Chemosensitivity and mechanosensitivity of nociceptors from incised rat hindpaw skin

Abstract

Background: The authors have demonstrated a decrease in pH in the incisional wound environment, suggesting a possible contribution of low pH to postsurgical pain. In this study, the authors characterized the acid-responsiveness of nociceptors innervating the plantar aspect of the rat hind paw 1 day after plantar incision and compared this to plantar skin from unincised control rats.

Methods: Using the rat glabrous in vitro skin-tibial nerve preparation, afferent nerve activities from single mechanosensitive nociceptors were recorded. Differences in mechanosensitivity, spontaneous activity, and chemosensitivity of units were evaluated. For chemosensitivity, acid-responsiveness of nociceptors to lactic acid (pH 5.5 to 6.5) was studied.

Results: C-fibers showed dose-dependent, sustained responses to lactic acid. A greater proportion of C-fibers from 2 mm or less from the incision was activated by pH 6.0 lactic acid (52.9%) compared to control (14.3%). Total evoked potentials during acid exposure were greater in C-fibers innervating 2 mm or less from the incision compared to those in unincised skin. The prevalence of acid responses and total evoked potentials during acid exposure in C-fibers innervating more than 2 mm from the incision were not different from control. Few A-fibers responded to lactic acid, with a range of pH 5.5 to 6.5 in both incision and control groups. Increased spontaneous activity and mechanosensitivity were also evident.

Conclusions: C-fibers in the vicinity of the incision showed qualitatively and quantitatively greater chemosensitivity to pH 6.0 lactic acid compared to control. This change was localized to 2 mm or less from the incision, suggesting increased chemosensitivity of nociceptive C-fibers 1 day after plantar incision.

Fig. 1

Sample recordings showing the experimental protocol used to test the acid- responsiveness of nociceptors one day after incision. Responses of two single C-fibers with receptive fields ≤ 2 mm from the incision to pH 6.0 lactic acid (A) and to control Krebs–Hensleit solution (B). The upper and lower panels in each figure 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. Black arrows indicate manual mechanical stimuli applied to the receptive fields of the unit. CV = conduction velocity.

Fig. 2

Location and percentage of nociceptors responsive to pH 6.0 lactic acid one day after incision. (A): Distribution of the receptive fields of C- and A-fibers with or without responsiveness to pH 6.0 lactic acid, for incised and sham control 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. (B, C): Percentage occurrence of acid-responsive units in C-fibers (B) and A-fibers (C) when tested with pH 6.0 lactic acid (* P < 0.05 vs. sham control, Fisher’s exact test). No units were excited by control Krebs–Hensleit solution. Inc = incision; Cont = control.

Fig. 3

Summary of the magnitude of the C-fiber response to pH 6.0 lactic acid. (A–C): Sample recordings from three single C-fibers innervating ≤ 2 mm from the incision (A), > 2 mm from the incision (B) and sham control skin (C). 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. (D–F): Mean spike density histograms of C-fibers innervating ≤ 2 mm from the incision (D, n = 9), > 2 mm from the incision (E, n = 3) and sham control skin (F, n =3). If the unit was spontaneously active before acid exposure, background activity was subtracted from the raw response data for each bin (bin width = 10 s). (G–I): Mean discharge rate of each acid-responsive C-fiber ≤ 2 mm from the incision (G), > 2 mm from the incision (H) and sham control (I) is shown. Five min periods before, during and after lactic acid application were used to calculate the mean discharge rate. Imp = impulse.

Fig. 4

Repeated application of pH 6.0 lactic acid to acid-responsive C-fibers ≤ 2 mm from the incision. (A): Sample recording from one single C-fiber. After a 5-min baseline, pH 6.0 lactic acid (15 mM; 32 °C) was repeatedly applied (three times) for 5 min at 5-min intervals, followed by 5-min washout. The upper and lower panels show the digitized oscilloscope tracings and spike density histograms (bin width = 10 s), respectively. Inset displays the action potential of this unit. CV = conduction velocity. (B): Mean discharge rate of each acid-responsive C-fiber ≤ 2 mm from the incision during repeated application of pH 6.0 lactic acid. The solid circles represent data from fiber in (A). Five min periods of baseline, acid application and washout were used to calculate the mean discharge rate. All five units were spontaneously active before acid exposure, and background activity was subtracted from the response data afterwards. Imp = impulse.

Fig. 5

Summary of chemical responses of nociceptors to lactic acid of three different pH levels. (A): Sample recordings from one single C-fiber innervating ≤ 2 mm from the incision. After a 5-min baseline, 15 mM lactic acid with pH 6.5, 6.0 and 5.5 was sequentially applied for 5 min, followed by 5-min washout. The interval between each acid application was 15 min. The upper and lower panels show the digitized oscilloscope tracings and spike density histograms (bin width = 10 s), respectively. Inset displays the action potential of this unit. CV = conduction velocity. (B, C): Prevalence of acid-responsive units in C-fibers (B) and A-fibers (C). (D): Mean discharge rate of each acid-responsive C-fiber during application of 15 mM lactic acid with three different pH levels. Imp = impulse.

Fig. 6

Spontaneous discharge of mechanosensitive nociceptors one day after plantar incision. (A): Distribution of the receptive fields of C- and A-fibers with or without spontaneous activity. Solid circles represent receptive fields of afferent units with spontaneous activity, and open circles represent those without spontaneous activity. (B): Prevalence of spontaneous discharge in C- and A-fibers (* P < 0.01 vs. sham control, Fisher’s exact test). (C): Relationship between spontaneous activity and acid sensitivity in C-fibers from incised rat. SA = spontaneous activity.

Fig. 7

Mechanical responses of afferent units one day after plantar incision. (A): Sample recordings of the mechanical responses of a control C-fiber to the ramp-shaped force stimulus. The upper and lower panels show the digitized oscilloscope tracings and the force stimulus applied, respectively. The mechanical threshold of this unit was 16.4 mN. Inset displays the action potential of this unit. CV = conduction velocity. (B): Responses of the same unit to the ascending series of sustained force stimuli, showing greater discharge response to higher force stimuli. The upper, middle and lower panels show the spike density histograms (bin width = 1 s), raw spike discharges and the force stimuli applied, respectively. (C, D): The percentage mechanosensitivity (C) and stimulus-response function (D) of C-fibers in sham control (n = 27), ≤ 2 mm from the incision (n = 24) and > 2 mm from the incision (n = 17) (* P < 0.05 vs. sham control, one-way ANOVA followed by Scheffé post hoc test). (E, F): The percentage of mechanosensitivity (E) and stimulus-response function (F) of A-fibers in control (n = 20), ≤ 2 mm from the incision (n = 12) and > 2 mm from the incision (n = 11) (* P < 0.05 vs. control, Fisher’s exact test).