Effect of capsaicin treatment on nociceptors in rat glabrous skin one day after plantar incision

Abstract

Dilute capsaicin produces a differential effect on incision-related pain behaviors depending upon the test; it reduces heat hyperalgesia and guarding pain but not mechanical hyperalgesia. This suggests that common mechanisms for heat hyperalgesia and guarding pain occur, and distinct mechanisms exist for mechanical hyperalgesia. The purpose of the present study was to evaluate the effect of capsaicin treatment on the activity of cutaneous nociceptors sensitized by incision to understand the mechanisms for the selective action of dilute capsaicin on incisional pain. We compared the effect of 0.05% capsaicin vs. vehicle treatment on pain behaviors after incision and on the activity of nociceptors from these same rats using the in vitro glabrous skin-nerve preparation. Immunohistochemical expression of protein gene product 9.5 (PGP9.5), neurofilament 200, calcitonin gene related peptide (CGRP) and isolectin B4 (IB4) in skin was also evaluated 1 week after 0.05% capsaicin infiltration. Infiltration of 0.05% capsaicin decreased CGRP and IB4/PGP9.5-immunoreactivity of nociceptors in skin. The same dose of capsaicin that inhibited heat hyperalgesia and guarding behavior interfered with chemo- and heat sensitivity of C-fibers. Neither mechanical hyperalgesia nor mechanosensitivity of nociceptors was affected by capsaicin, suggesting that the concentration of capsaicin used in this study did not cause fiber degeneration. These results demonstrate that nociceptors desensitized by capsaicin contribute to heat hyperalgesia and guarding pain after plantar incision. These putative TRPV1-expressing C-fibers are sensitized to heat and acid after incision, and the transduction of heat and chemical stimuli after plantar incision is impaired by dilute capsaicin.

Fig. 1

Effects of dilute capsaicin infiltration on afferent fiber immunoreactivity. A: Example for measuring immunoreactive area in skin sections. (Aa): Confocal image of a skin section for calcitonin gene related peptide (CGRP) fluorescent immunohistochemistry. Six adjacent squares (300 μm × 300 μm for each) were selected bordering the epidermis and dermis. (Ab,c): In each square, the total immunopositive area was determined by manually tracing CGRP-immunoreactive fibers. The relative area (RA) of immunoreactivity was calculated by dividing the total immunopositive area by the total skin area. B: CGRP fluorescent immunohistochemistry in plantar skin (Ba,b) and subcutaneous tissue containing large nerve bundles (Bc,d) 1 week after subcutaneous infiltration of vehicle versus 0.05% capsaicin. CGRP-immunolabeled fibers in plantar skin are indicated by arrows (Ba,b). C: Isolectin B4 (IB4) and protein gene product 9.5 (PGP9.5) fluorescent double labeling immunohistochemistry in plantar skin (Ca,b) and subcutaneous tissue (Cc,d) after vehicle versus capsaicin treatment. Double-labeled fibers (yellow) in the epidermal-dermal junction are indicated by arrows (Ca). D: Neurofilament 200 (N52) fluorescent immunohistochemistry in plantar skin (Da,b) and subcutaneous tissue (Dc,d) after vehicle vs. capsaicin treatment. E-H: The RA of immunoreactivity of CGRP- (E), IB4/PGP9.5- (F), N52- (G), and total PGP9.5-positive fibers (H) in vehicle-treated and 0.05% capsaicin-treated skin. Data are expressed as mean ± SE. *P < 0.05 vs. vehicle by unpaired t-test. Epi = epidermi; Der = dermis; n = nerve; a = artery; v = vein; veh = vehicle; cap = capsaicin.

Fig. 2

Effects of capsaicin treatment on pain behaviors one day after plantar incision in rats undergoing in vitro primary afferent recording. A: Guarding pain score was significantly smaller in capsaicin group (n = 23, ○) compared to vehicle group (n = 26, ●) (*P < 0.01 vs. vehicle by unpaired t-test). B: Withdrawal threshold to von Frey filament application. C: Paw withdrawal latency to radiant heat was significantly greater in capsaicin group compared to vehicle group (**P < 0.001 vs. vehicle by unpaired t-test). Results in A and C are expressed as means (circles) ± SE (error bars). The data in B are expressed as median (horizontal line) with 1^st and 3^rd quartiles (boxes), and 10^th and 90^th percentiles (whiskers).

Fig. 3

Receptive fields and percentage occurrence of mechanosensitive nociceptors. A: Sample recording traces showing the action potential evoked by electrical stimulation to identify the fiber type by conduction velocity. Arrows indicate stimulus artifacts. Inset displays an enlarged view of the action potential. The conduction velocity values of the C- and Aδ-fiber shown here were 0.71 and 2.94 m/s, respectively. CV = conduction velocity. B: Distribution of the receptive fields of C- and Aδ-fibers for capsaicin- and vehicle-treated rats. Each circle represents the center of a unit's mechanoreceptive filed. The receptive field area was not quantified. C: Conduction velocity distribution histograms. D: Percentage of occurrence of C- and Aδ-fibers identified.

Fig. 4

Effect of capsaicin treatment on spontaneous activity of mechanosensitive afferent fibers one day after plantar incision. A: A sample recording of ongoing activity in a C-fiber from a capsaicin-treated rat. The average firing frequency in this example was 0.76 imp/s. The upper and lower panels show the digitized oscilloscope trace and spike density histogram (bin width = 1 s), respectively. Inset displays an enlarged view of the action potential. CV = conduction velocity. B: Prevalence of spontaneously discharge in C- and Aδ-fibers. C: Average activity of spontaneously discharging fibers. Bars represent mean and whiskers represent SE. Imp = impulse; cap = capsaicin; veh = vehicle. D: Distribution of the receptive fields of C- and Aδ-fibers with or without spontaneous activity, for capsaicin- and vehicle-treated rats. Solid circles represent receptive fields of afferent units with spontaneous activity and open circles represent those without spontaneous activity.

Fig. 5

Effect of capsaicin treatment on heat sensitivity of afferent units one day after plantar incision. A: Representative recording traces showing heat response of C-fibers. Capsaicin treatment decreased heat-responsiveness of C-fibers compared to vehicle infiltration. The upper, middle and lower panels show the spike density histograms (bin width = 1 s), digitized oscilloscope tracings and the heat stimuli applied, respectively. Insets display single action potential. CV = conduction velocity. B: Prevalence of heat-responsive fibers in C- and Aδ-fibers. *P < 0.001 vs. vehicle by χ² test. C: Distribution of the receptive fields of C- and Aδ-fibers with or without heat sensitivity, for capsaicin- and vehicle-treated rats. Solid circles represent receptive fields of heat-responsive afferent units, and open circles represent those of non-responsive units.

Fig. 6

Capsaicin treatment decreases heat sensitivity of C-fibers one day after incision. A: The percentage of heat-responsive units at each temperature in C-fibers. A decreased percentage of fibers in capsaicin group (n = 25, ○) respond to 37-48 °C heat compared to those in vehicle group (n = 29, ●) (*P < 0.05; **P < 0.01 vs. vehicle by χ² test). B: Comparison of threshold temperature for activation of C-fibers between treatment groups. *P < 0.05 vs. vehicle by Mann-Whitney test. C: Comparison of total evoked action potentials by heat-responsive C-fibers between treatment groups. *P < 0.05 vs. vehicle by Mann-Whitney test. In B and C, the horizontal line represents median and vertical line the interquartile range. D: Relation between spontaneous activity and heat sensitivity in C-fibers of vehicle group. The horizontal line represents mean and vertical line the SE. *P < 0.05 vs. SA- by unpaired t-test. SA+ = fibers with spontaneous activity; SA- = fibers without spontaneous activity.

Fig. 7

Effect of capsaicin treatment on the acid-responsiveness of afferent units one day after plantar incision. A and B: Sample recordings from two single C-fibers from a capsaicin- (A) and a vehicle-treated rat (B). The upper and lower panels show the digitized oscilloscope tracings and spike density histograms (bin width = 10 s), respectively. Insets display the action potentials of these units. Artifacts produced by placing and removal of the metal ring are marked by two black arrowheads and two white arrowheads, respectively. CV = conduction velocity. C: Percentage occurrence of acid-responsive units in C- and Aδ-fibers when tested with pH 5.5 lactic acid. * P < 0.05 vs. vehicle by χ² test. D: acid-evoked discharge rate of each acid-responsive C-fiber during lactic acid application. Bars represent median and whiskers represent interquartile range. Imp = impulse; cap = capsaicin; veh = vehicle. E: Distribution of the receptive fields of C- and Aδ-fibers with or without responsiveness to pH 5.5 lactic acid, for capsaicin- and vehicle-treated rats. Each circle represents the center of a unit's mechanoreceptive field. Solid and open circles represent receptive fields of afferents with and without acid-responsiveness, respectively.

Fig. 8

Effect of capsaicin treatment on mechanosensitivity of afferents one day after plantar incision. A: Sample recording traces showing responses to mechanical stimuli. Both C- and Aδ-fibers from either capsaicin or vehicle treatment groups have force-related increase responses to the ascending series of mechanical stimuli (40, 80, and 120 mN). CV = conduction velocity. B and C: The percentage mechanosensitivity at each force (B) and stimulus-response function (C) of C-fibers in capsaicin (n = 38, ○) vs. vehicle group (n = 39, ●). D and E: The percentage of mechanosensitivity (D) and stimulus-response function (E) of Aδ-fibers in capsaicin (n = 29, ○) vs. vehicle group (n = 22, ●).