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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., (Copyright © 2023 United States Association for the Study of Pain, Inc. Published by Elsevier Inc. All rights reserved.)

Neuropathic pruritus conditions arise from structural and/or functional damage of the peripheral or central nervous system. Novel findings of pruritus specific mediators and pathways strengthen the specificity theory of pruritus transmission, however electrophysiological studies suggest that focal activation of nociceptors and distinct discharge patterns of primary afferents also contribute to the development of the sensation of pruritus. A complex interplay between excitatory and inhibitory interneurons at spinal level, non-neuronal cells and descending modulation from upper centers contributes to neuronal sensitization and clinically to the chronicity of pruritus, as well as accompanying phenomena such as alloknesis and hyperknesis. Several topical, systemic and non-pharmacological therapeutic approaches directed at distinct targets are currently available., Competing Interests: Conflict of interest statement Nothing declared., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

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., Competing Interests: All authors declare no conflict of interest.

Abstract: Ultraviolet B (UVB) irradiation induces hyperalgesia in human and animal pain models. We investigated mechanical sensitization, increase in axonal excitability, and spontaneous activity in different C-nociceptor classes after UVB in pig skin. We focused on units with receptive fields covering both irradiated and nonirradiated skin allowing intraindividual comparisons. Thirty-five pigs were irradiated in a chessboard pattern, and extracellular single-fibre recordings were obtained 10 to 28 hours later (152 fibers). Units from the contralateral hind limb served as a control (n = 112). Irradiated and nonirradiated parts of the same innervation territory were compared in 36 neurons; low threshold C-touch fibers (n = 10) and sympathetic efferents (n = 2) were unchanged, but lower mechanical thresholds and higher discharge frequency at threshold were found in mechanosensitive nociceptors (n = 12). Half of them could be activated with nonnoxious brush stimuli in the sunburn. Four of 12 mechanoinsensitive nociceptors were found sensitized to mechanical stimulation in the irradiated part of the receptive field. Activity-dependent slowing of conduction was reduced in the irradiated and in the nonirradiated skin as compared with the control leg, whereas increased ability to follow high stimulation frequencies was restricted to the sunburn (108.5 ± 37 Hz UVB vs 6.3 ± 1 Hz control). Spontaneous activity was more frequent in the sunburn (72/152 vs 31/112). Mechanical sensitization of primary nociceptors and higher maximum after frequency are suggested to contribute to primary hyperalgesia, whereas the spontaneous activity of silent nociceptors might offer a mechanistic link contributing to ongoing pain and facilitated induction of spinal sensitization., (Copyright © 2021 International Association for the Study of Pain.)

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., (© 2020 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.)

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., (© 2020 The Authors. European Journal of Pain published by John Wiley & Sons Ltd on behalf of European Pain Federation - EFIC®.)

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., (Copyright © 2020 International Association for the Study of Pain.)

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/m 2 ) in hairy skin compared to rectangular pulses (4 Hz, 5 mA, 53 ml/h/m 2 , 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., (© 2019 The Authors. European Journal of Pain published by John Wiley & Sons Ltd on behalf of European Pain Federation - EFICR®.)

Neuropathic itch is clinically important but has received much less attention as compared to neuropathic pain. In the past decade, itch-specific pathways have been characterized on a cellular and molecular level, but their exact role in the pathophysiology of neuropathic itch is still unclear. Traditionally, mutually exclusive theories for itch such as labeled line, temporal/spatial pattern, or intensity theory have been proposed, and experimental studies in mice mainly favor the specificity theory of itch. By contrast, results in humans also suggest a role for spatial and temporal patterns in neuropathic itch. Rarefication of skin innervation in neuropathy could provide a "spatial contrast" discharge pattern, and axotomy could induce de novo expression of the itch-specific spinal neuropeptide, gastrin-releasing peptide, in primary afferent nociceptors, thereby modulating itch processing in the dorsal horn. Thus, clinical neuropathy may generate itch by changes in the spatial and temporal discharge patterns of nociceptors, hijacking the labeled line processing of itch and abandoning the canonical scheme of mutual exclusive itch theories. Moreover, the overlap between itch and pain symptoms in neuropathy patients complicates direct translation from animal experiments and, on a clinical level, necessitates collaboration between medical specialities, such as dermatologists, anesthesiologists, and neurologists.

Secondary mechanical hyperalgesia to punctate mechanical stimuli and light touch (allodynia) are prominent symptoms in neuropathic pain states. In a combined microneurographic and psychophysical study, we investigated the role of mechano-insensitive (silent) nociceptors regarding induction. Electrical thresholds of mechano-sensitive and silent nociceptors were assessed by microneurography with two closely spaced intracutaneous electrodes (i.c.) and a transcutaneous configuration (t.c.) in the foot dorsum. For t.c. stimulation there was a marked difference between silent (median, quartiles; 60, 50-70 mA, n = 63) and mechano-sensitive fibers (3, 2-5 mA, n = 107). In silent fibers, thresholds were lower for i.c. stimulation (16, 14-19 mA, n = 8), but higher in mechano-sensitive units (6, 5-6 mA, n = 13). Corresponding psychophysical tests showed no difference between the stimulation configuration for pain thresholds, but lower thresholds for the i.c. stimulation in axon reflex erythema (12 vs. 21 mA), punctate hyperalgesia (9 vs. 15 mA) and allodynia (15 vs. 18 mA). Punctate hyperalgesia was evoked at very low stimulation frequencies of 1/20 Hz (7/7 subjects), whereas the induction of an axon reflex flare required stimulation at 1/5 Hz. Electrical stimulation which is sufficient to excite mechano-insensitive C nociceptors can induce secondary mechanical hyperalgesia even at low frequencies supporting a role of such low-level input to clinical pain states. Thus, differential nociceptor class-specific input to the spinal cord adds to the complexity of modulatory mechanisms that determine nociceptive processing in the spinal cord., (Copyright © 2018 IBRO. Published by Elsevier Ltd. All rights reserved.)

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., (© 2018 American Neurological Association.)

Nerve growth factor (NGF) injected into the human skin causes local hyperalgesia to mechanical and electrical stimuli lasting for weeks. Pig data suggested axonal sensitization of C-nociceptors as a contributing mechanism. Here, we recorded single C-nociceptors in 11 human subjects 3 weeks after intracutaneous injection of 1 μg NGF into the foot dorsum. For each identified unit, the receptive field was mapped and, whenever possible, we recorded 2 terminal branches of the same unit, 1 from the hyperalgesic NGF-site ("inside") and the other from the nonsensitized skin ("outside"). In the saline-treated control feet, mechano-insensitive nociceptors (CMi) were more abundant than at the NGF sites (36% vs 19%). Units with axonal properties of CMi fibres but displaying positive mechanical responses ("CMi-like") dominated at the NGF site (27% vs 6%). Moreover, axonal branches innervating the hyperalgesic skin displayed significantly lower electrical thresholds and less activity-dependent conduction velocity slowing when compared with "outside" or control skin. The "inside" branches also showed long-lasting after-discharges and less adaptation to repeated mechanical stimuli. NGF-induced long-term nociceptor hyperexcitability was maximum at the terminal branches directly treated with NGF. The sensitization included sensory and axonal components affecting both activation thresholds and supra-threshold responses. Our data suggest that a combination of sensory sensitization and axonal hyperexcitability is underlying the localized hyperalgesia by facilitating action potential generation and conduction. Axonal changes were also found in the asymptomatic skin surrounding the NGF-treatment sites, thereby possibly reflecting "nociceptive priming."

Introduction: The sodium channel Nav 1.9 is expressed in peripheral nociceptors and has recently been linked to human pain conditions, but the exact role of Nav 1.9 for human nociceptor excitability is still unclear., Methods: C-nociceptors from two patients with late onset of erythromelalgia-like pain, signs of small fiber neuropathy, and rare genetic variants of Nav 1.9 (N1169S, I1293V) were assessed by microneurography., Results: Compared with patients with comparable pain phenotypes (erythromelalgia-like pain without Nav-mutations and painful polyneuropathy), there was a tendency toward more activity-dependent slowing of conduction velocity in mechanoinsensitive C-nociceptors. Hyperexcitability to heating and electrical stimulation were seen in some nociceptors, and other unspecific signs of increased excitability, including spontaneous activity and mechanical sensitization, were also observed., Conclusions: Although the functional roles of these genetic variants are still unknown, the microneurography findings may be compatible with increased C-nociceptor excitability based on increased Nav 1.9 function.

Objectives: Nerve growth factor (NGF) is a protein important for growth and survival, but also for modulation of sensitivity of nociceptors and sympathetic neurons. The purpose of the present study was to investigate the effects of reduced NGF signaling in patients with hereditary sensory and autonomic neuropathies type V, congenital insensitivity to pain, caused by a mutation of the NGFβ gene, including a characterization of single nociceptive fibers using microneurography (MNG)., Materials and Methods: One homozygote and 2 heterozygote patients with this mutation were examined with electromyography/neurography, thermal testing, quantitative sudomotor axon reflex test, and electrically induced axon reflex erythema in addition to MNG., Results: Low quantitative sudomotor axon reflex test measurements of 0.02 (left foot) and 0.03 (right foot) μL/cm and elevated thermal thresholds for warmth and cold detection testing showed clear impairment of small nerve fibers, both sudomotor efferent and somatic afferent fibers, in the patient homozygote for the mutation. MNG from one of the heterozygote patients revealed changes in the small nociceptive fibers in skin, including abnormally low conduction velocity, spontaneous activity in A-δ fibers and C-nociceptors and abnormal or lacking response to heat., Discussion: The findings of grossly intact pain thresholds compared with anamnestic insensitivity of pain in deep somatic tissue such as bone suggest a gradient of impairment dependent on different NGF availability in various tissues. Even though these patients in some aspects report insensitivity to pain, they also report chronic spontaneous pain as their main symptom, strikingly highlighting differential mechanisms of insensitivity to evoked pain versus spontaneous pain.

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., (© 2015 World Institute of Pain.)

Seven patients diagnosed with erythromelalgia (EM) were investigated by microneurography to record from unmyelinated nerve fibers in the peroneal nerve. Two patients had characterized variants of sodium channel Nav1.7 (I848T, I228M), whereas no mutations of coding regions of Navs were found in 5 patients with EM. Irrespective of Nav1.7 mutations, more than 50% of the silent nociceptors in the patients with EM showed spontaneous activity. In the patient with mutation I848T, all nociceptors, but not sympathetic efferents, displayed enhanced early subnormal conduction in the velocity recovery cycles and the expected late subnormality was reversed to supranormal conduction. The larger hyperpolarizing shift of activation might explain the difference to the I228M mutation. Sympathetic fibers that lack Nav1.8 did not show supranormal conduction in the patient carrying the I848T mutation, confirming in human subjects that the presence of Nav1.8 crucially modulates conduction in cells expressing EM mutant channels. The characteristic pattern of changes in conduction velocity observed in the patient with the I848T gain-of function mutation in Nav1.7 could be explained by axonal depolarization and concomitant inactivation of Nav1.7. If this were true, activity-dependent hyperpolarization would reverse inactivation of Nav1.7 and account for the supranormal CV. This mechanism might explain normal pain thresholds under resting conditions.

Cutaneous pain sensations are mediated largely by C-nociceptors consisting of both mechano-sensitive (CM) and mechano-insensitive (CMi) fibres that can be distinguished from one another according to their characteristic axonal properties. In healthy skin and relative to CMi fibres, CM fibres show a higher initial conduction velocity, less activity-dependent conduction velocity slowing, and less prominent post-spike supernormality. However, after sensitization with nerve growth factor, the electrical signature of CMi fibres changes towards a profile similar to that of CM fibres. Here we take a combined experimental and modelling approach to examine the molecular basis of such alterations to the excitation thresholds. Changes in electrical activation thresholds and activity-dependent slowing were examined in vivo using single-fibre recordings of CM and CMi fibres in domestic pigs following NGF application. Using computational modelling, we investigated which axonal mechanisms contribute most to the electrophysiological differences between the fibre classes. Simulations of axonal conduction suggest that the differences between CMi and CM fibres are strongly influenced by the densities of the delayed rectifier potassium channel (Kdr), the voltage-gated sodium channels NaV1.7 and NaV1.8, and the Na+/K+-ATPase. Specifically, the CM fibre profile required less Kdr and NaV1.8 in combination with more NaV1.7 and Na+/K+-ATPase. The difference between CM and CMi fibres is thus likely to reflect a relative rather than an absolute difference in protein expression. In support of this, it was possible to replicate the experimental reduction of the ADS pattern of CMi nociceptors towards a CM-like pattern following intradermal injection of nerve growth factor by decreasing the contribution of Kdr (by 50%), increasing the Na+/K+-ATPase (by 10%), and reducing the branch length from 2 cm to 1 cm. The findings highlight key molecules that potentially contribute to the NGF-induced switch in nociceptors phenotype, in particular NaV1.7 which has already been identified clinically as a principal contributor to chronic pain states such as inherited erythromelalgia.

Action potential initiation and conduction along peripheral axons is a dynamic process that displays pronounced activity dependence. In patients with neuropathic pain, differences in the modulation of axonal conduction velocity by activity suggest that this property may provide insight into some of the pathomechanisms. To date, direct recordings of axonal membrane potential have been hampered by the small diameter of the fibers. We have therefore adopted an alternative approach to examine the basis of activity-dependent changes in axonal conduction by constructing a comprehensive mathematical model of human cutaneous C-fibers. Our model reproduced axonal spike propagation at a velocity of 0.69 m/s commensurate with recordings from human C-nociceptors. Activity-dependent slowing (ADS) of axonal propagation velocity was adequately simulated by the model. Interestingly, the property most readily associated with ADS was an increase in the concentration of intra-axonal sodium. This affected the driving potential of sodium currents, thereby producing latency changes comparable to those observed for experimental ADS. The model also adequately reproduced post-action potential excitability changes (i.e., recovery cycles) observed in vivo. We performed a series of control experiments replicating blockade of particular ion channels as well as changing temperature and extracellular ion concentrations. In the absence of direct experimental approaches, the model allows specific hypotheses to be formulated regarding the mechanisms underlying activity-dependent changes in C-fiber conduction. Because ADS might functionally act as a negative feedback to limit trains of nociceptor activity, we envisage that identifying its mechanisms may also direct efforts aimed at alleviating neuronal hyperexcitability in pain patients.

Anti-nerve growth factor (anti-NGF) treatment is analgesic in chronic inflammatory pain conditions without reducing inflammation. Hypothesizing that ongoing pain induced by inflammatory mediators is increased by long term sensitization of nociceptors, we combined the non-inflammatory NGF-sensitization model with an inflammatory ultraviolet-B (UV-B) model in human volunteers. UV-B irradiation of the skin presensitized with NGF 3 weeks before intensified the pre-existing NGF hyperalgesia during the inflammatory phase of UV-B and caused spontaneous pain in about 70% of the subjects. Pain levels paralleled the intensity of UVB inflammation. Hyperalgesia recorded on a VAS (0-100) was additive after combined NGF/UV-B treatment versus single NGF or UV-B treatment for mechanical impact and tonic heat stimuli, again paralleling the intensity of the UV-B inflammation. In contrast, ratings to tonic mechanical pressure (100 kPa for 10 seconds, peak VAS 58 ± 7 vs VAS 21 ± 5 [NGF] and VAS 12 ± 3 [UV-B]) and pinprick (150 mN for 5 seconds, peak VAS 33 ± 7 vs VAS 10 ± 2 [NGF] and VAS 8 ± 3 [UV-B]) increased in a supra-additive manner. This supra-additive effect faded 24 hours after irradiation, although heat sensitization remained increased. Hyperalgesia and spontaneous pain coexisted in NGF/UV-B treated skin but did not significantly correlate (r < -0.1 at day 1 and r < 0.2 at day 3). We conclude that NGF can sensitize nociceptive endings such that inflammatory mediators may cause sufficient excitation to provoke spontaneous pain. Our results suggest that neuronal sensitization and level of inflammation represent independent therapeutic targets in chronic inflammatory pain conditions., (Copyright © 2013 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved.)

Nerve growth factor (NGF) is involved in the long-term sensitization of nociceptive processing linked to chronic pain. Functional and structural ("sprouting") changes can contribute. Thus, humans report long-lasting hyperalgesia to mechanical and electrical stimulation after intradermal NGF injection and NGF-induced sprouting has been reported to underlie cancer bone pain and visceral pain. Using a human-like animal model we investigated the relationship between the structure and function of unmyelinated porcine nociceptors 3 weeks after intradermal NGF treatment. Axonal and sensory characteristics were studied by in vivo single-fiber electrophysiology and immunohistochemistry. C fibers recorded extracellularly were classified based on mechanical response and activity-dependent slowing (ADS) of conduction velocity. Intraepidermal nerve fiber (IENF) densities were assessed by immunohistochemistry in pigs and in human volunteers using the same NGF model. NGF increased conduction velocity and reduced ADS and propagation failure in mechano-insensitive nociceptors. The proportion of mechano-sensitive C nociceptors within NGF-treated skin areas increased from 45.1% (control) to 71% and their median mechanical thresholds decreased from 40 to 20 mN. After NGF application, the mechanical receptive fields of nociceptors increased from 25 to 43 mm(2). At the structural level, however, IENF density was not increased by NGF. In conclusion, intradermal NGF induces long-lasting axonal and mechanical sensitization in porcine C nociceptors that corresponds to hyperalgesia observed in humans. Sensitization is not accompanied by increased IENF density, suggesting that NGF-induced hyperalgesia might not depend on changes in nerve fiber density but could be linked to the recruitment of previously silent nociceptors., (Copyright © 2013 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved.)

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.

Unlabelled: Ultraviolet-B (UV-B) irradiation is a well-established inflammatory pain model inducing mechanical and thermal hyperalgesia, presumably mediated by released mediators that sensitize sensory nerve endings. Here, we used additional electrical stimulation to investigate axonal hyperexcitability. The lower leg of 13 volunteers was irradiated with 3-fold the minimum erythema UV-B dose and sensitization was recorded at days 1, 3, 7, and 14. Maximum heat pain (47°C, 5 seconds) developed at day 1 (visual analog scale [VAS: 0-100]; 59), was reduced at day 3 (VAS 43, P < .002), and was back to normal at day 7 (VAS 18). Mechanical impact pain (8 m/s), pinprick (150 mN), and pressure (100 kPa) hyperalgesia were maximum throughout days 1 to 3 (VAS 16, 8, and 12, respectively, P < .001) and back to normal at day 7. Suprathreshold transcutaneous electrical stimuli (1.5-fold pain threshold) were delivered in trains of 10 pulses at frequencies of 5 to 100 Hz. Electrical pain thresholds (determined at 2 Hz) decreased significantly (P < .002) and suprathreshold electrical pain increased by about 70% at days 1 to 3 after irradiation (VAS 36, P < .002). Electrical hyperalgesia did not correlate with mechanical sensitization but with reduced heat pain threshold and increased tonic heat pain (r = -.46 and .53; P < .05 and < .01), indicating that axonal hyperexcitability might contribute to heat hyperalgesia. Released inflammatory mediators (eg, prostaglandins) might sensitize both heat transducer molecules and axonal ion channels and receptors, which would explain the simultaneous development and close correlation between heat hyperalgesia and axonal hyperexcitability., Perspective: Local inflammation by UV-B irradiation sensitizes not only sensory endings, but also axons. Increased axonal excitability could contribute to inflammatory hyperalgesia by facilitating spike generation and increasing peak discharge frequencies of nociceptors. Thus, axonal channels and receptors crucial for this sensitization need to be identified to provide new therapeutic targets., (Copyright © 2013 American Pain Society. Published by Elsevier Inc. All rights reserved.)

Polyneuropathy can be linked to chronic pain but also to reduced pain sensitivity. We investigated peripheral C-nociceptors in painful and painless polyneuropathy patients to identify pain-specific changes. Eleven polyneuropathy patients with persistent spontaneous pain and 8 polyneuropathy patients without spontaneous pain were investigated by routine clinical methods. For a specific examination of nociceptor function, action potentials from single C-fibres including 214 C-nociceptors were recorded by microneurography. Patients with and without pain were distinguished by the occurrence of spontaneous activity and mechanical sensitization in C-nociceptors. The mean percentage of C-nociceptors being spontaneously active or mechanically sensitized was significantly higher in patients with pain (mean 40.5% and 14.6%, respectively, P=.02). The difference was mainly due to more spontaneously active mechanoinsensitive C-nociceptors (operationally defined by their mechanical insensitivity and their axonal characteristics) in the pain patients (19 of 56 vs 6 of 43; P=.02). The percentage of sensitized mechanoinsensitive C-nociceptors correlated to the percentage of spontaneously active mechanoinsensitive C-nociceptors (Kendall's tau=.55, P=.004). Moreover, spontaneous activity of mechanoinsensitive C-nociceptors correlated to less pronounced activity-dependent slowing of conduction (Kendall's tau=-.48, P=.009), suggesting that axons were included in the sensitization process. Hyperexcitability in mechanoinsensitive C-nociceptors was significantly higher in patients with polyneuropathy and pain compared to patients with polyneuropathy without pain, while the difference was much less prominent in mechanosensitive (polymodal) C-nociceptors. This hyperexcitability may be a major underlying mechanism for the pain experienced by patients with painful peripheral neuropathy., (Copyright © 2012 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved.)

Nerve growth factor (NGF) induces local hyperalgesia for a few days after intramuscular injection, but longer-lasting muscle pain upon systemic administration. As the muscle fascia is densely innervated by free nerve endings, we hypothesized a lasting sensitization of fascia nociceptors by NGF. We administered 1 μg NGF (dissolved in 100 μL saline) ultrasound-guided to the fascia of the Musculus erector spinae muscle at the lumbar level of 14 male volunteers and assessed hypersensitivity after 6 hours, and 1, 3, 7, 14, and 21 days. Pain upon mechanical stimuli (constant pressure and dynamic impact), upon exercise and electrically induced M. erector spinae contraction, and upon injection of 100 μL phosphate buffer pH4 (at day 7 and 14 only) to the fascia of both NGF- and saline-treated muscles, was investigated. Injections into the muscle fascia did not cause acute pain. Local heat pain thresholds were unchanged following NGF and saline (control) administration. NGF evoked a lasting (days 1-7) and significant reduction of pressure pain, pressure thresholds, exercise-evoked muscle pain, and hyperalgesia to impact stimuli (12 m/s). Pain upon injected protons was significantly elevated (P<0.04) for 2 weeks. NGF induced a sensitization of the muscle fascia to mechanical and chemical stimuli lasting for up to 2 weeks. As nociceptors in the fascia appear to be particularly prone to sensitization, they may contribute to acute or chronic muscle pain., (Copyright © 2012 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved.)

Multiple firing of C nociceptors upon a single electrical stimulus has been suggested to be a possible mechanism contributing to neuropathic pain. Because this phenomenon maybe based on a unidirectional conduction block, it might also be related to neuropathic changes without a direct link to pain. We investigated painful neuropathy patients using microneurography and analysed nociceptors for the occurrence of multiple spiking and spontaneous activity. In 11 of 105 nociceptors, double spiking was found, with 1 fibre even showing triple spikes on electrical stimulation. The interval between the main action potential and the multiple spikes ranged from 13 to 100 ms. There was a significant association between spontaneous activity and multiple spiking in C nociceptors, with spontaneous activity being present in 9 of 11 fibres with multiple spiking, but only in 21 of 94 nociceptors without multiple spiking (P<.005, Fisher exact test). Among the 75 C nociceptors without spontaneous activity, only 2 nociceptors showed multiple spiking. In 8 neuropathy patients without pain, double spiking was found only in 4 of 90 nociceptors. Multiple spiking of nociceptors coincides with spontaneous activity in nociceptors of painful neuropathy patients. We therefore conclude that rather than being a generic sign of neuropathy, multiple spiking is linked to axonal hyperexcitability and spontaneous activity of nociceptors. It is still unclear whether it also is mechanistically related to the clinical pain level., (Copyright © 2011 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved.)

Nerve growth factor (NGF) induces acute sensitization of nociceptive sensory endings and long-lasting hyperalgesia. NGF modulation of sodium channel expression might contribute to neurotrophin-induced hyperalgesia. Here, we investigated NGF-evoked changes of the activity-dependent slowing of conduction in porcine C-fibers. Animals received intradermal injections of NGF (2 μg or 8 μg) or saline in both hind limbs. Extracellular recordings from the saphenous nerves were performed 1 week later. Based on sensory thresholds and electrically induced activity-dependent slowing (ADS) of axonal conduction, C-fibers were classified as mechano-sensitive afferents, mechano-insensitive afferents, cold nociceptors, and sympathetic efferents. NGF (2 μg) increased conduction velocity in C-fibers from 1.0±0.05 m/s to 1.2±0.07 m/s. In mechano-insensitive afferents, NGF (8 μg) reduced activity-dependent slowing of conduction, from 5.3±0.2% to 3.2±0.5% (0.125-0.5 Hz stimulation) and from 28.5±1.3% to 20.9±1.9% (2 Hz stimulation), such that ADS no longer differentiated between mechano-sensitive and mechano-insensitive fibers. Accordingly, the number of fibers with pronounced ADS decreased but more units with pronounced ADS were mechano-sensitive. Spontaneously active C-fibers were increased above the control level (1%) by NGF 8 μg (8%). The results demonstrate that NGF changes the functional axonal characteristics of mechano-insensitive C-fibers and enhances spontaneous activity thereby possibly contributing to hyperalgesia., (Copyright © 2011 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved.)

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.

Microneurography has provided valuable data on single-fiber characteristics in healthy volunteers and patients, featuring a unique setting that allows linking discharge of single fibers to percept. This advantage is of particular value, when pain patients are examined. Latest results on specific changes of axonal excitability differentially expressed in various C-fiber classes have incited studies linking axonal excitability changes to the mechanism of chronic pain. These studies are mainly based on investigating patients rather than establishing animal models and have led to specific questions on the molecular mechanisms underlying axonal excitability changes. However, pharmacological interventions are limited in human microneurography and therefore an animal model is required. Ideally, the distribution of fiber classes and their sensory and axonal characteristics should be comparable to human. Here we report on corresponding C-fiber classes between human and pig. Nociceptive and non-nociceptive fiber classes found in pig correlate with human fiber classes, in both distribution and axonal excitability changes. It is suggested that the pig is an attractive model for studying modulation of excitability in nociceptive and non-nociceptive C-fiber classes that correspond to those in man.

Activity-dependent slowing of conduction velocity (ADS) differs between classes of human nociceptors. These differences likely reflect particular expression and use-dependent slow inactivation of axonal ion channels and other mechanisms governing axonal excitability. In this study, we compared ADS of porcine and human cutaneous C-fibers. Extracellular recordings were performed from peripheral nerves, using teased fiber technique in pigs and microneurography in humans. We assessed electrically-induced conduction changes and responsiveness to natural stimuli. In both species, the group of mechano-insensitive C-fibers showed the largest conduction slowing ( approximately 30%) upon electrical stimulation (2Hz for 3min). In addition, we found mechano-insensitive cold nociceptors in pig that slowed only minimally (<10% at 2Hz), and a similar slowing pattern was found in some human C-fibers. Mechano-sensitive afferents showed an intermediate conduction slowing upon 2Hz stimulation (pig: 14%, human 23%), whereas sympathetic efferent fibers in pig and human slowed only minimally (5% and 9%, respectively). In fiber classes with more pronounced slowing, conduction latencies recovered slower; i.e. mechano-insensitive afferents recovered the slowest, followed by mechano-sensitive afferents whereas cold nociceptors and sympathetic efferents recovered the fastest. We conclude that mechano-insensitive C-fiber nociceptors can be differentiated by their characteristic pattern of ADS which are alike in pig and human. Notably, cold nociceptors with a distinct ADS pattern were first detected in pig. Our results therefore suggest that the pig is a suitable model to study nociceptor class-specific changes of ADS., (Copyright 2009 International Association for the Study of Pain. All rights reserved.)

Tibia fracture in rats evokes nociceptive, vascular, and bone changes resembling complex regional pain syndrome (CRPS). Substance P (SP) signaling contributes to the hindpaw warmth, increased vascular permeability, and edema observed in this model, suggesting that neurogenic inflammatory responses could be enhanced after fracture. Four weeks after tibia fracture we measured SP and calcitonin gene-related peptide (CGRP) protein levels in the sciatic nerve and serum. Hindpaw skin extravasation responses and SP receptor (NK1), CGRP receptor (calcitonin receptor-like receptor, CRLR) and neutral endopeptidase (NEP) protein levels were also determined. Gene expression levels of these peptides, receptors, and peptidase were examined in the DRG and skin. Spontaneous and intravenous SP-evoked extravasation responses were increased ipsilateral, but not contralateral to the fracture. Fracture increased SP and CGRP gene expression in the ipsilateral L4,L5 DRG and neuropeptide protein levels in the sciatic nerve and in serum, but had no effect on electrically evoked SP and CGRP release. NK1 receptor expression was increased in the ipsilateral hindpaw skin keratinocytes and endothelial cells after injury, but CRLR and NEP expression were unchanged. Fracture also increased epidermal thickness, but had no effect on epidermal skin neurite counts. These results demonstrate that spontaneous and intravenous SP-evoked extravasation responses are enhanced in the ipsilateral hindlimb after fracture and that fracture chronically increases the expression of endothelial and keratinocyte NK1 receptors in the injured limb. We postulate that SP activation of these up-regulated NK1 receptors results in skin warmth, protein leakage, edema, and keratinocyte proliferation in the injured limb.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels contribute to stabilizing resting membrane potential, thus controlling neuron excitability. Subclasses of nociceptive neurons differ in their excitability, therefore, these channels could be a distinguishing marker. We investigated isolated dorsal root ganglion neurons from a non-rodent species, the pig, Sus scrofa domesticus. Single labeling revealed capsaicin-induced cobalt-uptake in 54.3% and transient receptor potential V1 (TRPV1) immunoreactivity in 55.1% of all neurons. Ruthenium red and capsazepine suppressed capsaicin-induced cobalt-uptake. HCN-1 and HCN-2 channel isoform immunoreactivity was detected in 82.6% and 88.3%, respectively, and binding of IB4 in 29.4% of all neurons. Double labeling revealed that out of the capsaicin-positive neurons, 42.3% were IB4-positive, 80.0% immunoreactive for the HCN-1, and 77.3% for the HCN-2 channel isoform, respectively. Neurons lacking HCN-1 or HCN-2 channel isoforms were mostly capsaicin-positive and IB4-negative. The soma size of neurons lacking HCN-1 and/or HCN-2 channels was small to medium. Western blot analysis showed protein products of sizes similar to those of HCN-1 and HCN-2 channel isoforms. Functionally, in patch-clamp experiments, some neurons were unresponsive to membrane hyperpolarization, thus, probably lacking HCN channels. In conclusion, in porcine dorsal root ganglion neurons there is a subset of capsaicin-positive, IB4-negative neurons lacking HCN-1 and/or HCN-2 channel isoforms.

Microneurography was used to record action potentials from afferent C-fibers in cutaneous fascicles of the peroneal nerve in healthy volunteers. Afferent fibers were classified according to their mechanical responsiveness to von Frey stimulation (75g) into mechano-responsive and mechano-insensitive nociceptors. Various concentrations of Endothelin1 (ET1) and Histamine were injected into the receptive fields of C-fibers. Activation and heat sensitization were monitored. Axon reflex flare and psychophysical ratings were assessed after injection of ET1 and codeine into the forearms after pre-treatment with an H1 blocker or sodium chloride. 65% of mechanosensitive nociceptors were activated by ET1. One-third showed long lasting responses (>15min). In contrast, none of thirteen mechano-insensitive fibers were activated. Sensitization to heat was observed in 62% of mechanosensitive and in 46% of mechano-insensitive fibers. Injection of ET1 produced a widespread axon reflex flare, which was suppressed by pre-treatment with an H1 receptor blocker. In addition, pain sensations were induced more often than itching by ET1 in contrast to codeine. No wheal was observed after injection of ET1. Both itching and pain were decreased after H1 blocker treatment. In summary: (1) In humans ET1 activates mechanosensitive, but not mechano-insensitive, nociceptors. (2) Histamine released from mast cells is not responsible for all effects of ET1 on C-nociceptors. (3) ET1 could have a differential role in pain compared to other chemical algogens which activate additionally or even predominantly mechano-insensitive fibers.

Primary hyperalgesia to mechanical and thermal stimuli are major clinical symptoms of inflammatory pain and can be induced experimentally by ultraviolet-B (UV-B) irradiation in humans. We set-up a pig model in order to have more options for pharmacological intervention on primary hyperalgesia. Pig skin was irradiated with a dose one- to threefold higher than the minimum erythema dose (MED) and investigated for mechanical and heat responsiveness 24 and 48 h post UV-B treatment. C-fiber activation upon mechanical and thermal stimulation was assessed indirectly by extent of the axon reflex erythema (flare) measured by laser Doppler imaging. Mechanical stimulation with von Frey filaments (100 mN) induced flare responses in UV-B treated skin at 24 and 48 h, but no effect was measured in normal untreated skin. Increased mechanical stimulation (600 mN) elicited a small flare response in normal skin in an area of 1.8 cm(2) on average that was extending about 2.5 cm(2) in the UV-B irradiated sites. Thermal stimuli provoked in normal pig skin flare areas of approximately 2 cm(2) (45 degrees C, 10 s) and 4.5 cm(2) (47 degrees C, 10 s) which increased to about 3.5 cm(2) (45 degrees C) and 5.5 cm(2) (47 degrees C) following UV-B irradiation at 24 and 48 h. No significant differences of mechanically or thermally induced hypersensitivity were seen between 24 and 48 h after irradiation. We conclude that UV-B induced mechanical and heat sensitization of primary afferent nociceptors can be assessed in pig skin, providing a new human-like model of primary hyperalgesia. Sensitization of primarily mechano-insensitive (silent) nociceptors, which are underlying the flare response in humans, most probably contributes to the observation presented here.

Sympathetically maintained pain could either be mediated by ephaptic interactions between sympathetic efferent and afferent nociceptive fibers or by catecholamine-induced activation of nociceptive nerve endings. We report here single fiber recordings from C nociceptors in a patient with sympathetically maintained pain, in whom sympathetic blockade had repeatedly eliminated the ongoing pain in both legs. We classified eight C-fibers as mechano-responsive and six as mechano-insensitive nociceptors according to their mechanical responsiveness and activity-dependent slowing of conduction velocity (latency increase of 0.5+/-1.1 vs. 7.1+/-2.0 ms for 20 pulses at 0.125 Hz). Two C-fibers were activated with a delay of several seconds following strong endogenous sympathetic bursts; they were also excited for about 3 min following the injection of norepinephrine (10 microl, 0.05%) into their innervation territory. In these two fibers, a prolonged activation by injection of low pH solution (phosphate buffer, pH 6.0, 10 microl) and sensitization of their heat response following prostaglandin E2 injection were recorded, evidencing their afferent nature. Moreover, their activity-dependent slowing was typical for mechano-insensitive nociceptors. We conclude that sensitized mechano-insensitive nociceptors can be activated by endogenously released catecholamines and thereby may contribute to sympathetically maintained pain. No evidence for ephaptic interaction between sympathetic efferent and nociceptive afferent fibers was found.

The discovery of a specialized neuronal pathway for itch has markedly improved our understanding of itch processing under physiological conditions. However, the complex interactions of pain and itch are only partly understood. This review focuses on the neurophysiological mechanisms involved in clinical and experimental itch conditions. There is emerging evidence that similar patterns of peripheral and central sensitization occur in chronic pain and chronic itch conditions. It will be of major interest to reveal whether the underlying mechanism for sensitization in the itch and pain pathways are also similar, as this might have major implications for therapy.

The mechanisms for the induction of primary mechanical hyperalgesia are unclear. We analyzed the neurogenic axon reflex erythema (flare) following phasic mechanical stimulation in normal and in UV-B irradiated skin. In a cross-over double blind design (n = 10), low dose of systemic lidocaine suppressed mechanical hyperalgesia in sunburned skin and in the mechanically induced flare. Phasic mechanical stimulation, even at painful intensities, did not evoke a flare reaction in normal skin. However, stimulation within the UV-B burn dose-dependently provoked an immediate flare reaction. Systemic lidocaine suppressed the mechanically induced flare as well as the mechanical hyperalgesia in sunburned skin, while leaving the impact-induced ratings in normal skin unchanged. Systemic lidocaine reduced these effects of sensitization, but did not reduce ratings in normal skin. As mechanically insensitive ("sleeping") nociceptors have been shown to mediate the axon-reflex in human skin, sensitization of this class of nociceptors might contribute also to the UV-B-induced primary mechanical hyperalgesia.

Microelectrode recordings of impulse activity in nociceptive C fibres were performed in cutaneous fascicles of the peroneal nerve at the knee level in healthy human subjects. Mechano-heat responsive C units (CMH), mechano-insensitive but heat-responsive (CH) as well as mechano-insensitive and heat-insensitive C units (CM(i)H(i)) were identified. A subgroup of the mechano-insensitive units was readily activated by histamine. We studied the responsiveness of these nociceptor classes to injection of 20 microl 5 mM adenosintriphosphate (ATP) using saline injections as control. Because of mechanical distension during injection, which typically activates mechano-responsive C fibres, interest was focused on responsiveness to ATP after withdrawal of the injection needle. Post-injection responses were observed in 17/27 (63%) mechano-responsive units and in 14/22 (64%) mechano-insensitive units. Excitation by ATP occurred in 9/11 CH units and in 5/11 CM(i)H(i) units. ATP responsive units were found both within the histamine-responsive and the histamine-insensitive group of mechano-insensitive fibres. ATP responses appeared with a delay of 0-180 s after completion of injection; responses were most pronounced during the first 1-3 min of activation, and irregular ongoing activity was observed for up to 10 or even 20 min. ATP responses were dose-dependent, concentrations lower than 5 mM gave weaker responses. No heat or mechanical sensitisation was observed in any of the major fibre classes. In conclusion, we have shown that ATP injections at high concentrations activate C-nociceptors in healthy human skin, without preference for mechano-responsive or mechano-insensitive units. ATP did not sensitise human C fibres for mechanical or heat stimuli. We discuss how various mechanisms might contribute to the observed responses to ATP.

Interleukin-6 (IL-6) contributes to increased pain and hyperalgesia in inflamed tissue. We have investigated the effects of IL-6, alone or in combination with its soluble receptor (sIL-6R), on the sensitivity of nociceptors to noxious heat, using dermal microdialysis. Plasmapheresis membranes were inserted into the abdominal skin of adult male Wistar rats (n=46) and perfused with modified Ringer solution. After three control samples (20 min each), the skin area above the membrane was heated to 48 degrees C for 20 min. The stimulation was followed by two washout samples. The calcitonin gene-related peptide (CGRP) content of the dialysate was measured with an enzyme immunoassay. Heat stimulation provoked a significant CGRP increase in the dialysate. Intradermal application of IL-6 (200 ng ml-1) did not significantly alter heat-induced CGRP release. However, a significant sensitisation of the heat-induced CGRP release was observed when sIL-6R (25 ng ml-1) was applied, either alone or in combination with IL-6. Neutralisation of endogenous IL-6 with a sheep anti-rat IL-6 serum did not alter heat-induced CGRP release, but abolished the sIL-6R-mediated sensitising effect. We show that IL-6 in combination with its soluble receptor can sensitise nociceptors to heat and provide evidence for the constitutive expression of the signalling molecule gp130, but not of the IL-6-membrane-bound (specific) receptor, in nociceptors.

The majority of somatosensory DRG neurons express GABAA receptors (GABAAR) and depolarise in response to its activation based on the high intracellular chloride concentration maintained by the Na-K-Cl cotransporter type 1 (NKCC1). The translation of this response to peripheral nerve terminals in people is so far unclear. We show here that GABA (EC50 = 16.67μM) acting via GABAAR produces an influx of extracellular calcium in approximately 20% (336/1720) of isolated mouse DRG neurons. In contrast, upon injection into forearm skin of healthy volunteers GABA (1mM, 100μl) did not induce any overt sensations nor a specific flare response and did not sensitize C-nociceptors to slow depolarizing electrical sinusoidal stimuli. Block of the inward chloride transporter NKCC1 by furosemide (1mg/100μl) did not reduce electrically evoked pain ratings nor did repetitive GABA stimulation in combination with an inhibited NKCC1 driven chloride replenishment by furosemide. Finally, we generated a sustained period of C-fiber firing by iontophoretically delivering codeine or histamine to induce tonic itch. Neither the intensity nor the duration of histamine or codeine itch was affected by prior injection of furosemide. We conclude that although GABA can evoke calcium transients in a proportion of isolated mouse DRG neurons, it does not induce or modify pain or itch ratings in healthy human skin even when chloride gradients are altered by inhibition of the sodium coupled NKCC1 transporter. [ABSTRACT FROM AUTHOR]

TRPM3 is a calcium-permeable cation channel expressed in a range of sensory neurons that can be activated by heat and the endogenous steroid pregnenolone sulfate (PS). During inflammation, the expression and function of TRPM3 are both augmented in somatosensory nociceptors. However, in isolated dorsal root ganglion (DRG) neurons application of inflammatory mediators like prostaglandins and bradykinin (BK) inhibit TRPM3. Therefore, the aim of this study was to examine the effect of preceding activation of cultured 1 day old mouse DRG neurons by the inflammatory mediator BK on TRPM3-mediated calcium responses. Calcium signals were recorded using the intensity-based dye Fluo-8. We found that TRPM3-mediated calcium responses to PS were enhanced by preceding application of BK in cells that responded to BK with a calcium signal, indicating BK receptor (BKR) expression. The majority of cells that co-expressed TRPM3 and BKRs also expressed TRPV1, however, only a small fraction co-expressed TRPA1, identified by calcium responses to capsaicin and supercinnamaldehyde, respectively. Signaling and trafficking pathways responsible for sensitization of TRPM3 following BK were characterized using inhibitors of second messenger signaling cascades and exocytosis. Pharmacological blockade of protein kinase C, calcium–calmodulin-dependent protein kinase II and diacylglycerol (DAG) lipase did not affect BK-induced sensitization, but inhibition of DAG kinase did. In addition, release of calcium from intracellular stores using thapsigargin also resulted in TRPM3 sensitization. Finally, BK did not sensitize TRPM3 in the presence of exocytosis inhibitors. Collectively, we show that preceding activation of DRG neurons by BK sensitized TRPM3-mediated calcium responses to PS. Our results indicate that BKR-mediated activation of intracellular signaling pathways comprising DAG kinase, calcium and exocytosis may contribute to TRPM3 sensitization during inflammation. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Gain-of-function mutations in the tetrodotoxin (TTX) sensitive voltage-gated sodium channel (Nav) Nav1.7 have been identified as a key mechanism underlying chronic pain in inherited erythromelalgia. Mutations in TTX resistant channels, such as Nav1.8 or Nav1.9, were recently connected with inherited chronic pain syndromes. Here, we investigated the effects of the p.M650K mutation in Nav1.8 in a 53 year old patient with erythromelalgia by microneurography and patch-clamp techniques. Recordings of the patient’s peripheral nerve fibers showed increased activity dependent slowing (ADS) in CMi and less spontaneous firing compared to a control group of erythromelalgia patients without Nav mutations. To evaluate the impact of the p.M650K mutation on neuronal firing and channel gating, we performed current and voltage-clamp recordings on transfected sensory neurons (DRGs) and neuroblastoma cells. The p.M650K mutation shifted steady-state fast inactivation of Nav1.8 to more hyperpolarized potentials and did not significantly alter any other tested gating behaviors. The AP half-width was significantly broader and the stimulated action potential firing rate was reduced for M650K transfected DRGs compared to WT. We discuss the potential link between enhanced steady state fast inactivation, broader action potential width and the potential physiological consequences. [ABSTRACT FROM AUTHOR]

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-β-S. Notably, S1P3 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 S1P3-/- mice, whereas in control S1P1 floxed (S1P1fl/fl) mice and mice with a nociceptor-specific deletion of S1P1-/- receptor (SNS-S1P1-/-), 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 S1P3 receptors present in nociceptors, and point to these receptors as valuable therapeutic targets for post-traumatic pain. [ABSTRACT FROM AUTHOR]

Background. The endogenous peptide bradykinin (BK) is an inflammatory mediator that induces nociceptor activation and sensitization as well as protein extravasation and vasodilation. Objective. To test the hypothesis if sympathectomy affects BK-induced inflammation in humans. Methods. Dermal microdialysis was employed on the volar forearm in 10 patients (21–41 years) with regional hyperhidrosis before and three months after preganglionic endoscopic transthoracic sympathetic clipping (ETSC) at the T2 or T3 level and in 10 healthy volunteers (22–36 years). After 60 minutes perfusion with Ringer's solution microdialysis fibers were perfused with BK 10−7 M and 10−5 M for 30 minutes followed by 30 minutes Ringer's solution again. To assess protein extravasation dialysate protein content was measured photometrically and Laser-Doppler imaging was used to quantify axonreflex vasodilation. Results. Baseline flux values after ETSC were higher as compared with controls and preoperative values (anova, Bonferroni post hoc test, P < 0.05), but neither BK 10−7 M nor 10−5 M led to significant vasodilation. Baseline dialysate protein did not significantly differ between groups. BK 10−5 M induced protein extravasation while BK 10−7 M was ineffective, and BK 10−5 M induced protein extravasation was significantly enhanced after ETSC ( P < 0.001). Conclusions. Forearm skin perfusion is increased after ETSC on the T2 or T3 level indicating decreased sympathetic activity while BK-induced protein extravasation was increased. These results show that preganglionic sympathectomy does not diminish bradykinin-induced protein extravasation as found for postganglionic sympathectomy in rats. [ABSTRACT FROM AUTHOR]

Abstract: Decades of pain research have succeeded in elucidating complex mechanisms of acute activation and chronic sensitization of nociceptors leading to pain. In contrast, itch conditions have received less attention and even basic mechanisms for the induction of itch are still unclear. In this review we describe itch-specific pathways, but also evidence for a modified pattern theory of pruritus offering independent mechanisms for the itch induction. Traditionally pain and itch have been regarded as antagonistic as painful stimuli such as scratching suppress itch and opioids suppress pain, but generate itch. However, concerning mechanisms of sensitization to itch or pain, surprisingly similar patterns have been observed lately in both inflamed tissue and in the spinal cord. These similarities open up two highly interesting perspectives: the role of well established analgesic therapeutic concepts can be validated in chronic itch conditions and on the other hand investigations of sensitization in easily accessible pruritic skin may help to validate concepts of nociception in humans. These perspectives illustrate that itch and pain research no longer follows separate paths, but can be advantageously interconnected. [Copyright &y& Elsevier]

As volunteers can easily communicate quality and intensity of painful stimuli, human pain models appear to be ideally suited to test analgesic compounds, but also to study pain mechanisms. Acute stimulation of nociceptors under physiologic conditions has proven not to be of particular use as an experimental pain model. In contrast, if the experimental models include sensitization of the peripheral or central pain processing they may indeed mimic certain aspects of chronic pain conditions. Peripheral inflammatory conditions can be induced experimentally with sensitization patterns correlating to clinical inflammatory pain. There are also well-characterized models of central sensitization, which mimic aspects of neuropathic pain patients such as touch evoked allodynia and punctate hyperalgesia. The main complaint of chronic pain patients, however, is spontaneous pain, but currently there is no human model available that would mimic chronic inflammatory or neuropathic pain. Thus, although being helpful for proof of concept studies and dose finding, current human pain models cannot replace patient studies for testing efficacy of analgesic compounds. [ABSTRACT FROM AUTHOR]

Activation of protease-activated receptors (PAR) can induce vasodilation (VD) and increase of vascular permeability either directly by stimulating endothelial cells or indirectly via activation of nociceptors and subsequent release of neuropeptides (neurogenic inflammation). We aimed to estimate the relative contribution of the two pathways for stimulation with endogenous activators of PAR-2 (trypsin) and of PAR-1, 3 and 4 (thrombin) using in vivo dermal microdialysis in rats. Protein extravasation (PE) was assessed by increase of protein concentration in the dialysate, and VD was quantified by laser Doppler scanning. Both trypsin (10−8−10−4 M) and thrombin (10−6, 10−5.5 and 10−5 M) provoked PE and local VD in a dose-dependent manner. Trypsin (10−4 M)-induced PE was inhibited by 87.2 ± 21% due to the substance P (SP) NK1 receptor antagonist SR140333. VD was blocked by 58.15 ± 10.1% in response to the calcitonin gene-related peptide (CGRP) receptor antagonist CGRP8-37. By contrast, CGRP8-37 did not affect thrombin-induced VD, while blockade of SP receptors prevented the PE elicited only by low doses of thrombin (10−6 M), being ineffective at higher thrombin concentrations. In conclusion, intradermal trypsin elicits a neurogenic inflammation in rat, probably mediated via PAR-2 activation on nociceptors and subsequent SP and CGRP release. Thrombin-induced PE and VD are mediated mainly by a non-neurogenic mechanism. [ABSTRACT FROM AUTHOR]