squeezing small areas of skin with fine, smooth-tipped forceps (up to 10 stimuli per fiber, each of <1 s duration, repeated once every 5-10 s) and marked on the skin with ink. A glass probe with a smooth, curved tip (1-mm-diam) was also used similarly. Mechanical threshold was determined using calibrated monofilaments (Semmes-Weinstein; Stoelting, Chicago, IL). Threshold was defined as the minimum force that elicited impulses in 50% of trials. Once a unit had been characterized in this preliminary manner, no further stimuli were applied for 10-20 min to insure recovery from the preliminary testing protocol. All the nociceptors that were studied in detail had cutaneous receptive fields overlying the bones of the foot. Some nociceptors had to be discarded because the tissues beneath their receptive fields were unstable. These were usually on the tips and sides of digits, where controlled mechanical stimulation was impossible.

The responses of each unit were evaluated with graded mechanical stimuli (5, 10, 20, 30, 45, 60, and 90 g; equivalent to 49, 98 196, 294, 441, 588, and 882 mN) that were applied to the most sensitive region of a fiber's receptive field. Ramp-and-hold stimuli (ramp rate 75 g/s [735 mN/s], hold time 4.0 s, interstimulus interval 8.0 s) were delivered with a computer-controlled linear motor (Neurologic, Bloomington, IN; Barlow 1991) under force feedback regulation (model 501 motor controller; Biocommunication Electronics, Madison, WI) in order of increasing intensity. The probe tip of the stimulator was interchangeable, allowing stimuli to be delivered with probes of variable contact area (Garell et al. 1996). In the current study cylindrical probes with a flat tip of area 1.0 and 0.1 mm2 were used. Stimuli were always applied to the same spot within a fiber's receptive field. The adaptational properties of each unit were also studied. Stimuli of intensity 25, 50, and 100 g (245, 490, and 980 mN) were applied for 2 min with the 0.1-mm2 probe in order of increasing intensity. For the 2-min, 100-g stimuli, tissue compliance was measured by recording the probe's vertical position just before stimulus onset and again just before stimulus offset. These two values were subtracted to derive the amount of tissue displacement per 100-g stimulus force. To prevent nociceptor sensitization or desensitization, stimulus trials were widely separated in time (10-20 min). None of the units became spontaneously active during testing, indicating that sensitization did not occur within the time taken to evaluate the receptor properties of a fiber (~60-80 min). However, in those experiments in which more than one nociceptor was studied per animal, care was taken to ensure that their receptive fields were separated to preclude any possible effects of extended stimulation at one site. After the mechanical stimulation testing, the heat sensitivity of each unit was evaluated using an ascending series of temperatures (40-50°C in 2°C steps, from a baseline of 35°C), each applied for 5 s, with a 25-s intertrial interval.

At the end of the experiment, the animals were killed with an overdose of anesthetic (100 mg/kg iv pentobarbital sodium).

Human psychophysics

Twelve subjects were initially tested to reproduce the psychophysical findings reported by Adriansen et al. (1984) and to establish that pain scores increased significantly over time (temporal summation). Mechanical stimuli were applied to the skin of the digital web between the thumb and first finger of a subject's left hand. Here, the innervation is purely cutaneous/subcutaneous, because the tissue does not overlay deep tissues, such as bone or muscle. Each subject's hand was supported, palmar surface downward, with a contoured, hard plastic mold designed to separate the thumb and first finger. The mold also provided a firm base against which stimuli could be delivered, thus making the force-displacement relationship comparable with that of the animal experiments. Mechanical stimuli were delivered in a manner identical with those used in the rat experiments (25, 50, and 100 g applied for 2 min with a 0.1-mm2 probe). Two methods of intensity rating were used---intermittent and continuous. For intermittent rating, the subject used a potentiometer connected to a pair of finger holders. "No pain" was represented by apposing digits 1 and 2 ("closing" the device), whereas "intolerable pain" was defined as maximal separation of the two digits. Subjects rated their pain intensity during the first 10 s of the stimulus presentation and were then instructed to close the finger-span device. Subsequently, they were given acoustic cues every 15 s as a signal to rate their perceived pain intensity at that moment, and they then closed the finger-span device until the next cue. For continuous ratings, the subjects used a 100-point visual analog scale (VAS) presented on a computer screen. A computer mouse was used to adjust the height of an indictor bar on a vertical scale. Descriptors such as faint, mild, and moderate along the length of the scale were used to aid subjects in rating the intensity of their pain.

Eleven subjects also participated in a third experiment, when the contribution of afferent fiber categories to the mechanically evoked temporal summation of pain perception was investigated. Sequential conduction block of the large- and then small-diameter myelinated fibers was achieved by compression of the left superficial radial nerve (MacKenzie et al. 1975; Torebjörk and Hallin 1973). At the beginning of the experiment, a pair of stainless steel electrodes were fixed to the skin overlying the first dorsal interosseous muscle. The electrodes were of 7-mm2 contact area. They were separated by 10 mm from their centers and made electrical contact with the skin with conductive jelly (EC2 electrode cream; Grass, West Warwick, RI). Trains of constant current stimuli (200 Hz for 50-100 ms, pulse width 0.5 or 1.0 ms, repeated every 2 s) were applied between the electrodes with a photoelectric isolation unit (model SIU 7, Grass). Stimulus intensity was gradually increased (usually 1-5 mA) until the subjects reported a sharp pricking or stinging sensation typical of first pain. This sensation is considered to be mediated by finely myelinated (Adelta ) nociceptors (Campbell and LaMotte 1983; Collins et al. 1960; Lewis and Pochin 1937; Lundberg et al. 1992; Torebjörk and Ochoa 1990). Loss of the perception of electrically evoked first pain was used to indicate conduction block of the Adelta fibers. This method appears to be superior to that commonly used---the loss of cool perception---because there is evidence that the Adelta nociceptors are more resistant to compression block than are the Adelta cold-sensitive thermoreceptors and would therefore still be capable of impulse conduction at the point at which cool perception was lost (Ziegler et al. 1997).

Initial pain ratings were recorded to graded mechanical stimuli (5-90 g) delivered with a probe of 0.1-mm2 tip area. Ratings were obtained with a VAS presented on a computer screen. Continuous ratings of the pain evoked by a 50-g stimulus delivered for 2 min were also obtained. Differential conduction block of the fibers of the superficial radial nerve was achieved by placing a 2.5-cm wide band over the wrist, with 700-g weights attached at each end (total weight 1.4 kg). Psychophysical evaluation was repeated when the subjects could no longer feel light stroking of the skin with a camel's hair brush and then again when the sensation of electrically evoked first pain disappeared. Conduction in unmyelinated fibers was always confirmed by determining that warmth perception was still present. Seven of the subjects who participated in the nerve block experiments also provided verbal descriptors of the quality of their pain sensations. The sensory-descriptive component of the McGill short-form questionnaire was used. After each series of magnitude scaling and temporal summation testing, subjects were asked which of the following adjectives described their sensations: "throbbing," "shooting," "stabbing," "sharp," "cramping," "gnawing," "hot-burning," or "aching." Subjects also rated the magnitude of each applicable sensation by describing it as "mild," "moderate," or "severe."

Data analysis

Electrophysiological data were displayed on an oscilloscope, recorded on magnetic tape, and digitized with a computer interface (model 1401 plus; Cambridge Electronic Design, Cambridge, UK). Neural records were sampled at 50 kHz, and records of force and tissue displacement were sampled at 1 kHz. These data were analyzed off-line, using Spike 2 display and analysis software (Cambridge Electronic Design). Unpaired t-tests were used to make statistical comparisons between two different receptor types, and an ANOVA was used to make comparisons among multiple receptor types. In the case of comparing latencies, when the data did not pass a normality test at the 95% level, a Mann-Whitney rank-sum test was used.

Pain rating data were evaluated with repeated-measures ANOVA. To evaluate the effects of time on pain scores evoked by sustained mechanical stimulation, a repeated-measures ANOVA was used that included a specific test for a linear trend over time. Tukey's LSD test was used to compare groups to one another if the ANOVA revealed significant differences between groups. P < 0.05 was considered significant.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

General properties of rat glabrous skin nociceptors

Recordings were made from 40 nociceptors innervating the plantar skin of the rat hindpaw. Twenty of these were A-fibers and, 20 were C-fibers; none was spontaneously active. The conduction velocities (cv) of the A-fibers ranged from 5.0 to 32.0 m/s (16.5 ± 9.4 m/s, mean ± SD), those of the C-fibers were 0.8-1.5 m/s (1.1 ± 0.2 m/s). Mean monofilament threshold of the A-fiber nociceptors was 926 kPa (820 ± 379 kPa, median ± SD; range 520-1639; 100 kPa = 1 bar = 10 g/mm2), and that of the C fibers was 777 kPa (764 ± 432 kPa, range 156-1639). In general, tibial nerve nociceptor-receptive fields were similar to those described previously (Leem et al. 1993; Lynn and Shakhanbeh 1988). A-fibers had small multispot receptive fields separated by insensitive zones (n = 8) or were a single sensitive spot (n = 12). C-fiber-receptive fields were single spots. Sixteen of the C-fibers responded to heat as well as mechanical stimuli and were classified as C mechanoheat nociceptors. The remaining four were excited only by noxious mechanical stimuli and were designated mechanical nociceptors. Only one of the A-fibers was heat-sensitive in the range tested (up to 50°C), the remainder being exclusively mechanosensitive (high-threshold mechanoreceptors; Burgess and Perl 1967).

Responses to mechanical stimuli and adaptive properties of nociceptors

The responses of the A- and C-fiber nociceptors to mechanical stimulation with discrete forces applied with probes of 1- and 0.1-mm2 contact area are shown in Fig. 1. Nociceptor discharge frequency increased with stimulus intensity and also showed the expected relationship between firing and probe size. A-fiber nociceptors were more responsive than C-fiber nociceptors; that is, for a given force and probe size the mean firing rate of the A-fiber nociceptors was higher than that of the C-fiber nociceptors. In these respects rat glabrous skin nociceptors are similar to cat hairy skin nociceptors (Garell et al. 1996).



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Fig. 1. Mean responses of all A-fibers (A, n = 20) and all C-fibers (B, n = 20) to mechanical stimulation of their receptive fields (intensity 5-90 g, ramp rate 75 g/s, hold time 4.0 s, intertrial interval 8.0 s) applied with probes of 1- () and 0.1-mm2 (open circle ) tip area. Bars indicate SD.

The responses of units to sustained mechanical stimulation fell into one of two categories (Fig. 2): 1) those units that rapidly adapted to the stimulus (Fig. 2, B and C), or 2) units that adapted much more slowly (Fig. 2A). All the C fibers exhibited rapidly adapting responses to suprathreshold stimuli (e.g., Fig. 2C). After the dynamic response associated with stimulus onset, impulse generation was irregular and seldom exceeded 1 Hz (Fig. 3C). A-fiber nociceptors responded similarly to C-fiber units (A-rapidly adapting nociceptors, A-HT[RA], n = 10; Figs. 2B and 3B) or showed sustained firing throughout the duration of the mechanical stimulus (A-slowly adapting nociceptors, A-HT[SA], n = 10; Figs. 2A and 3A). Despite prominent differences in the adaptive properties of A-fiber nociceptors, neither their cvs (A-HT[RA] nociceptors: 19.2 ± 8.6 m/s, A-HT[SA] nociceptors 15.3 ± 7.3 m/s) nor their thresholds to monofilament stimulation (threshold for A-HT[RA] nociceptors 1005 ± 441 kPa; threshold for A-HT[SA] nociceptors 848 ± 317 kPa) were significantly different (cv: P = 0.3, unpaired t-test; threshold: P > 0.4, unpaired t-test).



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Fig. 2. Recordings of a slowly adapting A-fiber (A-HT[SA]) nociceptor (A, conduction velocity [cv] 18.5 m/s), a rapidly adapting A-fiber (A-HT[RA]) nociceptor (B, cv 7.4 m/s), and a C-fiber nociceptor (C, cv 1.2 m/s) to sustained mechanical stimulation with a probe of contact area 0.1 mm2 (diam 0.36 mm). Instantaneous frequency was determined by calculating the reciprocal of successive spike intervals.



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Fig. 3. Average responses to sustained mechanical stimulation of A-HT[SA] nociceptors (A, n = 10), A-HT[RA] nociceptors (B, n = 10), and C-fiber nociceptors (C, n = 20). Stimuli were applied for 2 min with a probe of tip area 0.1 mm2. Number of impulses evoked per 1-s bin were counted and plotted as spike density histograms. Inset: discharge rates of all A-fiber nociceptors in response to a 25-g, 120-s mechanical stimulus applied with a probe of tip area 0.1 mm2. The criteria used to classify units as A-HT[SA] or A-HT[RA] nociceptors was an instantaneous firing frequency of >= 5 Hz at 22.5 s after stimulus onset.

The major difference in nociceptor response across fibers to sustained mechanical stimulation was not dependent on differences in receptive field site; thus, it was not due to systematic variations in tissue compliance. Tissue displacement was measured for each stimulation site with the 100-g, 2-min stimulus. There were no significant differences among the mean tissue compliances of receptive fields of A-HT[RA] nociceptors (315 ± 87 µm/100 g, mean ± SD), A-HT[SA] nociceptors (344 ± 39µm/100 g), or C-fiber nociceptors (297 ± 70 µm/100g; P > 0.2, ANOVA).

The classification of A-fibers into slowly adapting and rapidly adapting groups was based on their instantaneous firing rate evoked by the 25-g, 2-min stimulus. Firing frequency was measured well after the dynamic component of their response was over (25 s after stimulus onset). The firing frequencies of all the slowly adapting A-fiber nociceptors were >= 5 Hz; those of the rapidly adapting nociceptors were much lower, usually <1 Hz. When the A-fiber nociceptors were divided into A-HT[RA] and A-HT[SA] nociceptors and their mechanical intensity-coding properties replotted, an obvious difference between the two groups was observed (Fig. 4). The response of A-HT[RA] nociceptors to stimulation with a probe of tip area 0.1 mm2 plateaued at stimulus intensities close to pain threshold reported for humans (median 35 g, 1st quartile; 25 g, 3rd quartile, 65 g with a probe of 0.1 mm2 tip area, Greenspan and McGillis 1991, 1994); whereas A-HT[SA] nociceptors continued to increase their discharge monotonically as stimulus intensity increased above pain threshold.



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Fig. 4. Average intensity responses of A-HT[SA] (A) and A-HT[RA] (B) nociceptors using probes of contact area of 1 () and 0.1 mm2 (open circle ). Bars indicate SD.

One frequent observation (shown in Figs. 2 and 3) was an abrupt increase in firing rate of a unit during the plateau phase of stimulation. This was observed in 14 units (6 C fibers, 6 A-HT[RA] nociceptors, and 2 A-HT[SA] nociceptors). There was no tendency for such units to differ in conduction velocity, threshold, or suprathreshold sensitivity from nociceptors that did not exhibit such increases in discharge rate. The increase in firing frequency was usually observed with the more intense stimuli (50 and 100 g), and its latency from stimulus onset was dependent on stimulus intensity (latency for 50-g stimulus: 70.4 ± 32.5 s, mean ± SD; latency for 100-g stimulus: 50.8 ± 24.9 s; P < 0.05, Mann-Whitney rank-sum test).

Human psychophysics and the effects of differential nerve block

Pain was reported by four of the subjects for the 25-g stimulus; by seven of them for the 50-g stimulus, and by all subjects for the 100-g stimulus (n = 12). Subjective magnitude scaling of the pain intensity showed that pain scores increased significantly over time for both the 50-g (P < 0.02, ANOVA) and 100-g forces (P < 0.001, ANOVA; Fig. 5). No significant increase in pain occurred with the 25-g force (P > 0.13, ANOVA). There was no significant difference between pain scores, depending on which method of scaling was used (intermittent versus continuous; P > 0.4, 2-factor ANOVA). After the initial dynamic response associated with stimulus onset, pain scores tended to increase smoothly (Fig. 5B), and there were no abrupt increases in pain ratings that may have been associated with rapid increases in nociceptor firing.



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Fig. 5. Mean pain ratings during mechanical stimulation of digital web skin for 2 min at 25 (), 50 (open circle ), and 100 g (). A: forces were applied with a probe of 0.1-mm2 tip area. Pain was rated using a finger-span device at 15-s intervals. Asterisks indicate pain scores significantly higher than the first rating (P < 0.05, ANOVA). Bars indicate SE. B: mean pain scores, as described in A. Pain intensity was recorded continuously with a mouse-controlled, computerized visual analog scale (VAS). B: arrow indicates stimulus offset.

Compression of the superficial radial nerve produced sequential conduction block of myelinated fibers. Loss of the perception of light touch occurred within 24-51 min (mean 39 min), and loss of the sensation of electrically evoked first pain took an additional 17-45 min (mean 27 min). The mean latency to achieve full A-fiber block was 64 min (range 41-90 min). Abolition of conduction in Abeta mechanoreceptors caused a significant increase in magnitude estimates of mechanical pain (Fig. 6A; P < 0.001, 2-factor ANOVA). Whereas magnitude estimates of the pain evoked by the same stimuli obtained after Adelta fiber block were not significantly different from controls (P > 0.05, 2-factor ANOVA). The rate of temporal summation of pain caused by sustained stimulation was assessed by fitting a power function to each subject's ratings versus time for the first 30 s of stimulation (i.e., during the time of most rapid perceptual change). The exponents of these power functions were not significantly different between control conditions and during conduction block of the Abeta fibers (t = 1.23; P > 0.20), or between control and Adelta block conditions (t = 0.69; P > 0.50; Fig. 6B). Thus, although the absolute pain ratings were significantly higher during the Abeta fiber block, the rate of pain increment over time was not found to be significantly different. Verbal descriptors taken after each series of psychophysical evaluations for seven subjects showed that the quality of the evoked sensations changed from sharp (42% of subjects) and stabbing (57% of subjects) during the control and Abeta block testing, to throbbing (71% of subjects), aching (42% of subjects), and, occasionally, burning (28% of subjects) after Adelta block.



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Fig. 6. The effects of conduction block on the magnitude scaling and temporal summation of mechanically evoked pain. A: magnitude scaling of the pain evoked by graded mechanical stimulation of digital web skin with a probe of contact area 0.1 mm2 (), and the effects of sequential conduction block of the large- (open circle ) and then small- () diameter myelinated nerve fibers on those ratings. Bars indicate SE. B: temporal summation of pain perception in response to maintained mechanical stimulation (50 g, 120 s), and the effects of progressive radial nerve block on the evoked pain. Pain ratings were obtained with a 100-point VAS presented on a computer screen.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

We have described the stimulus-response properties of rat glabrous skin nociceptors to mechanical stimuli and also their adaptive responses to sustained mechanical stimulation. The differential coding of both stimulus intensity and stimulus area (probe size) of A- and C-fibers is similar to cat hairy skin nociceptors (Garell et al. 1996) and monkey cutaneous nociceptors (Slugg et al. 1994), indicating that rat glabrous skin is a suitable mammalian model for mechanical nociception. There is also a similar distribution of nociceptor mechanical thresholds in rat, primate, and man (Lynn 1994) which further underscores the validity of this preparation. This is not the case for all aspects of nociception, because heat responses from rat myelinated nociceptors are rarely found, if at all, whereas in the monkey they are the norm. Among the A-fiber nociceptor population there is a group the mechanical adaptation properties of which are distinctly different from those of other A-fiber nociceptors and from C-fiber nociceptors. Although the responses of these units, which we termed A-HT[SA] nociceptors, are somewhat better related to human pain intensity than others, their discharge still adapts during stimulation while perceived pain increases. These A-HT[SA] units continued to increase their discharge in an approximately linear fashion as stimulus intensity increased; the remainder of the A-fiber nociceptor population (A-HT[RA] nociceptors) had stimulus-response functions that plateaued at stimulus intensities close to the average threshold for pain in humans (Greenspan and McGillis 1991, 1994). This plateau response and the rapid adaptation to long-duration mechanical stimuli indicates that these A-HT[RA] nociceptors are unlikely to contribute much to the painfulness of such stimuli.

Adaptation is a general property of sensory receptors (Adrian 1928), and it would have been surprising if we had observed units that increased their firing during sustained stimulation. In this respect peripheral mechanisms would be unlikely to explain the increase in pain that sustained mechanical stimulation causes. However, we observed primary afferent properties that could contribute to pain intensity increases with sustained stimulation. Thirty-five percent of nociceptors showed transient increases in their firing rates during long-duration stimulation (e.g., Fig. 2, A and B). This delayed excitation could contribute to temporal summation if several units, each showing delayed excitation at different latencies, converged onto the same central neuron that also received unmyelinated fiber inputs. However, it is unclear whether sufficient summation occurs for such events to influence CNS responses, and ultimately, perception. We did not observe any units that were excited at long latency by subthreshold stimuli (see Fig. 3, inset) as reported by White et al. (1991) in rat hairy skin. The difference between the results of White et al. (1991) and those reported here is partly technical. Even our weakest stimulus (25 g) was suprathreshold for almost all units. However, it may be that the delayed nociceptor excitation reported here is mechanistically similar to the long-latency excitation seen with subthreshold stimuli described by White et al. (1991).

The delayed excitation of some nociceptors may have been caused by one or more chemical mediators released from the surrounding tissues (e.g., prostaglandins, bradykinin), although this is unlikely, given the short duration of the effect. In addition, the long-latency, subthreshold excitation phenomenon reported by White et al. (1991) was not affected by prostaglandin E2, a known nociceptor-sensitizing agent (see Kress and Reeh 1996 and Levine and Taiwo 1994 for recent reviews). It remains to be determined whether other putative chemical modulators of nociceptor sensitivity, such as neuropeptides, produce similar effects on nociceptor responses as the kinins or prostanoids. Such agents may cause effects similar to the short-duration firing increases that were observed, given their short half-lives.

That pain perception still continued to increase during sustained stimulation when conduction in the Abeta fibers of the superficial radial nerve was blocked argues against the theory that perceptual temporal summation results from gradually changing Abeta -fiber input relative to Adelta - and C-fiber input (Adriansen et al. 1984). Indeed, pain perception approximately doubled after Abeta block, consistent with the well-known phenomenon of mechanoreceptor-mediated inhibition (Brown et al. 1973; Handwerker et al. 1975; Hillman and Wall 1969). Temporal summation was still evident when only the unmyelinated fibers were capable of impulse conduction. This summation could have both peripheral and central components. Delayed excitation of mechanically insensitive C-fiber nociceptors, including C-heat nociceptors, by tonic pressure has recently been reported in humans (Schmelz et al. 1997), with such units showing increasing firing rates during a response. Reeh et al. (1987) have reported recruitment of Adelta nociceptors in the rat's tail by long-duration mechanical stimuli applied with a large probe outside their receptive fields. We were unable to reproduce this finding, because, occasionally, stimuli were inadvertently applied to areas just outside a unit's receptive field because of small lateral movements of the stimulator's probe between stimulus blocks. Under these conditions the fiber under study was never excited by the stimulus. Also, there are substantial differences in method between the stimuli applied by Reeh et al. (1987) and those used in the current study, which may underlie the differing results. Summation of inputs by central neurons is also likely to be involved in mediating the increases in pain sensation that tonic mechanical stimuli of noxious intensity causes. Wind-up (Mendell and Wall 1965) of deep dorsal horn neurons, caused by summation of slow potentials from unmyelinated inputs, increases their responsiveness to subsequent afferent inputs for tens of seconds or even minutes (Woolf and King 1987; Woolf et al. 1988). There is also evidence of summation of A-fiber inputs. Cervero et al. (1988) reported that some dorsal horn neurons (both "wide dynamic range" and nociceptive-specific) increase their discharge rate during long-duration mechanical stimuli, regardless of whether they receive C-fiber inputs or not. Perhaps these cells, whose responses parallel our psychophysical data, receive strong inputs from A-HT[SA] nociceptors, many of which show the delayed excitation phenomenon.


    ACKNOWLEDGMENTS

This work was supported by National Science Foundation Grant IBN-9696127 and National Institute of Neurological Disorders and Stroke Grant NS-28559 to J. D. Greenspan. D. Andrew was a Senior International Scholar of the Fulbright Commission during this research.

Current address of D. Andrew: Division of Neurosurgery, Barrow Neurological Institute, 350 W. Thomas Rd., Phoenix, AZ 85013.


    FOOTNOTES

Address reprint requests to J. D. Greenspan, Dept. OCBS, Room 5-A-12, University of Maryland Dental School, 666 W. Baltimore St., Baltimore, MD 21201.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 26 August 1998; accepted in final form 16 July 1999.


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ABSTRACT
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0022-3077/99 $5.00 Copyright © 1999 The American Physiological Society