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Research Paper

Slow depolarizing stimuli differentially activate mechanosensitive and silent C nociceptors in human and pig skin

Rukwied, Romana; Thomas, Christiana; Obreja, Otiliaa; Werland, Fionaa; Kleggetveit, Inge Petterc; Jorum, Ellenb; Carr, Richard W.a; Namer, Barbarad,e; Schmelz, Martina,*

Author Information
doi: 10.1097/j.pain.0000000000001912

1. Introduction

Classification of primary afferents has been traditionally based on morphology and sensory function; however, recent analysis of single-cell expression patterns of neurons23,62,66 provided data from a different perspective suggesting a new classification with 10 to 14 molecularly defined subpopulations of sensory afferents.31,32 Unfortunately, there is still a gap between neuronal classes defined by the expression patterns in their somata and functional classes determined by their sensory characteristics and spinal connectivity. Moreover, species differences add another level of complexity when translating functional and molecular findings obtained in rodents to neuronal classes in human.21,47,67 Subpopulations of primary afferents can elegantly be linked to their sensory function by optogenetic approaches in rodents, given that specific neuronal markers allow for their specific activation.11,44 In humans, by contrast, other techniques may be used such as microstimulation of single characterized myelinated mechanoreceptors.3,48,60,61 However, electrical stimulation of unmyelinated C fibers is problematic as several axons of these fibers are located in close association within a Remak bundle. The administration of C-fiber-specific chemical agonists, as demonstrated, for example, by histamine iontophoresis to activate C fibers involved in itch,50 is not available yet for other classes of C nociceptors. Recently, transcutaneous electrical sine wave stimulation in humans was shown to selectively activate C fibers.24 However, both mechanosensitive and mechanoinsensitive (“silent”) nociceptors were activated by this stimulus. These 2-fiber classes can be differentiated by their characteristic axonal properties. In particular, activity-dependent slowing of conduction has been used to characterize functional classes of nociceptors, originally demonstrated in the rat19 and later shown to separate silent from mechanosensitive nociceptors in humans52,53 and pig.40 Functionally, the spontaneous activity in silent but not mechanosensitive nociceptors has been found to correlate with ongoing pain in patients with chronic pain conditions,28,55 and silent nociceptors evoke the axon reflex erythema in humans.51 By contrast, discharge in mechanosensitive nociceptors has been linked to heat pain threshold and suprathreshold pain ratings of contact heat30,59 in humans and might thereby play a role in inflammatory heat hyperalgesia. Thus, a differential stimulation protocol for activation of these nociceptor classes would be useful to investigate their functional role in clinical pain conditions.

We hypothesized that long-lasting depolarizing stimuli (500 ms) would generate bursts of action potentials (APs) like in tetrodotoxin (TTX)-sensitive dorsal root ganglion cells in humans10 and rodents1 probably based on ramp currents through TTX-sensitive voltage-gated sodium channel subtypes NaV1.79 or NaV1.3.15 Modelling results suggest predominant expression of NaV1.7 in mechanosensitive and NaV1.8 in silent nociceptors.45,56,57 Thus, mechanosensitive nociceptors might show more pronounced bursting upon long-lasting depolarization than silent nociceptors, considering that NaV1.7 can produce substantial ramp currents during slow depolarizations due to characteristic slow-inactivation kinetics.9 However, thresholds for the first AP were similar between mechanosensitive and silent nociceptors upon 4-Hz sine wave stimulation24 speaking against a major role of ramp currents for this stimulation profile. We combined psychophysics and axon reflex measurements in volunteers with single-fiber recordings of classified nociceptors to investigate different response pattern between the fiber classes.

2. Methods

Ethical approval for animal experimental procedures was issued by the Ethics committee of the regional government (Karlsruhe, Baden-Wuerttemberg, Germany). Procedures followed the guidelines for animal research of the European Union.16 Ethical approval for human studies was obtained from the regional ethics committees of the Universities of Heidelberg and Nuremberg.

2.1. Experimental protocols

For surgery, pigs (n = 35; 21-26 kg) were premedicated as described previously40 with intramuscular injection of azaperone (Stresnil; Janssen Pharmaceutica, Beerse, Belgium) 2 mg/kg, atropine (Eifelfango, Bad Neuenahr, Germany) 0.015 mg/kg, and midazolam 1 mg/kg. General anesthesia was induced with Propofol (Fresenius, Bad Homburg, Germany) 2 mg/kg intravenously and maintained with pentobarbital (Narcoren; Merial, Halbergmoos, Germany) 8 to 14 mg/kg/h. Pigs were intubated, ventilated, and vital parameters (heart rate, O2 saturation, rectal temperature) were monitored.

2.1.1. Extracellular single-fiber recordings in pig

Saphenous nerves were exposed at midthigh over a length of about 6 cm, and the teased fiber technique7,20 was used to extracellularly record APs from single nerve fibers (for detail see Ref. 40). Signals were amplified (Model 5113; Ametek Inc, TN), audio monitored, filtered (Model 3364; Krohn-Hite Corp, Brockton, MA), and displayed on an oscilloscope. An electrical search strategy was used to identify individual C units. Electrical stimuli (20 mA; 0.5 ms) were generated at 0.25 Hz by a constant current stimulator (DS7A; Digitimer Ltd, Hertfordshire, United Kingdom) and applied to the skin through 2 noninsulated microneurography electrodes (FHC Inc, Bowdoin, ME). Needles were inserted intradermally at sites where time-locked, electrically evoked APs with long latencies (∼100-200 ms) could be elicited. Current intensity was adjusted at 1.5 times the electrical threshold. The shortest distance between the stimulation needles and the recording electrode was measured and divided by the latency recorded after a 2-min pause to calculate the resting conduction velocity (CV). All fibers in this study had CV values <2 m/s. Action potentials were amplified, processed online, and displayed on a computer using DAPSYS 8.0, a joint hardware and software system designed for real-time acquisition, window discrimination, and latency measurements of the APs (for technical details, see www.dapsys.net).

2.1.1.1. Characterization of C-fiber classes

Single C fibers were classified according to their responses to mechanical stimulation (brush, v.Frey filaments 10-600 mN) into 3 classes: low-threshold mechanosensitive (LT) afferents (brush positive), mechanosensitive (CM) nociceptors (brush negative, mechanical threshold between 10 and 150 mN), and mechanoinsensitive (CMi) nociceptors (mechanically insensitive). Slowing of conduction to repetitive electrical stimulation at 2 Hz for 3 minutes was assessed after a 2 minutes pause to clearly identify CMi-units.40

2.1.2. Electrical stimulation with half-sine wave

Half-sine wave pulses of 500-ms duration (1 Hz) were generated at intensities of 0.01 to 10 mA by a constant current stimulator (A 395; WPI, Sarasota, FL) controlled by DAPSYS 8 (www.dapsys.net). Stimuli were delivered by a pair of L-shaped blunted bipolar platinum–iridium electrodes (diameter 0.4 mm, distance 2 mm; Cephalon, Nørresundby, Denmark) placed on a length of 3 mm onto the skin surface (suppl. Figure 1, available at http://links.lww.com/PAIN/B12) within the innervation territory of characterized C fibers. Initially, amplitudes of 0.2, 0.4, 0.6, 0.8, and 1 mA were applied with one repetition for each stimulation intensity and interstimulus intervals of 10 seconds. If the units were already activated by the lowest intensity, we also tested 0.01, 0.02, 0.04, 0.06, 0.08, and 0.1 mA. In case of negative responses to 1 mA, higher stimulation intensities of 2, 4, 6, 8, and 10 mA were applied.

Apart from half-sine wave stimulation threshold, we assessed the time point of the 500-ms stimulation period at which the first AP had been generated (“time to first AP,” Table 1) and calculated the first derivative at this time (“slope at first AP,” Table 1) as well as the charge delivered (area under the curve of the half-sine stimulation from start to first spike initiation). We also recorded the peak discharge frequency of APs by calculating the mean of the 3 highest instantaneous frequencies with at least 5 APs generated by the half-sine stimulus and determined the corresponding time of the half-sine cycle (“peak frequency” and “time of peak frequency,” Table 1).

Table 1
Table 1:
Summary of different characteristics of C-fiber classes in the pig (total number of neurons in first row).

2.1.3. Microneurography in healthy subjects

Single nerve fiber recordings were obtained from superficial peroneal nerve fascicles in 9 healthy human subjects (5 women, 4 men, 28 ± 7 years) and nociceptors classified according their CV, their responsiveness to mechanical stimuli (“marking”), and their activity-dependent latency shift upon repetitive electrical stimulation, as described before in detail.65 Half-sine wave pulses were delivered within the innervation territory of the characterized C nociceptors through epicutaneous bipolar electrodes using a Digitimer DS5 constant current stimulator (Welwyn Garden City, Hertfordshire, United Kingdom) and pulse generator (NI USB-6221; National Instruments, TX) controlled by Dapsys 8 (www.dapsys.net) at intensities of 0.1-, 0.2-, 0.4-, 0.6-, 0.8-, and 1-mA amplitude. Nerve fiber activation was assessed by the characteristic slowing of CV (“marking”).

2.1.4. Psychophysics and axon reflex flare in healthy subjects

Half-sine wave pulses were delivered through epicutaneous bipolar electrodes at intensities of 0.2-, 0.4-, 0.8-, and 1-mA amplitude to the volar forearm skin of 15 male and 15 female healthy volunteers (28 ± 4 years), as described above. In 14 subjects, pain (numeric rating scale [NRS], 0-10) ratings were assessed in 2 sessions with 1-week interval and 5 repetitions for each stimulus intensity. In the first of the 5 repetitions, the intensities were applied in ascending order to reaccommodate the subjects to the stimuli. In the following 4 repetitions, stimulus intensities were applied in random order (single blinded, randomization generated in Excel with Excel's sorting feature and RAND formula). Subjects were also asked to describe the quality and the temporal profile of the sensation in their own terms. In addition, perception and pain thresholds to half-sine wave pulses were recorded in 24 healthy subjects (9 men, 15 women; 39 ± 15 years) by increasing the half-sine current in steps of 0.01 mA in 10-second intervals. Volunteers were instructed to indicate when the stimulus was felt and when it was perceived as painful (measurements were performed in duplicate).

In addition, axon reflex flare responses were compared between sine and half-sine stimuli directed to the volar forearm in 10 subjects (7 men, 3 women; 37 ± 14 years). Superficial blood flow of the forearm was repetitively recorded by laser Doppler imaging (Moor Instruments Ltd, Millwey, United Kingdom) within a skin area of 7.5 × 15 cm requiring 1 minute for image capture. Following skin blood flow baseline recording, half-sine (10 half-sine pulses, 0.4 mA at 2-second intervals) and 4-Hz sine wave stimulation (0.2 mA, 20 seconds) were administered subsequently at a distance of 8 cm. The stimulation order was randomized and timed such that the delay between stimulation and laser Doppler recording was matched between the 2 stimulation sites. Laser Doppler blood flow imaging was continued for 10 minutes, and the area of the widespread axon reflex vasodilation was analyzed offline. We assessed the number of pixels that exceeded the baseline blood flux by twofold SD around the individual stimulation sites and calculated the area. Pain intensity perceived by the subject during half-sine and sine wave stimulation was recorded (numerical rating scale, 0-10).

2.2. Statistics

Statistical tests were performed in STATISTICA 7.1 (StatSoft Inc, Tulsa, OK). Responses to half-sine waves (electrical thresholds, peak frequencies, delay of first evoked AP, etc.) were compared between C-fiber classes by one-way analysis of variance. Bonferroni-corrected t-tests were used for post hoc analysis. Linear correlations were calculated between stimulation intensity and discharge or latency of first evoked AP. Group data are presented as mean ± SD or arithmetic mean from averaged data ± SEM.

3. Results

3.1. In vivo recordings in the pig

We obtained recordings from 28 mechanosensitive nociceptors (CV 1.02 ± 0.29 m/s; mean ± SD), 19 mechanoinsensitive “silent” nociceptors (CV 0.87 ± 0.32 m/s), and 14 low-threshold mechanosensitive C fibers (“C-touch”) (CV 1.41 ± 0.84 m/s). The electrical thresholds assessed by rectangular pulses (0.5 ms duration) applied through bipolar intracutaneous needle electrodes did not differ between C-touch and mechanosensitive nociceptors (1.9 ± 2.7 mA vs 1.7 ± 0.8 mA) but were higher in silent nociceptors (5.8 ± 4.5 mA; P < 0.01, Bonferroni-corrected post hoc test).

Half-sine wave stimulation activated all C-touch fibers and mechanosensitive nociceptors with thresholds being significantly lower in the C-touch fibers (0.11 ± 0.18 vs 0.42 ± 0.30 mA; P < 0.01, Bonferroni, Table 1). Mechanosensitive nociceptors intensity-dependently responded with increasing numbers of APs (Fig. 1A, r = 0.37, linear correlation; P < 0.001) in the stimulation interval between 0.2 and 1 mA. Based on their lower activation thresholds, all C-touch fibers responded to the half-sine waves between 0.2 and 1 mA, but their vigorous response recorded even at 0.2 mA did not increase further with higher current intensity up to 1 mA (r = 0.10, P > 0.2).

Figure 1.
Figure 1.:
(A) Specimen of a mechanosensitive nociceptor and a low-threshold mechanosensitive C fiber (C-touch) responding with action potential discharge to 500-ms half-sine electrical stimulation with increasing amplitude between 0.2 and 1.0 mA (each amplitude was tested twice) in their receptive field (upper panels). The conduction delay to single suprathreshold electrical rectangular pulses has been subtracted such that the delay between start of the half-sine stimulus and the initiation of action potentials is shown. In the lower panel, the number of action potentials provoked by increasing amplitudes is shown for mechanosensitive nociceptors (red) and C-touch fibers (black, dotted line represents linear correlation). (B) Specimen of a silent nociceptor responding with action potentials discharge to 500-ms half-sine electrical stimulation as in (A), but at intensities delivered from 2 to 10 mA. In the lower panel responses of all recorded silent units (n = 19) are shown. Note that most of the fibers remained unresponsive even to 10 mA (dotted line represents linear correlation).

By contrast, silent nociceptors had much higher thresholds to half-sine wave stimulation with 14/19 being unresponsive even at amplitudes of 10 mA (Fig. 1B). The remaining 5 silent fibers had thresholds of 0.8, 1, 4, 6, and 8 mA and responded in intensity-dependent manner (r = 0.19, P < 0.02).

At low stimulation intensities between 0.01- and 0.1-mA mechanosensitive nociceptors showed considerable activation around 0.1 mA but only spurious discharges at intensities below 0.1 mA (Fig. 2). By contrast, C-touch fibers displayed a clear intensity-dependent increase of discharge in this stimulation range (0.01-0.1 mA). Notably, only 1 of 14 C-touch units had activation thresholds above 0.2 mA. Correspondingly, on a single fiber-level C-touch fibers had the highest linear correlations between stimulus intensity and evoked AP number in the lower stimulation range (r = 0.86 ± 0.07 [mean ± SD]; 9 significant correlations in 14 C-touch fibers: P = 0.001 ± 0.002) as compared to the higher stimulation range of 0.2 to 1 mA (r = 0.75 ± 0.14 [mean ± SD]; 6 significant correlations in 14 C-touch fibers: P = 0.014 ± 0.02). Single mechanosensitive nociceptors showed the opposite pattern with higher linear correlation coefficient in the stimulation range between 0.2 and 1 mA (r = 0.86 ± 0.1, n = 19) compared with low stimulation intensities between 0.01 and 0.1 mA (r = 0.79 ± 0.1, n = 6).

Figure 2.
Figure 2.:
Linear correlation (dotted line) between low current intensities of the half-sine wave stimulus (0.01-0.1 mA) and the number of evoked action potentials in mechanosensitive nociceptors (red) and C-touch fibers (black). Note that most mechanosensitive nociceptors responded with considerable activation at around 0.1 mA, whereas most of the C-touch fibers are already activated at around 0.02 mA.

At suprathreshold stimulation intensities (0.2-1 mA), the delay from the start of the half-sine stimulus to the initiation of the first AP progressively declined for both, C-touch fibers (r = −0.17, P < 0.01) and mechanosensitive nociceptors (r = −0.3, P < 0.0001, suppl. Figure 2A and C, available at http://links.lww.com/PAIN/B12). The charge delivered at the time of initiation of the first AP did not increase for higher stimulation intensities (suppl. Figure 2B and D, available at http://links.lww.com/PAIN/B12). These findings indicate that the threshold for AP generation during half-sine wave stimulation is determined mainly by the applied charge.

The delay between onset of half-sine wave stimulation and initiation of the first AP at threshold intensity was shorter in C-touch and mechanosensitive nociceptors (81.4 ± 42 and 97.2 ± 35 ms; Table 1 “time to first AP”) as compared to silent nociceptors (154.4 ± 51 ms, n = 5; threshold intensity 9.5 mA, P < 0.01 Bonferroni post hoc). The slope of the stimulus (first derivative) at which the first AP was induced was significantly lower in C-touch and mechanosensitive fibers (4.1 ± 8 and 9 ± 10 mA/s) as compared to the 5 activated silent nociceptors (31 ± 2 mA/s, P < 0.001 Bonferroni). Peak frequencies did not differ between C-touch and mechanosensitive nociceptors (51.8 ± 23.5 vs 52.0 ± 58.1 Hz) but were lower for silent nociceptors (27.6 ± 6.6 Hz; n = 5; P < 0.05 Bonferroni post hoc, Table 1). The delay from start of the stimulus to peak frequency did not differ between C-touch, mechanosensitive, and silent nociceptors (163.3 ± 107 ms, 171.9 ± 101 ms, 196.5 ± 52 ms; n.s. Table 1).

It is important to note that the response patterns of the electrically evoked discharge upon increased stimulation intensity fundamentally differed between the 1-Hz half-sine and the previously reported24 repetitive 4-Hz stimulation. Stronger half-sine stimulation evoked discharges at higher instantaneous frequencies with silent nociceptors requiring about 10-fold higher currents (Figs. 3A and B). By contrast, single sine wave stimulation at 4 Hz typically evokes just one phase-locked AP. Upon increasing the intensity of repetitive 4-Hz stimulation, the number of successful stimulations increased such that the response finally reaches an instantaneous frequency of 4 Hz (Figs. 3C and D). The activation thresholds for the 4-Hz stimulus were found only slightly higher in silent vs mechanosensitive nociceptors.24

Figure 3.
Figure 3.:
Comparison of instantaneous discharge frequencies to half-sine wave (A and C) and sine wave stimuli (B and D) in a mechanosensitive (red, top panels) and a mechanoinsensitive (blue, lower panels) C nociceptor. Single half-sine wave pulses intensity dependently evokes bursts of action potentials. Note that mechanoinsensitive (“silent”) nociceptors require about 10-fold higher currents for activation (C). At low current intensity (0.2 mA), sinusoidal stimuli of 4 Hz and 3-second duration evoked a single action potential. At higher intensities (0.3-0.4 mA) both C-nociceptor classes tend to show regular 4-Hz discharges (usually phase locked to the sine wave). Bursts of action potentials—as seen upon single half-sine wave stimuli—were observed in mechanosensitive nociceptors upon repetitive and suprathreshold sinusoidal pulses (0.5 mA, B).

3.2. Human microneurography

We recorded 24 nerve fibers, classified as mechanosensitive C nociceptors (CM, n = 13) and mechanoinsensitive (silent) C nociceptors (CMi, n = 11). Responsiveness to increasing intensities of electrical half-sine stimulation (0.1-1 mA) was assessed through bipolar epicutaneous electrodes placed inside the innervation territories of the recorded units. Half-sine wave induced activation of the unit under study was verified by induction of an increased response latency (“marking”)58 to subsequent regular square pulse stimulation (specimen of mechanosensitive nociceptors and a silent nociceptors depicted in Fig. 4). About 85% (n = 11) of mechanosensitive nociceptors were activated by the half-sine stimulus with a mean threshold of 0.3 ± 0.1 mA. Increasing stimulation intensities of the half-sine stimulus evoked markings of increasing magnitude (specimen Fig. 4A) of on average 1.4 ms at 0.1 mA (n = 11) up to 9.4 ms at 1 mA (tested in only n = 7 for technical reasons). Notably, 2 mechanosensitive nociceptors, however, remained unresponsive even to 1 mA. By contrast, only 1 of 11 silent nociceptors was activated by the maximum stimulation intensity (specimen Fig. 4B). This silent nociceptor was activated at a threshold of 0.8 mA and showed a slight marking of 1 ms that is expected after firing of 1 to 2 APs (not shown).

Figure 4.
Figure 4.:
Specimen of 2 microneurographic recordings showing single action potentials evoked by intracutaneous electrical square pulses given every 4 seconds in the innervation territory in human foot dorsum. Subsequent traces are shown from top to bottom. In (A) action potentials of a mechanosensitive nociceptor (red) and in (B) of a mechanosensitive (red) and a silent nociceptor (blue) in a combined recording are depicted. Note that the response latency of the units remains unchanged when there is no extra activation. Each additional stimulation with transcutaneous electrical half-sine wave stimuli of 0.2 to 1 mA 2000 ms before the next square pulse stimulation is marked by a black circle. The mechanosensitive nociceptors show abrupt increases of response latency of the square wave induced action potential after stimulation with the half-sine wave pulse followed by subsequent normalization of response latency (“marking”). The extent of the marking increases for higher half-sine stimulation intensities indicating that a higher number of action potentials were evoked by the stimulus. The silent nociceptor is not activated even by the highest half-sine wave stimulation intensity applied (1 mA).

3.3. Human psychophysics and axon reflex flare response

Transcutaneous half-sine wave stimulation of 0.2- to 1-mA amplitude delivered to the volar forearm in healthy volunteers (n = 30) evoked intensity-dependent pain (Fig. 5). Perception thresholds to half-sine wave stimuli were on average 0.06 ± 0.03 mA and pain thresholds assessed at 0.18 ± 0.1 mA (n = 24, data not shown). No significant differences in the intensity response profile were recorded between female and male subjects (Fig. 5A). Pain upon half-sine wave stimulation was estimated at 0.2 mA with NRS 1.4 ± 1 and was maximum at 1 mA (NRS 3.5 ± 0.5, n = 30). Repetition of the half-sine wave stimuli delivered 5 times led to a mild adaptation, but pain levels were stable between 2 assessment sessions with a one-week interval (Fig. 5B). The subjects described the sensation induced by low half-sine stimulus intensities (0.2 and 0.4 mA) predominantly as “sharp” and “pricking” pain without any concomitant perception of “touch” or “vibration,” whereas for higher intensities (0.8 and 1 mA), “burning” and “wrenching” dominated. No subject felt the stimulation as a sudden pulse. Short-lasting burning aftersensations (<5 seconds) were reported in 16% of the higher intensity stimuli, but not at the lower intensities.

Figure 5.
Figure 5.:
Half-sine wave stimulations of 0.2 to 1 mA intensity-dependently induced pain ratings (numeric rating scale from 0 to 10) in men and women without significant sex differences (A). Across 5 repetitions of the electrical stimulation, pain intensity slightly decreases, but no decrease was observed when 2 sessions were performed at an interval of 1 week (B).

The kinetics of axon reflex development was compared between the 2 stimulation protocols in 10 volunteers. Four-Herz sine wave pulses (0.2 mA delivered for 20 seconds) evoked an axon reflex flare of 2.9 ± 0.7 cm2 area within one minute, reaching a maximum response of 3.2 ± 0.7 cm2 at minute 2 (Fig. 6) confirming previous results.24 By contrast, 10 half-sine wave pulses (0.4 mA delivered in 2-second intervals) induced a rather localized vasodilation of 1 ± 0.2 cm2 within 1 minute and 1.7 ± 0.4 cm2 at minute 2. Maximum responses were recorded with a delay of 3 to 4 minutes reaching 1.9 ± 0.5 cm2. Instantaneous flare area was significantly smaller when compared with sine wave stimulation (P < 0.00005, analysis of variance) at minute 1 to 4 (P < 0.01, Bonferroni post-hoc). However, 5 minutes after the stimulation flare areas were similar at both sites (2.2 ± 0.6 cm2 sine wave and 1.8 ± 0.5 cm2 at the half-sine wave site [n.s.]). Notably, pain ratings did not differ significantly between the stimulation modes (NRS 3 ± 0.5 vs NRS 3.2 ± 0.4, not shown). Occasionally, subjects reported an itch after the half-sine stimulation (n = 4) accompanied by wheal and erythema development indicative for histamine release upon mast cell degranulation.

Figure 6.
Figure 6.:
Time course of axon reflex erythema following transcutaneous electrical stimulation with 4 Hz sine wave (0.2 mA, 20 seconds) or half-sine wave stimulation (0.4 mA at 2-second intervals for 20 seconds) recorded from 10 age- and sex-matched volunteers (mean ± SEM). The stimulation period is marked by a gray vertical bar. Half-sine wave pulses did not evoke an instantaneous axon reflex flare response as compared to sine wave stimulation (ANOVA, LSD post hoc tests; *P < 0.05; ** P < 0.01; P < 0.001***). ANOVA, analysis of variance.

4. Discussion

Slow depolarizing stimuli applied transcutaneously for 500 ms in a half-sine shape activate mechanosensitive nociceptors intensity-dependently when applied in a range between 0.2 and 1 mA. The activity of mechanosensitive nociceptors corresponds to the intensity-dependent pain evoked in volunteers by the same stimulation. By contrast, most of the silent nociceptors could not be activated and had activation thresholds >10 mA (tested in pig only). Very few silent C fibers (n = 5) could be activated in the pig with excitation thresholds between 0.8 and 8 mA. In human microneurography, only one of 11 silent CMi-nociceptors was activated upon half-sine wave stimulation in contrast to 85% of mechanosensitive CM nociceptors. Therefore, recruitment of silent nociceptors is unlikely to substantially contribute to the pain ratings evoked by half-sine wave stimulation. Another class of C fibers, the low-threshold mechanosensitive afferents (LTM, C-touch fibers)8 had even lower activation thresholds to the half-sine wave stimuli as compared with mechanosensitive nociceptors and showed an intensity-dependent activation in a stimulation range between 0.01 and 0.1 mA. As the pain threshold of our volunteers was reached at about 0.2 mA, it is unlikely that C-touch fibers critically contribute to the pain sensation. Our data therefore support the conclusion that half-sine wave stimuli (500 ms, 0.2-1 mA) evoke graded pain in human skin by activating mechanosensitive nociceptors without major contribution of silent nociceptors and could therefore be used for subclass- selective functional tests in humans.

4.1. Differential functional role of silent and mechanosensitive nociceptors in humans

Mechanosensitive C nociceptors have been shown to determine heat pain threshold and suprathreshold contact heat pain.30,59 Moreover, their sensitization can account for heat hyperalgesia found after a mild burn.29 By contrast, silent nociceptors have been linked to the development of the axon reflex flare,51 and their spontaneous activity has been found to correlate with intensity of spontaneous pain in chronic neuropathic pain patients.28,54 Thus, differential activation of these nociceptor classes seems to be of clinical interest to validate their contribution under specific pathologic conditions. Traditionally, graded mechanical and heat stimuli have been used to assess nociceptive sensitivity of human skin.13,14,33,46,64 Based particularly on threshold assessment, these tests provide an excellent basis for the early diagnosis of small fiber neuropathies6 but leave suprathreshold encoding largely untested. For suprathreshold nerve fiber excitation, which may play a critical role in the pathogenesis of neuropathic pain, strong mechanical or continuous heat stimuli have to be administered. However, suprathreshold mechanical stimulation obviously coactivates also low-threshold A and C fibers in addition to A-delta and C nociceptors. An electrical stimulation paradigm may be considered as alternative approach because it circumvents the signal transduction process for mechanical or heat stimulation by direct axonal excitation. Delivering electrical pulses, for instance, by using intraepidermal concentric bipolar needle electrodes with an outer diameter of 1.2 mm, very locally applied stimuli induced APs in intraepidermal endings of A-delta and C fibers.43 Small stimulation surfaces of the electrodes could be advantageous when considering sensitivity of functional assessment, particularly in patients with small fiber neuropathy, as it limits spatial summation. Hence, under pathologic conditions, reduced heat pain is reported when small skin areas of around 1 mm2 are stimulated.38 Correspondingly, after a >80% denervation of epidermal innervation density by topical capsaicin heat pain thresholds are unchanged when measured with a 9-cm2 thermode,26 but heat sensitivity is clearly reduced in the same model when tested with a 9-mm2 probe.27 A focal electrical stimulation paradigm, for functional nociceptor investigation, should therefore be particularly sensitive in patients with reduced peripheral nerve fiber density.

4.2. Mechanisms of burst vs single-action potential response upon slow depolarization

Sine wave stimulation at 4 Hz has been shown to activate silent and mechanosensitive C nociceptors in human and pigs.24 Each cycle of sinusoidal field stimulation evokes a single, time-locked C-compound AP.24 By contrast, a 500-ms half-sine stimulus (ie, 1-Hz sinusoidal pulse) elicits unsynchronized and intensity-dependent bursts of activity in single nociceptors (Fig. 1A). The latency for the first AP induced by the half-sine stimulus is about 80 to 90 ms (C-touch and mechanosensitive nociceptor, Table 1) and more variable than the synchronous response at about 60 ms observed during 4-Hz sinusoidal stimulation.24 Irrespective of this temporal difference between the 2 stimulus paradigms, it is striking that at 4 Hz both mechanosensitive and silent nociceptors were activated with roughly the same threshold (0.05 mA).24 By contrast, upon half-sine wave stimulation, most of the silent nociceptors were found unresponsive even up to 10 mA, resulting in a more than 20-fold higher threshold than mechanosensitive nociceptors. It is unlikely that spatial distribution of the nociceptor classes, ie, differences in the depth of the nociceptor endings within the skin, can account for the different activation thresholds recorded from mechanosensitive and silent nociceptors for the half-sine wave stimulation, given that these units have similar activation thresholds at 4-Hz sine wave stimuli. Rather, transcutaneous placement of the stimulating electrodes may be considered, as these have to be placed precisely within the receptive field of the unit and at the site of lowest electrical excitation threshold. Otherwise, a decreased sensitivity to the half-sine stimulus could be recorded for this unit. Obviously, this would apply equally to mechanosensitive and silent nociceptors. Also physical properties of the membrane or the expression pattern of axonal ion channels could underlie differential nociceptor activation upon half-sine wave stimulation. The efficacy of slow depolarizing stimuli to generate an AP is expected to decrease with reduced slope of the ramp stimulus,34 probably due to closed state inactivation kinetics of voltage-sensitive sodium channels,2 and this would explain the delayed and rather unsynchronized AP generation upon half-sine stimulation. On the other hand, ramp currents through NaV1.7 or NaV1.3 are expected to amplify slowly depolarizing stimuli,15,63 although results from inducible pluripotent stem cell–derived nociceptors did not support a major role for NaV1.7 in subthreshold activation.35 Lower levels of NaV1.7 in silent nociceptors have been speculated to contribute to marked activity-dependent conduction slowing in human nerve recordings45,56,57 and slower conduction velocities in rat DRG neurons.12 However, there are numerous mechanisms that reduce excitability upon neuronal activity, such as hyperpolarization, accumulation of intracellular sodium, and sodium channel inactivation.56 Differences in passive membrane properties may also contribute to the differential nociceptor activation. For example, higher baseline membrane resistance due to lower background potassium conductance will render the membrane time constant longer and thereby more excitable at lower stimulus frequency.68 The phase of the half-sine stimulus for the induction of the first AP at threshold level was significantly shorter for mechanosensitive nociceptors as for the few silent nociceptors that could be activated (Table 1). This would be consistent with both, higher expression of NaV1.7 and a better match of membrane constant of mechanosensitive nociceptors.

4.3. Psychophysics of pain, axon reflex erythema, and contribution of C-touch fibers

Given that the 4 Hz sine wave stimulation activates mechanosensitive and silent nociceptors in combination and the 500 ms half-sine stimulus preferably excites mechanosensitive nociceptors, it is tempting to compare the reports of the subjects concerning the quality and intensity of pain. However, this comparison between the stimulus modalities does not provide a clear distinction of sensation: in both cases, the predominant descriptor was “burning” pain and the intensity almost identical. Moreover, weak half-sine wave pulses were more often described as “pricking,” and it seems that the temporal profile of the nociceptor discharge influences the quality of sensation. Although the 4 Hz sine wave stimulation simply causes a 4-Hz discharge in the activated C fibers,24 the 500-ms half-sine wave protocol intensity-dependently results in increasing discharge frequencies of single mechanosensitive nociceptors. Surprisingly, this major difference in the temporal discharge profile does not translate into distinct differences in the reports of pain quality and intensity. This might reflect the limitations of our qualitative descriptors that are based on experiences with natural stimulation and do not correspond to the artificial discharge patterns created by our selective electrical stimulation paradigms. For instance, radiant heat laser stimulation evokes a predominant “pricking” and “burning” pain36; nonpainful electrical stimulation (1 pulse, 0.05-ms duration, twice detection threshold) with broad electrodes (diameter 5 mm) was predominantly felt as “shock,” “touch,” or “tingling” sensation, whereas painful intraepidermal stimulation (2 pulses, 100 Hz, 0.05-ms pulse duration, twofold threshold) primarily evoked “pricking” through A-delta nociceptors.37 When intraepidermal electrical stimulation for A-delta (3 pulses at 50 Hz) and C fibers (triangular wave 0.6-ms anodal, 3 pulses 50 Hz) applied to the hand dorsum were compared, mainly a “pricking” sensation was reported for both protocols, but the C fiber stimulation also led to sensations of “light touch,” “tingling,” “warm,” and “burning.”43 Hence, although “pricking” and “burning” pain appears indicative for A-delta and C-nociceptor activation, respectively, our psychophysical results do not allow to differentiate between mechanosensitive and silent C nociceptor activation upon half-sine and sine wave stimulation. Nonetheless, our subjects did not report “pulses” or “shocks” when stimulated with half-sine wave stimuli speaking against a relevant activation of A fibers. In addition to the temporal aspects of nociceptor discharges, it is also important to consider the skin area being activated by the electrodes when recording the quality and intensity of pain sensation. A-delta fiber stimulation was reported to cause mainly “sharp,” “shooting,” and “stabbing” sensations at the sole of the foot.17 However, augmented thickness of the horny layers contributes to lower pain ratings at the heel as compared to the sole,18 or in glabrous as compared to hairy skin.25 For deep somatic fibers, pain quality evoked by electrical stimulation of muscle vs fascia could be differentiated by the subjects,49 but the concomitant muscle twitch might have contributed to the estimates.

Our data show that neither pain intensity rating nor pain quality report is sufficiently sensitive to discriminate predominant mechanosensitive from combined mechanosensitive plus silent nociceptor input. By contrast, the objectively assessed axon reflex erythema developed differentially after sine and half-sine wave stimulation: a widespread instantaneous axon reflex erythema only developed after sine wave stimulation. Half-sine stimulation provoked an axon reflex vasodilation with a delay of several minutes, probably resulting from mast cell degranulation and consecutive histamine release. Thus, based on our nerve fiber recordings, the few silent nociceptors that are expected to be activated by the half-sine pulses are evidently too weak to cause an immediate widespread axon reflex flare as observed upon sine wave stimulation.

Low-threshold mechanosensitive C fibers (LTM, “C-touch”) have been described in humans decades ago but were broadly ignored until recently when their role in “social touch” was substantiated.4,5,22,39,42 Their activation does not provoke an overt and dominant sensation or is mirrored by an activation of the primary sensory cortex, but their functional role has been linked to hedonic aspects processed in the insular cortex,41 and major implications of this fiber class during human development has been discussed.8 Here, we recorded a profound activation of this fiber class during half-sine wave pulse administration, but the stimulus–response curve of activation did not match the profile for the induction of pain. Moreover, most of the C-touch fibers supposedly were activated already at a stimulus intensity that did not cause an overt sensation. Thus, we assume that this fiber class does not contribute to the pain sensation reported by the subjects or even might have reduced it, as shown before.22 An interesting implication of our present finding is the use of half-sine wave stimulus at intensity levels that are subthreshold for an overt sensation but that already activate C-touch fibers. Such a stimulation pattern, providing C-touch fiber activation without concomitant A fiber activation, may be used to investigate cortical networks specifically activated during hedonic low-threshold C fiber recruitment. It would be highly interesting whether the insular activation without contribution of primary sensory cortex observed in a patient with deficient A fibers41 could be also induced with low intensity half-sine wave stimulation in healthy volunteers.

In summary, our data demonstrate the use of transcutaneous electrical stimulation with 500-ms half-sine profile to selectively evoke pain through mechanosensitive C nociceptors in human skin. The additional activation of low threshold mechanoreceptors also could provide a paradigm to selectively investigate C-touch fibers when administering particularly low stimulation intensities around 0.01 mA. It will be of major interest to use this stimulus protocol to functionally identify skin denervation in small fiber neuropathy but also to identify the role of mechanosensitive vs silent nociceptors in neuropathic or inflammatory chronic itch and pain.

Conflict of interest statement

The authors have no conflict of interest to declare.

Appendix A. Supplemental digital content

Supplemental digital content associated with this article can be found online at http://links.lww.com/PAIN/B12.

Acknowledgements

The authors thank Anja Bistron and Elmar Forsch for their technical expertise. This work was supported by grants from Deutsche Forschungsgemeinschaft SFB 1158 to M. Schmelz and R.W. Carr, FOR 2690 to M. Schmelz and R. Rukwied, and project 397846571 to R. Rukwied and Na 970 3-1 to B. Namer. B. Namer is supported by a grant from the Interdisciplinary Center for Clinical Research within the faculty of Medicine at the RWTH Aachen University.

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Keywords:

Nociceptor class; Electrical stimulation; Transcutaneous; Suprathreshold activation; Microneurography

Supplemental Digital Content

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