Maximum axonal following frequency separates classes of cutaneous unmyelinated nociceptors in the pig.

KEY POINTS: C-nociceptors are generally assumed to have a low maximum discharge frequency of 10-30 Hz. However, only mechano-insensitive 'silent' C-nociceptors cannot follow electrical stimulation at 5 Hz (75 pulses) whereas polymodal C-nociceptors in the pig follow stimulation at up to 100 Hz without conduction failure. Sensitization by nerve growth factor increases the maximum following frequency of 'silent' nociceptors in pig skin and might thereby contribute in particular to intense pain sensations in chronic inflammation. A distinct class of C-nociceptors with mechanical thresholds >150 mN resembles 'silent' nociceptors at low stimulation frequencies in pigs and humans, but is capable of 100 Hz discharge and thus is suited to encode painfulness of noxious mechanical stimuli. ABSTRACT: Using extracellular single-fibre recordings from the saphenous nerve in pig in vivo, we investigated peak following frequencies (5-100 Hz) in different classes of C-nociceptors and their modulation by nerve growth factor. Classes were defined by sensory (mechano-sensitivity) and axonal characteristics (activity dependent slowing of conduction, ADS). Mechano-insensitive C-nociceptors (CMi) showed the highest ADS (34% ± 8%), followed only 66% ± 27% of 75 pulses at 5 Hz and increasingly blocked conduction at higher frequencies. Three weeks following intradermal injections of nerve growth factor, peak following frequency increased specifically in the sensitized mechano-insensitive nociceptors (20% ± 16% to 38% ± 23% response rate after 72 pulses at 100 Hz). In contrast, untreated polymodal nociceptors with moderate ADS (15.2% ± 10.2%) followed stimulation frequencies of 100 Hz without conduction failure (98.5% ± 6%). A distinct class of C-nociceptors was exclusively sensitive to strong forces above 150 mN. This class had a high ADS (27.2% ± 7.6%), but displayed almost no propagation failure even at 100 Hz stimulation (84.7% ± 17%). Also, among human mechanosensitive nociceptors (n = 153) those with thresholds above 150 mN (n = 5) showed ADS typical of silent nociceptors. C-fibres with particularly high mechanical thresholds and high following frequency form a distinct nociceptor class ideally suited to encode noxious mechanical stimulation under normal conditions when regular silent nociceptors are inactive. Sensitization by nerve growth factor increases maximum discharge frequency of silent nociceptors, thereby increasing the frequency range beyond their physiological limit, which possibly contributes to excruciating pain under inflammatory conditions.

Transcutaneous Slowly Depolarizing Currents Elicit Pruritus in Patients with Atopic Dermatitis.

Slowly depolarizing currents applied for one minute have been shown to activate C-nociceptors and provoke increasing pain in patients with neuropathy. This study examined the effect of transcutaneous slowly depolarizing currents on pruritus in patients with atopic dermatitis. C-nociceptor-specific electrical stimu-lation was applied to areas of eczema-affected and non-affected skin in 26 patients with atopic dermatitis. Single half-sine wave pulses (500 ms, 0.2-1 mA) induced itch in 9 patients in eczema-affected areas of the skin (numerical rating scale 5 ± 1), but pain in control skin (numerical rating scale 6 ± 1).Sinusoidal stimuli (4 Hz, 10 pulses, 0.025-0.4 mA) evoked itch in only 3 patients in eczema-affected areas of the skin but on delivering pulses for one minute (0.05-0.2 mA) 48% of the patients (n= 12) reported itch with numerical rating scale 4 ± 1 in areas of eczema-affected skin. The number of patients reporting itch in eczema-affected areas of the skin increased with longer stimulation (p < 0.005). These results demonstrate a reduced adaptation of peripheral C-fibres conveying itch in patients with atopic dermatitis. Sensitized spinal itch processing had been suggested before in atopic dermatitis patients, and this could be present also in our patients who therefore might benefit from centrally acting antipruritic therapy.

Tuning in C-nociceptors to reveal mechanisms in chronic neuropathic pain.

OBJECTIVE: Develop and validate a low-intensity sinusoidal electrical stimulation paradigm to preferentially activate C-fibers in human skin. METHODS: Sinusoidal transcutaneous stimulation (4Hz) was assessed psychophysically in healthy volunteers (n = 14) and neuropathic pain patients (n = 9). Pursuing laser Doppler imaging and single nociceptor recordings in vivo in humans (microneurography) and pigs confirmed the activation of "silent" C-nociceptors. Synchronized C-fiber compound action potentials were evoked in isolated human nerve fascicles in vitro. Live cell imaging of L4 dorsal root ganglia in anesthetized mice verified the recruitment of small-diameter neurons during transcutaneous 4-Hz stimulation of the hindpaw (0.4mA). RESULTS: Transcutaneous sinusoidal current (0.05-0.4mA, 4Hz) activated "polymodal" C-fibers (50% at ∼0.03mA) and "silent" nociceptors (50% at ∼0.04mA), intensities substantially lower than that required with transcutaneous 1-ms rectangular pulses ("polymodal" ∼3mA, "silent" ∼50mA). The stimulation induced delayed burning (nonpulsating) pain and a pronounced axon-reflex erythema, both indicative of C-nociceptor activation. Pain ratings to repetitive stimulation (1 minute, 4Hz) adapted in healthy volunteers by Numeric Rating Scale (NRS) -3 and nonpainful skin sites of neuropathic pain patients by NRS -0.5, whereas pain even increased in painful neuropathic skin by approximately NRS +2. INTERPRETATION: Sinusoidal electrical stimulation at 4Hz enables preferential activation of C-nociceptors in pig and human skin that accommodates during ongoing (1-minute) stimulation. Absence of such accommodation in neuropathic pain patients suggest axonal hyperexcitability that could be predictive of alterations in peripheral nociceptor encoding and offer a potential therapeutic entry point for topical analgesic treatment. Ann Neurol 2018;83:945-957.

Effects of Current Density on Nociceptor Activation Upon Electrical Stimulation in Humans.

OBJECTIVES: Mechano-insensitive ("silent") nociceptors contribute to neuropathic pain. Their activation causes an axon-reflex erythema, but their high electrical excitation thresholds complicate their assessment, particularly in painful neuropathy. We therefore developed electrical stimulation paradigms for brief nociceptor activation and explored their sensitivity for clinical trials. METHOD: The local ethics committee approved the study protocol, and 14 healthy subjects were enrolled. Electrical stimuli were administered to ventral forearm and dorsum of the foot via self-adhesive 3 × 10 mm electrodes and a pair of blunted 0.4-mm-diameter platinum/iridium pin electrodes. Pain thresholds were determined and nociceptors activated at 1.5-fold pain threshold by 5 blocks delivering 10 pulses each and at randomized frequencies of 5 to 10 to 20 to 50 to 100 Hz, respectively. Axon reflex erythema and pain were recorded. RESULTS: Increased frequencies dose-dependently increased pain (P < 0.0001). Pin electrode stimulation was more painful than adhesive electrode stimulation (P < 0.04) particularly at the feet. Axon reflex erythema was significantly smaller at the feet than at the forearm (P < 0.0001). At both skin sites, pin electrode stimuli evoked significantly larger erythema (P < 0.05). CONCLUSIONS: Electrical stimulation at high current density using pin electrodes is a sensitive method for investigating "silent" nociceptors, which might therefore preferably be applied in neuropathic pain conditions.

Comparison of nerve growth factor-induced sensitization pattern in lumbar and tibial muscle and fascia.

INTRODUCTION: Nerve growth factor (NGF) induces profound hyperalgesia. In this study we explored patterns of NGF sensitization in muscle and fascia of distal and paraspinal sites. METHODS: We injected 1 µg of NGF into human (n = 8) tibialis anterior and erector spinae muscles and their fasciae. The spatial extent of pressure sensitization, pressure pain threshold, and mechanical hyperalgesia (150 kPa, 10 s) was assessed at days 0.25, 1, 3, 7, 14, and 21. Chemical sensitization was explored by acidic buffer injections (pH 4, 100 µl) at days 7 and 14. RESULTS: The mechanical hyperalgesia area was larger in tibial fascia than in muscle. Pressure pain thresholds were lower, tonic pressure pain ratings, and citrate buffer evoked pain higher in fascia than in muscle. CONCLUSIONS: Spatial mechanical sensitization differs between muscle and fascia. Thoracolumbar fasciae appear more sensitive than tibial fasciae and may be major contributors to low back pain, but the temporal sensitization profile is similar between paraspinal and distal sites. Muscle Nerve 52: 265-272, 2015.

Sphingosine-1-phosphate-induced nociceptor excitation and ongoing pain behavior in mice and humans is largely mediated by S1P3 receptor.

The biolipid sphingosine-1-phosphate (S1P) is an essential modulator of innate immunity, cell migration, and wound healing. It is released locally upon acute tissue injury from endothelial cells and activated thrombocytes and, therefore, may give rise to acute post-traumatic pain sensation via a yet elusive molecular mechanism. We have used an interdisciplinary approach to address this question, and we find that intradermal injection of S1P induced significant licking and flinching behavior in wild-type mice and a dose-dependent flare reaction in human skin as a sign of acute activation of nociceptive nerve terminals. Notably, S1P evoked a small excitatory ionic current that resulted in nociceptor depolarization and action potential firing. This ionic current was preserved in "cation-free" solution and blocked by the nonspecific Cl(-) channel inhibitor niflumic acid and by preincubation with the G-protein inhibitor GDP-ß-S. Notably, S1P(3) receptor was detected in virtually all neurons in human and mouse DRG. In line with this finding, S1P-induced neuronal responses and spontaneous pain behavior in vivo were substantially reduced in S1P(3)(-/-) mice, whereas in control S1P(1) floxed (S1P(1)(fl/fl)) mice and mice with a nociceptor-specific deletion of S1P(1)(-/-) receptor (SNS-S1P(1)(-/-)), neither the S1P-induced responses in vitro nor the S1P-evoked pain-like behavior was altered. Therefore, these findings indicate that S1P evokes significant nociception via G-protein-dependent activation of an excitatory Cl(-) conductance that is largely mediated by S1P(3) receptors present in nociceptors, and point to these receptors as valuable therapeutic targets for post-traumatic pain.

Mechano-insensitive nociceptors are sufficient to induce histamine-induced itch.

The nerve fibres underlying histamine-induced itch have not been fully elucidated. We blocked the lateral femoral cutaneous nerve and mapped the skin area unresponsive to mechanical stimulation, but still sensitive to electrically induced pain. Nerve block induced significantly larger anaesthetic areas to mechanical (100 mN pin-prick, 402 ± 61 cm²; brush, 393 ± 63 cm²) and heat pain stimuli (401 ± 53 cm²) compared with electrical stimulation (352 ± 62 cm², p < 0.05), whereas the anaesthetic area tested with 260 mN (374 ± 57 cm²) did not differ significantly. Histamine was applied by iontophoresis (7.5 mC) at skin sites in which mechanical sensitivity was blocked, but electrical stimulation was still perceived 30 min after the nerve block (n = 9). In these areas iontophoresis of histamine provoked itching in 8/9 subjects with a mean maximum of 4.6 ± 1 (on an 11-point rating scale). Histamine-induced itch can thus be perceived at skin sites where input from mechano-sensitive polymodal nociceptors is blocked. In conclusion, input from mechano-insensitive nociceptors is sufficient to generate histamine-induced itch.

Optimized Electrical Stimulation of C-Nociceptors in Humans Based on the Chronaxie of Porcine C-Fibers.

Classically, to electrically excite C-nociceptors, rectangular pulses are used with a duration close to the estimated chronaxie of C-fibres (about 2 ms). Recent results using slow depolarizing stimuli suggest longer chronaxies. We therefore set out to optimize C-fiber stimulation based on recordings of single C-nociceptors in-vivo and C-fiber compound-action-potentials (C-CAP) ex-vivo using half-sine shaped stimuli of durations between 1 and 250ms. Single fiber (n = 45) recording in pigs revealed high chronaxie values for C-touch fibers (15.8 ms), polymodal- (14.2 ms) and silent-nociceptors (16.8 ms). Activation thresholds decreased 2 to 3-fold in all fibre classes when increasing the duration of half-sine pulses from 1 to 25 ms (P < .05). C-CAPs strength-duration curves of the pig saphenous nerve (n = 7) showed the highest sensitivity for half-sine durations between 10 and 25 ms. Half-maximum currents for C-CAPS were reduced 3-fold compared to rectangular pulses (P < .01) whereas the opposite was found for A-fiber compound action potentials. Psychophysics in humans (n = 23) revealed that half-sine stimulus durations >10 ms reduced detection thresholds, pain thresholds, and stimulus current amplitudes required to generate a pain rating of 3 on an 11-point Numeric Rating Scale (NRS) as compared to 1 ms rectangular pulses (P < 0.05). Increasing the duration from 1 to 25 ms led to a 4-fold amplitude reduction for pain-thresholds and stimuli caused an axon-reflex flare. Excitability of single polymodal nociceptors in animals paralleled human psychophysics and we conclude optimized half-sine pulses facilitate C-nociceptor activation. PERSPECTIVE: Electrical stimulation with longer lasting half-sine wave pulses preferentially activates C-nociceptors and changes in the strength duration curve may identify nociceptor hyperexcitability in patients with neuropathic pain.

Human pain ratings to electrical sinusoids increase with cooling through a cold-induced increase in C-fibre excitability.

ABSTRACT: Low-frequency sinusoidal current applied to human skin evokes local axon reflex flare and burning pain, indicative of C-fibre activation. Because topical cooling works well as a local analgesic, we examined the effect of cooling on human pain ratings to sinusoidal and rectangular profiles of constant current stimulation. Unexpectedly, pain ratings increased upon cooling the skin from 32 to 18°C. To explore this paradoxical observation, the effects of cooling on C-fibre responses to stimulation with sinusoidal and rectangular current profiles were determined in ex vivo segments of mouse sural and pig saphenous nerve. As expected by thermodynamics, the absolute value of electrical charge required to activate C-fibre axons increased with cooling from 32°C to 20°C, irrespective of the stimulus profile used. However, for sinusoidal stimulus profiles, cooling enabled a more effective integration of low-intensity currents over tens of milliseconds resulting in a delayed initiation of action potentials. Our findings indicate that the paradoxical cooling-induced enhancement of electrically evoked pain in people can be explained by an enhancement of C-fibre responsiveness to slow depolarization at lower temperatures. This property may contribute to symptoms of enhanced cold sensitivity, especially cold allodynia, associated with many forms of neuropathic pain.

The differential effects of two sodium channel modulators on the conductive properties of C-fibers in pig skin in vivo.

BACKGROUND: Axonal sodium channels are attractive targets for chronic pain treatment, and recent evidence suggests that specific targeting of the slow inactivation of sodium channels (NaV) might exert analgesic effects. Using a human-like animal model, the pig, we compared changes in the conductive properties of different C-fiber classes on acute administration of lidocaine (nonselective NaV blocker) and lacosamide (selective enhancer of NaV slow inactivation). METHODS: Single-fiber extracellular recordings from saphenous nerves were performed. We classified C-fibers according to mechanical responsiveness and amount of activity-dependent slowing (ADS) of conduction velocity. Lidocaine (4 mM; 100 µL), lacosamide (4 mM; 100 µL), or saline was injected intradermally at the stimulation site, and changes of fibers' conductive properties were assessed. RESULTS: Conduction latencies evoked by lidocaine were more prominent in mechanosensitive (5.5%± 2.1%) than in mechano-insensitive nociceptors (2.5% ± 1%), whereas lacosamide increased conduction latencies to a greater extent in the mechano-insensitive (3% ± 1%) than in mechanosensitive C-nociceptors (2% ± 0.9%). Lidocaine, but not lacosamide, increased electrical thresholds in all mechanosensitive, but not in the mechano-insensitive, C-fibers. Lacosamide blocked conduction and, in addition, reduced ADS in mechano-insensitive nociceptors significantly more than in mechanosensitive nociceptors (ΔADS: 2.4% ± 0.5% vs 1.6% ± 0.5%), whereas lidocaine had opposite effects. Saline had no significant effect on the conductive properties of C-fibers. CONCLUSION: Local application of test compounds in pig skin allows for functional assessment of steady-state and use-dependent modulation of sodium channels in nociceptive and nonnociceptive C-fibers. Increased analgesic specificity might derive from selective enhancement of slow inactivation of sodium channels.

Local hyperexcitability of C-nociceptors may predict responsiveness to topical lidocaine in neuropathic pain.

We explored whether increased C-nociceptor excitability predicts analgesic effects of topical lidocaine in 33 patients with mono- (n = 15) or poly-neuropathy (n = 18). Excitability of C-nociceptors was tested by transcutaneous electrical sinusoidal (4 Hz) and half sine wave (single 500 ms pulse) stimulation delivered to affected and non-affected sites. Analgesic effects of 24 hrs topical lidocaine were recorded. About 50% of patients reported increased pain from symptomatic skin upon continuous 4 Hz sinusoidal and about 25% upon 500 ms half sine wave stimulation. Electrically-evoked half sine wave pain correlated to their clinical pain level (r = 0.37, p < 0.05). Lidocaine-patches reduced spontaneous pain by >1-point NRS in 8 of 28 patients (p < 0.0001, ANOVA). Patients with increased pain to 2.5 sec sinusoidal stimulation at 0.2 and 0.4 mA intensity had significantly stronger analgesic effects of lidocaine and in reverse, patients with a pain reduction of >1 NRS had significantly higher pain ratings to continuous 1 min supra-threshold sinusoidal stimulation. In the assessed control skin areas of the patients, enhanced pain upon 1 min 4 Hz stimulation correlated to increased depression scores (HADS). Electrically assessed C-nociceptor excitability identified by slowly depolarizing electrical stimuli might reflect the source of neuropathic pain in some patients and can be useful for patient stratification to predict potential success of topical analgesics. Central neuronal circuitry assessment reflected by increased pain in control skin associated with higher HADS scores suggest central sensitization phenomena in a sub-population of neuropathic pain patients.

Pituitary adenylate cyclase activating polypeptide: an important vascular regulator in human skin in vivo.

Pituitary adenylate cyclase-activating peptide (PACAP) is an important neuropeptide and immunomodulator in various tissues. Although this peptide and its receptors (ie, VPAC1R, VPAC2R, and PAC1R) are expressed in human skin, their biological roles are unknown. Therefore, we tested whether PACAP regulates vascular responses in human skin in vivo. When injected intravenously, PACAP induced a significant, concentration-dependent vascular response (ie, flush, erythema, edema) and mediated a significant and concentration-dependent increase in intrarectal body temperature that peaked at 2.7°C. Topical application of PACAP induced marked concentration-dependent edema. Immunohistochemistry revealed a close association of PACAP-immunoreactive nerve fibers with mast cells and dermal blood vessels. VPAC1R was expressed by dermal endothelial cells, CD4+ and CD8+ T cells, mast cells, and keratinocytes, whereas VPAC2R was expressed only in keratinocytes. VPAC1R protein and mRNA were also detected in human dermal microvascular endothelial cells. The PACAP-induced change in cAMP production in these cells demonstrated VPAC1R to be functional. PACAP treatment of organ-cultured human skin strongly increased the number of CD31+ vessel cross-sections. Taken together, these results suggest that PACAP directly induces vascular responses that may be associated with neurogenic inflammation, indicating for the first time that PACAP may be a crucial vascular regulator in human skin in vivo. Antagonists to PACAP function may be beneficial for the treatment of inflammatory skin diseases with a neurogenic component.

Microinjection of pruritogens in NGF-sensitized human skin.

Single intradermal injections of nerve growth factor (NGF) evoke prolonged but temporally distinct sensitization patterns to somatosensory stimuli. Focal administration of the non-histaminergic pruritogen cowhage but not histamine resulted in elevated itch at day 21 after NGF administration. Here, we injected bovine adrenal medulla peptide 8-22 (BAM8-22), ß-alanine (ß-ALA) and endothelin-1 (ET-1) into NGF-treated skin of 11 healthy volunteers and investigated the corresponding itch/pain and flare reactions. ß-ALA was the weakest pruritogen, while BAM8-22 and ET-1 were equally potent as histamine. NGF did not sensitize itch or flare reactions induced by any compound, but injection and evoked pain were increased at day 21 and 49. The involvement of histamine H1 receptors in itch was explored in eight subjects after oral cetirizine. ET-1-induced itch and flare were significantly reduced. BAM8-22 and ß-ALA itch were not affected, but flare responses after BAM8-22 reduced by 50%. The results indicate that a single NGF injection does not sensitize for experimentally induced itch but increases pain upon pruritogen injection. In healthy humans, pruritic and algetic processing appear differentially regulated by NGF. However, in patients suffering chronic itch, prolonged elevation of NGF-levels under inflammatory conditions may contribute to elevated itch.

Nerve growth factor sensitizes nociceptors to C-fibre selective supra-threshold electrical stimuli in human skin.

BACKGROUND: Intradermal injection of 1 µg nerve growth factor (NGF) causes sustained nociceptor sensitization. Slowly depolarizing electrical current preferentially activates C-nociceptors. METHODS: We explored the differential contribution of A-delta and C-nociceptors in NGF-sensitized skin using slowly depolarizing transcutaneous electrical current stimuli, CO2 laser heat, mechanical impact, and A-fibre compression block. In 14 healthy volunteers, pain rating was recorded on a numeric scale at days 1-14 after NGF treatment. Ratings during A-fibre conduction block were investigated at days 3 and 7 post-NGF. RESULTS: Pain ratings to electrical, CO2 heat and mechanical impact stimuli were enhanced (>30%, p < .0005, ANOVA) at NGF-injection sites. Axon reflex erythema evoked by electrical stimulation was also larger at NGF-injection sites (p < .02, ANOVA). Diminution of pain during continuous (1 min) sinusoidal current stimulation at 4 Hz was less pronounced after NGF (p < .05, ANOVA). Pain ratings to electrical sinusoidal and mechanical impact stimuli during A-fibre conduction block were significantly elevated at the NGF sites compared to NaCl-treated skin (p < .05, ANOVA). CONCLUSIONS: NGF-induced sensitization of human skin to electrical and mechanical stimuli is primarily driven by C-nociceptors with little contribution from A-delta fibres. Less-pronounced accommodation during ongoing sinusoidal stimulation suggests that NGF could facilitate axonal spike generation and conduction in primary afferent nociceptors in humans. Further studies using this sinusoidal electrical stimulation profile to investigate patients with chronic inflammatory pain may allow localized assessment of skin C-nociceptors and their putative excitability changes under pathologic conditions. SIGNIFICANCE: The application of novel slowly depolarizing electrical stimuli demonstrated a predominant C-nociceptor sensitization in NGF-treated skin. Increased pain ratings, larger axon reflex erythema and less accommodation of C-fibres to ongoing sinusoidal stimulation all indicated an enhanced nociceptor discharge after NGF. A-fibre conduction block had little effect on electrical and mechanical hyperalgesia skin in NGF-treated compared to NaCl-treated skin. This electrical stimulus profile may be applicable for patients with chronic inflammatory pain, allowing localized assessment of skin C-nociceptors and their putative excitability changes under pathologic conditions.

Nerve growth factor-evoked nociceptor sensitization in pig skin in vivo.

Peripheral sensitization of skin nociceptors by nerve growth factor (NGF) was explored in pig skin in vivo. As an objective output measure, the area of axon-reflex-mediated erythema was assessed upon mechanical, thermal, chemical, and electrical stimuli delivered at 1, 3, and 7 days after i.d. injection of 1 microg NGF into the pig's back skin (n = 8). Pretreatment with NGF provoked a sensitization to mechanical (600 mN), thermal (10 sec 49 degrees C) and chemical (15 microl, pH 3) stimuli that lasted for 7 days. No sensitization, however, was found in response to weak mechanical (100 mN), weak thermal (10 sec 45 degrees C), or electrical stimuli. Irrespective of the skin pretreatment (NGF or PBS vehicle control), the area of electrically induced erythema decreased upon repetition (days 1-7) by 70% (P < 0.05). Sensitization of sensory endings by NGF upon mechanical, heat, and chemical stimuli suggests recruitment of sensory transducer molecules [e.g., TRPV1, acid-sensing ion channels (ASICs)]. In contrast, the gradual decrease in electrically induced erythema over 7 days might be attributable to axonal desensitization and possibly activity-dependent down-regulation of sodium channels. Thus, long-lasting sensitization processes of nociceptor endings or axonal sodium channel desensitization mechanisms can be explored in the pig as a translational experimental animal model.

Electrically Evoked Itch in Human Subjects.

Administration of chemicals (pruritogens) into the skin evokes itch based on signal transduction mechanisms that generate action potentials mainly in mechanically sensitive and insensitive primary afferent C-fibers (pruriceptors). These signals from peripheral neurons are processed in spinal and supra-spinal centers of the central nervous system and finally generate the sensation of itch. Compared to chemical stimulation, electrical activation of pruriceptors would allow for better temporal control and thereby a more direct functional assessment of their activation. Here, we review the electrical stimulation paradigms which were used to evoke itch in humans in the past. We further evaluate recent attempts to explore electrically induced itch in atopic dermatitis patients. Possible mechanisms underlying successful pruritus generation in chronic itch patients by transdermal slowly depolarizing electrical stimulation are discussed.

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

ABSTRACT: High-threshold mechanosensitive and mechanoinsensitive ("silent") nociceptors have similar electrical thresholds for transcutaneous sine wave stimulation at 4 Hz that selectively activates cutaneous C nociceptors in human skin. Their fundamentally different functions particularly in chronic pain warrant differential stimulation protocols. We used transcutaneously delivered slow depolarizing stimuli (half-sine, 500 ms duration, 0.01-1 mA) in humans to assess intensity-response relations for the induction of pain psychophysically and recorded activation of mechanosensitive and silent nociceptors in healthy volunteers by microneurography. Differential C-fiber activation was confirmed in single-fiber recordings in pig allowing for stimulation amplitudes up to 10 mA. Perception and pain thresholds to half-sine wave pulses were 0.06 ± 0.03 mA and 0.18 ± 0.1 mA, respectively, and caused pain in an amplitude-dependent manner (n = 24). When matched for pain intensity, only sine wave stimulation induced an instant widespread axon reflex erythema (n = 10). In human microneurography, half-sine stimulation activated mechanosensitive nociceptors (n = 13), but only one of 11 silent nociceptors. In pig skin, the amplitude-dependent activation of mechanosensitive nociceptors was confirmed (0.2-1 mA, n = 28), and activation thresholds for most silent nociceptors (n = 13) were found above 10 mA. Non-nociceptive low-threshold mechanosensitive C fibers (n = 14) displayed lower activation thresholds for half-sine wave stimuli with an amplitude-dependent discharge increase between 0.01 and 0.1 mA. We conclude that transcutaneous electrical stimulation with 500-ms half-sine wave pulses between 0.2 and 1 mA causes amplitude-dependent pain by preferential activation of mechanosensitive C nociceptors.

Sympathetic efferent neurons are less sensitive than nociceptors to 4 Hz sinusoidal stimulation.

BACKGROUND: Sinusoidal current stimuli preferentially activate C-nociceptors. Sodium channel isoforms NaV1.7 and NaV1.8 have been implicated in this. Sympathetic efferent neurons lack NaV1.8 and were explored upon sinusoidal activation. METHODS: Quantitative Sudomotor Axon Reflex Test (QSART) was performed in hairy (n = 16) and glabrous (n = 12) skin. Responses of sympathetic efferents (n = 10) and nociceptive afferents (n = 21) to sinusoidal current stimulation (4 Hz, 0.05-0.15 mA) were recorded in humans by microneurography (n = 11). Activation of sympathetic units upon supra-threshold sinusoidal currents (>0.8 mA) was recorded in pigs (n = 8). RESULTS: Sinusoidal stimuli (4 Hz, 0.4 mA) evoked weak sweat output (30 ml/h/m2 ) in hairy skin compared to rectangular pulses (4 Hz, 5 mA, 53 ml/h/m2 , p < .00001, ANOVA). No change in sweat output was recorded from glabrous skin to sine wave stimuli. Sinusoidal current at intensities ranging from 0.05 to 0.15 mA activated almost all (85%) nociceptors but only 40% of sympathetic units in human. Stimuli lead to a significantly lower activation in sympathetic versus nociceptive fibres as measured by activity-dependent slowing (ADS) of conduction (sympathetic efferents average ADS 100 ± 0.2% vs. C-nociceptors average ADS 113 ± 4%, p < .003, ANOVA). CONCLUSIONS: Sympathetic efferent neurons are less apt to convert slow depolarizations into action potentials as compared to nociceptors. Distinctive sodium channel expression patterns between nociceptors and sympathetic efferent neurons may account for this difference. Sinusoidal stimulation therefore provokes weak sweat responses and provides no alternative for clinical assessment of autonomic function. SIGNIFICANCE: C-nociceptors in hairy skin are activated by 4 Hz sinusoidal current stimulation at lower intensities than myelinated fibres. Sympathetic efferent neurons-albeit also unmyelinated-are less responsive to sinusoidal activation than nociceptors within the same skin area. Cutaneous sympathetic efferent neurons apparently are less apt than nociceptors to convert slow depolarization into action potentials.

TTX-Resistant Sodium Channels Functionally Separate Silent From Polymodal C-nociceptors.

Pronounced activity-dependent slowing of conduction has been used to characterize mechano-insensitive, "silent" nociceptors and might be due to high expression of NaV1.8 and could, therefore, be characterized by their tetrodotoxin-resistance (TTX-r). Nociceptor-class specific differences in action potential characteristics were studied by: (i) in vitro calcium imaging in single porcine nerve growth factor (NGF)-responsive neurites; (ii) in vivo extracellular recordings in functionally identified porcine silent nociceptors; and (iii) in vitro patch-clamp recordings from murine silent nociceptors, genetically defined by nicotinic acetylcholine receptor subunit alpha-3 (CHRNA3) expression. Porcine TTX-r neurites (n = 26) in vitro had more than twice as high calcium transients per action potential as compared to TTX-s neurites (n = 18). In pig skin, silent nociceptors (n = 14) characterized by pronounced activity-dependent slowing of conduction were found to be TTX-r, whereas polymodal nociceptors were TTX-s (n = 12) and had only moderate slowing. Mechano-insensitive cold nociceptors were also TTX-r but showed less activity-dependent slowing than polymodal nociceptors. Action potentials in murine silent nociceptors differed from putative polymodal nociceptors by longer duration and higher peak amplitudes. Longer duration AP in silent murine nociceptors linked to increased sodium load would be compatible with a pronounced activity-dependent slowing in pig silent nociceptors and longer AP durations could be in line with increased calcium transients per action potential observed in vitro in TTX-resistant NGF responsive porcine neurites. Even though there is no direct link between slowing and TTX-resistant channels, the results indicate that axons of silent nociceptors not only differ in their receptive but also in their axonal properties.

Comparison of electrically induced flare response patterns in human and pig skin.

OBJECTIVE: We compared the characteristics of neurogenic flare responses in human and pig skin to establish a translational research animal model. MATERIAL AND SUBJECTS: Eight domestic pigs and six male subjects were investigated. TREATMENT: Electrical pulses were delivered transcutaneously with increasing current intensities, pulse frequencies and pulse widths. METHODS: Inflammatory skin responses were recorded by laser Doppler imaging and analyzed by ANOVA and Fisher's (LSD) post hoc test. RESULTS: Transcutaneous stimuli of 5 mA onward induced a significant flare development in humans. In the pig, significantly lower currents of 2.5 mA already induced a flare response. Smaller flare sizes of about 3.5 cm(2) were analyzed. The flare continuously declined despite ongoing stimulation. CONCLUSIONS: Lower excitation thresholds and smaller receptive fields of nociceptors can be suggested in pigs. Impaired neuropeptide release, altered vesicle replenishment, different neuropeptide sensitivity, or insufficient peripheral decoding of action potentials may contribute to steadily decreasing flare responses. These attributes may be objectives of pre-clinical anti-hyperalgesic studies and their accurate analysis in pigs reveals a particularly sensitive translational animal model for nociceptor researches.