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Metadata standards are well-established for many types of electrophysiological methods but are still lacking for microneurographic recordings of peripheral sensory nerve fibers in humans. Finding a solution for daily work in the laboratory is a complex process. We have designed templates based on odML and odML-tables to structure and capture metadata and provided an extension to the existing GUI to enable database searching.

In a healthy state, pain plays an important role in natural biofeedback loops and helps to detect and prevent potentially harmful stimuli and situations. However, pain can become chronic and as such a pathological condition, losing its informative and adaptive function. Efficient pain treatment remains a largely unmet clinical need. One promising route to improve the characterization of pain, and with that the potential for more effective pain therapies, is the integration of different data modalities through cutting edge computational methods. Using these methods, multiscale, complex, and network models of pain signaling can be created and utilized for the benefit of patients. Such models require collaborative work of experts from different research domains such as medicine, biology, physiology, psychology as well as mathematics and data science. Efficient work of collaborative teams requires developing of a common language and common level of understanding as a prerequisite. One of ways to meet this need is to provide easy to comprehend overviews of certain topics within the pain research domain. Here, we propose such an overview on the topic of pain assessment in humans for computational researchers. Quantifications related to pain are necessary for building computational models. However, as defined by the International Association of the Study of Pain (IASP), pain is a sensory and emotional experience and thus, it cannot be measured and quantified objectively. This results in a need for clear distinctions between nociception, pain and correlates of pain. Therefore, here we review methods to assess pain as a percept and nociception as a biological basis for this percept in humans, with the goal of creating a roadmap of modelling options.

Background Migraine shows a cyclic pattern with an inter-ictal-, a pre-ictal, an ictal- and a post-ictal phase. We aimed to examine changes in psychophysical parameters during the migraine cycle. Methods The perception of nociceptive and non-nociceptive stimuli and an electrically induced axon-reflex-erythema were assessed in 20 healthy controls and 14 migraine patients on five consecutive days according to different phases of the migraine cycle. Pain was rated three times during a 10-second electrical stimulus. The size of the axon-reflex-erythema was determined using laser-Doppler-imaging. Intensity and hedonic estimates of odours presented by Sniffin’ Sticks were rated. Results In healthy controls, no significant changes over the test days were observed. In migraine patients pain thresholds at the head decreased with an ictal minimum. Less habituation after five seconds of stimulation at the head was found pre-ictally, whereas reduced habituation to 10-second electrical stimulation was present in all phases. The axon-reflex-erythema size showed an inter-ictal-specific minimum at the head. odours were perceived ictally as more unpleasant and intense. Conclusions Somatosensory functions, pain thresholds and habituation as predominantly central parameters, axon-reflex-erythema as a peripheral function of trigeminal neurons and odour perception as a predominantly extra-thalamic sensation change specifically over the migraine cycle indicating complex variations of neuronal signal processing.

openMNGlab is an open-source software framework for data analysis, tailored for the specific needs of microneurography - a type of electrophysiological technique particularly important for research on peripheral neural fibers coding. Currently, openMNGlab loads data from Spike2 and Dapsys, which are two major data acquisition solutions. By building on top of the Neo software, openMNGlab can be easily extended to handle the most common electrophysiological data formats. Furthermore, it provides methods for data visualization, fiber tracking, and a modular feature database to extract features for data analysis and machine learning.

openMNGlab is an open-source software framework for data analysis, tailored for the specific needs of microneurography – a type of electrophysiological technique particularly important for research on peripheral neural fibers coding. Currently, openMNGlab loads data from Spike2 and Dapsys, which are two major data acquisition solutions. By building on top of the Neo software, openMNGlab can be easily extended to handle the most common electrophysiological data formats. Furthermore, it provides methods for data visualization, fiber tracking, and a modular feature database to extract features for data analysis and machine learning.

Frontiers in molecular neuroscience 14, 709228 (2021). doi:10.3389/fnmol.2021.709228 special issue: "Brain Disease Mechanisms", Published by Frontiers Research Foundation, Lausanne

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

The endogenous metabolite methylglyoxal (MG) accumulates in diabetic patients with neuropathic pain. Methylglyoxal could be a mediator of diabetes-induced neuropathic pain through TRPA1 activation and sensitization of the voltage-gated sodium channel subtype 1.8. In this study, we tested the algogenic and sensitizing effect of MG in healthy human subjects using intracutaneous microinjections. The involvement of C fibers was assessed through selective A-fiber nerve block, axon-reflex-erythema, and through single nerve fiber recordings in humans (microneurography). Involvement of the transduction channels TRPA1 and TRPV1 in MG-induced pain sensation was investigated with specific ion channel blockers. We showed for the first time in healthy humans that MG induces pain, axon-reflex-erythema, and long-lasting hyperalgesia through the activation of C nociceptors. Predominantly, the subclass of mechano-insensitive C fibers is activated by MG. A fibers contribute only negligibly to the burning pain sensation. Selective pharmacological blockade of TRPA1 or TRPV1 showed that TRPA1 is crucially involved in MG-induced chemical pain sensation and heat hyperalgesia. In conclusion, the actions of MG through TRPA1 activation on predominantly mechano-insensitive C fibers might be involved in spontaneously perceived pain in diabetic neuropathy and hyperalgesia as well as allodynia.

One of the important questions in the research on neural coding is how the preceding axonal activity affects the signal propagation speed of the following one. We present an approach to solving this problem by introducing a multi-level spike count for activity quantification and fitting a family of linear regression models to the data. The best-achieved score is R2=0.89 and the comparison of different models indicates the importance of long and very short nerve fiber memory. Further studies are required to understand the complex axonal mechanisms responsible for the discovered phenomena.

One of the important questions in the research on neural coding is how the preceding axonal activity affects the signal propagation speed of the following one. We present an approach to solving this problem by introducing a multi-level spike count for activity quantification and fitting a family of linear regression models to the data. The best-achieved score is R2=0.89 and the comparison of different models indicates the importance of long and very short nerve fiber memory. Further studies are required to understand the complex axonal mechanisms responsible for the discovered phenomena.

BackgroundApart from the most prominent symptoms in Autism spectrum disorder (ASD), namely deficits in social interaction and repetitive behavior, patients often show abnormal sensory reactivity to environmental stimuli. Especially potentially painful stimuli are reported to be experienced in a different way compared to healthy persons.MethodsIn our present study, we present an ASD patient carrying compound heterozygous mutations in the voltage-gated sodium channel (VGSC) Nav1.8, which is preferentially expressed in sensory neurons. We expressed both identified mutations, p.I1511M and p.R512X, in a heterologous expression system and investigated their biophysical properties using patch-clamp recordings. ResultsThe results of these experiments suggest that both mutations lead to different degrees of loss-of-function of Nav1.8. Behavioral experiments in a Nav1.8 loss-of-function mouse model additionally revealed Nav1.8 may play a role in autistic behavior. LimitationsOur study did not verified the functions of p.I1511M and p.R512X mutations in vivo. The mice with these mutations can be constructed to verify the mutation functions in the future investigations. In addition, since we have only found one ASD patient which may relate to SCN10A mutations, the role of Nav1.8 in ASD needs to be confirmed in more cases. Moreover, the cellular mechanism underlying the effect of Nav1.8 on ASD needed to be explored in future studies.ConclusionsOur results present Nav1.8 as a protein potentially involved in ASD pathophysiology and may therefore offer new insights to the genetic basis of this disease.

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 even 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 pig and human, 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 for 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 condition 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. This article is protected by copyright. All rights reserved.

Migraine attacks can be accompanied by many different symptoms, some of them appearing within 24 hours before the onset of the headache. In previous work, reduced habituation to an electrical pain stimulus at the head was observed in the pre-ictal phase within 24 hours before the headache attack. Based on these results, this work presents an application to track influence factors on migraine attacks and an Arduino-based control unit which replaces the traditional approach of manual electrical stimulation. The usability of both components of the project was evaluated in separate user studies. Results of the usability study show a good acceptance of the systems with a mean SUS score of 92.4. Additionally, they indicate that the developed control unit may substitute the current manual electrical stimulation. Overall, the designed system allows standardized repeatable measurements and is a first step towards the home-use of a device for establishing a new method for migraine prediction.

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.

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."

Supplemental Digital Content is Available in the Text. Human sodium channel NaV1.7 in induced pluripotent stem cell–derived sensory neurons sets the action potential threshold but does not support subthreshold depolarizations., The chronic pain syndrome inherited erythromelalgia (IEM) is attributed to mutations in the voltage-gated sodium channel (NaV) 1.7. Still, recent studies targeting NaV1.7 in clinical trials have provided conflicting results. Here, we differentiated induced pluripotent stem cells from IEM patients with the NaV1.7/I848T mutation into sensory nociceptors. Action potentials in these IEM nociceptors displayed a decreased firing threshold, an enhanced upstroke, and afterhyperpolarization, all of which may explain the increased pain experienced by patients. Subsequently, we investigated the voltage dependence of the tetrodotoxin-sensitive NaV activation in these human sensory neurons using a specific prepulse voltage protocol. The IEM mutation induced a hyperpolarizing shift of NaV activation, which leads to activation of NaV1.7 at more negative potentials. Our results indicate that NaV1.7 is not active during subthreshold depolarizations, but that its activity defines the action potential threshold and contributes significantly to the action potential upstroke. Thus, our model system with induced pluripotent stem cell–derived sensory neurons provides a new rationale for NaV1.7 function and promises to be valuable as a translational tool to profile and develop more efficacious clinical analgesics.

Background Etomidate is a preferred drug for the induction of general anesthesia in cardiovascular risk patients. As with propofol and other perioperatively used anesthetics, the application of aqueous etomidate formulations causes an intensive burning pain upon injection. Such algogenic properties of etomidate have been attributed to the solubilizer propylene glycol which represents 35% of the solution administered clinically. The aim of this study was to investigate the underlying molecular mechanisms which lead to injection pain of aqueous etomidate formulations. Results Activation of the nociceptive transient receptor potential (TRP) ion channels TRPA1 and TRPV1 was studied in a transfected HEK293t cell line by whole-cell voltage clamp recordings of induced inward ion currents. Calcium influx in sensory neurons of wild-type and trp knockout mice was ratiometrically measured by Fura2-AM staining. Stimulated calcitonin gene-related peptide release from mouse sciatic nerves was detected by enzyme immunoassay. Painfulness of different etomidate formulations was tested in a translational human pain model. Etomidate as well as propylene glycol proved to be effective agonists of TRPA1 and TRPV1 ion channels at clinically relevant concentrations. Etomidate consistently activated TRPA1, but there was also evidence for a contribution of TRPV1 in dependence of drug concentration ranges and species specificities. Distinct N-terminal cysteine and lysine residues seemed to mediate gating of TRPA1, although the electrophile scavenger N-acetyl-L-cysteine did not prevent its activation by etomidate. Propylene glycol-induced activation of TRPA1 and TRPV1 appeared independent of the concomitant high osmolarity. Intradermal injections of etomidate as well as propylene glycol evoked severe burning pain in the human pain model that was absent with emulsification of etomidate. Conclusions Data in our study provided evidence that pain upon injection of clinical aqueous etomidate formulations is not an unspecific effect of hyperosmolarity but rather due to a specific action mediated by activated nociceptive TRPA1 and TRPV1 ion channels in sensory neurons.

Introduction Migraine is characterized by cycling phases (interictal, preictal, ictal and postictal) with differing symptoms, while in chronic tension type headache pain phases are fluctuating. The question we asked is whether these phases are associated with changes in parameters of somatosensation and axon-reflex erythema. Methods Patients with episodic migraine and chronic tension type headache were examined psychophysically in the interictal, preictal and ictal phase and healthy subjects on five different test days. Thresholds and suprathreshold ratings of pressure and electrical pain were assessed on three different regions of the head. In migraine patients and in healthy controls, electrically induced axon-reflex erythema was measured in the area of the first trigeminal branch. All migraine patients filled out questionnaires about prodromal symptoms at every visit. Results The axon-reflex erythema was always larger in patients with migraine in contrast to healthy subjects. The pressure pain threshold was lower in migraine patients and chronic tension type headache in comparison to healthy subjects. Electrical pain thresholds did not differ between headache patients and healthy subjects and showed no changes between the phases. However, suprathreshold pain ratings showed less habituation solely in the preictal phase of migraine. The number of prodromal symptoms in migraine patients was increased in the preictal and ictal phase. Discussion Reduced habituation was the unique sign of the preictal phase in migraine patients, independently of prodromal symptoms, whereas a larger axon-reflex erythema and higher pressure pain sensitivity are constitutional and non-phase dependent properties of migraine. Reduced inhibitory mechanisms in the preictal phase may contribute to trigger headache attacks in migraine.

Develop and validate a low-intensity sinusoidal electrical stimulation paradigm to preferentially activate C-fibers in human skin.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).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.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.

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.

Background Inflammatory mediators activate and sensitize nociceptors. Tissue acidosis with low pH of 5.5 often accompanies inflammation and could enhance inflammatory pain and sensitization. Methods Action potentials from single mechano-responsive (CM) and mechano-insensitive (CMi) C-nociceptors of cutaneous fascicles of the peroneal nerve in healthy volunteers were recorded by microneurography. Low pH solutions with and without prostaglandin E2 (PGE2) were injected twice (with an interval of approximately 5 min) into two spots of the receptive fields of C-fibres. Heat thresholds of the C-fibres were obtained before and after each injection. Results Injections of the low pH solutions immediately induced phasic responses in CM nociceptors, whereas CMi fibres responded after a delay of several seconds with a sustained response. More CMi fibres than CM fibres showed ongoing discharge after low pH injection, but the duration and intensity of the responses to the first low pH injection did not differ between them. Upon repetition, duration and intensity of the pH responses increased more than twofold in CMi fibres only. Furthermore, combined application of pH and PGE2 sensitized the response in CMi fibres only. In contrast, heat activation thresholds were sensitized by the combination of low pH and PGE2 in both fibre classes. Conclusions Our results confirm nociceptor class independent heat sensitization by PGE2 which is probably mediated by transient receptor potential vanilloid 1 phosphorylation. However, prolonged and increased pain responses in humans upon low pH/PGE2 stimulation appear to be primarily dependent on CMi fibres, whereas CM nociceptors appear crucial for phasic responses.

The nonselective sodium channel blocker lidocaine is widely used as a local anesthetic but also systemically for treatment of postoperative and neuropathic pain. Voltage-gated sodium channels are crucial for action potential generation and conduction, and their availability controls the amount of activity-dependent conduction velocity slowing. This important axonal property, as assessed by microneurography, is used to differentiate human mechanoinsensitive (silent) nociceptors from the classical polymodal nociceptors. In the current study, microneurography was used to assess axonal properties of the 2 main nociceptor classes in humans, before and after intradermal injection of lidocaine .1% or control saline solution in the receptive field. In mechanosensitive nociceptors, lidocaine reduced baseline conduction velocity and turned activity-dependent slowing into speeding of conduction. In contrast, mechanoinsensitive fibers were not affected in their baseline conduction velocity or their activity-dependent slowing, but probability of conduction block with repetitive stimulation increased. Recovery cycles showed reduced hyperpolarization in all C-fiber classes after lidocaine injections. These results support our hypothesis that sodium channel subtypes are differentially expressed in the 2 nociceptor classes of mechanosensitive C-fibers (CMs) and mechanoinsensitive C-fibers (CMis). Perspective This study reveals that microneurography can be used to assess pharmacological effects on single C-fibers directly in humans.

Acidosis occurs in a variety of pathophysiological and painful conditions where it is thought to excite or contribute to excitation of nociceptive neurons. Despite potential clinical relevance the principal receptor for sensing acidosis is unclear, but several receptors have been proposed. We investigated the contribution of the acid-sensing ion channels, transient receptor potential vanilloid type 1 (TRPV1) and transient receptor potential ankyrin type 1 (TRPA1) to peripheral pain signaling. We first established a human pain model using intraepidermal injection of the TRPA1 agonist carvacrol. This resulted in concentration-dependent pain sensations, which were reduced by experimental TRPA1 antagonist A-967079. Capsaicin-induced pain was reduced by the TRPV1 inhibitor BCTC. Amiloride was used to block acid-sensing ion channels. Testing these antagonists in a double-blind and randomized experiment, we probed the contribution of the respective channels to experimental acidosis-induced pain in 15 healthy human subjects. A continuous intraepidermal injection of pH 4.3 was used to counter the buffering capacity of tissue and generate a prolonged painful stimulation. In this model, addition of A-967079, BCTC or amiloride did not reduce the reported pain. In conclusion, target-validated antagonists, applied locally in human skin, have excluded the main hypothesized targets and the mechanism of the human acidosis-induced pain remains unclear. Perspective An acidic milieu is a trigger of pain in many clinical conditions. The aim of this study was to identify the contribution of the currently hypothesized sensors of acid-induced pain in humans. Surprisingly, inhibition of these receptors did not alter acidosis-induced pain.

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.

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).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.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.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.

Introduction Objective diagnosis of small fiber impairment is difficult. Methods We used the quantitative sudomotor axon reflex test (QSART) and axon-reflex-flare-test in the foot and thigh of 46 patients with peripheral neuropathy to assess C-fiber function in addition to conventional neurography and thermal threshold testing. Results In all patients, small fiber impairment was suspected because of abnormal warmth detection thresholds (76% of all tested) and/or pain in the feet. A total of 83% had reduced axon-reflex flare areas and 17% lower QSART scores. Patients with pure small fiber neuropathy had higher rates of reduced flare areas (87.5%) and sweating rates (25.5%). There was no difference between patients with and without pain regarding thermotesting and axon-reflex testing. Conclusions Both axon-reflex tests are helpful to identify objectively patients with small fiber impairment. Afferent and efferent C-fiber classes can be impaired differently. These tests detect small fiber impairment, but they cannot differentiate between painful and nonpainful neuropathy. Muscle Nerve 47: 357–363, 2013

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.

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

Background Anewexperimental protocol of electrically induced pain and hyperalgesia was established to examine orally administered drugs. In a randomized, double-blind, placebo-controlled cross-over study this experimental protocol was used to assess the effects of paracetamol. Methods Twenty-four subjects were enrolled in this study. The magnitude of pain, axon reflex flare, and areas of pin-prick hyperalgesia and touch-evoked allodynia were assessed in two consecutive sessions; prior to, and 2 h after drug administration. This protocol was repeated after 1 week. Subjects were randomized to receive either paracetamol (2 g) or a placebo. Results In comparison to the placebo arm there were no significant effects of paracetamol on pain, hyperalgesia, allodynia, or axon reflex flare. Pain and flare responses were highly reproducible on the same day (r = 0.77 and r = 0.79, respectively), and after 1 week (r = 0.6 and r = 0.71, respectively). The correlation between areas of hyperalgesia and allodynia was, however, significantly improved when the protocol was repeated on the same day (r = 0.8 and r = 0.75), as opposed to after a week (r = 0.54 and r = 0.53). Discussion The electrical pain model is a well established method for the assessment of intravenously applied analgesics. In order to assess effects of orally applied drugs the model had to be modified: for the assessment of hyperalgesia and allodynia a protocol repeating the model within 1 day proved to have advantages over repetition after 1 week.

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 (

The size of the neurogenic axon reflex flare (ARFS) has been proposed to serve as a non-invasive measure of C-fiber neuropathies. This idea is based on the observation that ARFS is often reduced in patients with small-fiber neuropathies. In this study, we compared ARFS and electrically evoked axon reflex sweating with intraepidermal nerve fiber density (IENF) in patients with peripheral neuropathy in order to validate these methods against an objective standard method of diagnosing small-fiber neuropathy. ARFS was significantly correlated with IENF, while axon reflex sweating was not correlated to IENF. We conclude that measurement of ARFS is a potential objective non-invasive diagnostic tool for analysis of C-fiber function in patients with small-fiber neuropathies.

Physiological changes in the nervous system occur with ageing. Both a decline of function and a decrease in the number of C-fibres in the skin have been reported for healthy aged subjects. With the use of microneurographic recordings from single C-fibres in humans we have compared the sensory and axonal properties of these neurones in young and aged healthy subjects. A total of 146 C-fibres were recorded from the common peroneal nerve in young subjects (mean age 24.7 years) and 230 C-fibres were recorded in aged subjects (mean age 56.2 years). In aged subjects, changes were found in the composition of the C-fibre population and in sensory and axonal properties. The relative incidence of afferent to efferent C-fibres was relatively constant independent of the age of subjects. The ratio of mechano-responsive to mechano-insensitive nociceptors was approximately 8 : 2 in the young controls while in aged subjects it was 7 : 3. In aged subjects 13% of the fibres showed atypical discharge characteristics, while this was not observed in young subjects. Spontaneous activity, sensitization and loss of sensory function were found regularly. Changes in functions of the conductile membrane were also observed in fibres from aged subjects. The degree of activity-dependent conduction velocity slowing in response to high frequency stimulation (2 Hz) was more pronounced, while the normalization of conduction velocity subsequent to high frequency stimulation was protracted. We found that both sensitization and desensitization or degeneration of afferent C-fibres occur with age, but are still rare compared to patients with neuropathy. The changes in the axonal properties of C-fibres in aged subjects are compatible with hypoexcitability of the fibres. These findings are important for the understanding and differential diagnoses regarding pathological processes and normal ageing.

Cowhage and histamine, both applied via spicules, were used to induce itch. The quality and intensity of the sensations, axon reflex flare, sympathetic skin vasoconstrictions and the interference of scratching with itch processing were studied. Axon reflex flare reactions were measured by laser Doppler imaging and reflex vasoconstrictions in the finger were recorded by laser Doppler flowmetry. Magnitude of itch sensations was assessed on an electronic visual analogue scale while the skin was intermittently scratched proximal to the application site. The quality of itch was assessed with a questionnaire. Only histamine produced an axon reflex flare. Histamine itch increased faster, but recovered more slowly after scratching, by which it was more effectively suppressed. Cowhage induced a sharper itch sensation and stronger vasoconstrictor reflexes. These findings support the notion that both agents activate different pathways. The differences in sympathetic reflex induction and in the modulation by scratching indicate differential central nervous processing.

Local cold injury often induces hypersensitivity to cold and cold allodynia. Sensitisation of TRPM8 or TRPA1 could be the underlying mechanisms. This was evaluated by psychophysics and axon-reflex-flare induction following topical menthol and cinnamaldehyde application in cold injury patients and healthy subjects. The patients had no signs of neuropathy except cold allodynia. We applied 20% cinnamaldehyde and 40% menthol solutions in the cold-allodynic area of the patients and in a corresponding area in healthy subjects and obtained sensory ratings during application. Thermotesting and Laser Doppler Imaging were performed before and after exposure to the compounds. Menthol did not induce axon-reflex-erythema in patients or in controls. After menthol cold pain threshold was decreased in healthy subjects; however, no further sensitisation was observed in the patients moreover in some patients an amelioration of their cold allodynia was observed. Cinnamaldehyde-induced pain sensation did not differ between patients and controls. Heat pain thresholds following cinnamaldehyde were lowered to a similar extent in patients and controls (43-39.8 and 44-39 degrees C) and also the axon-reflex-flare responses were comparable. No evidence for sensitisation of responses to TRPM8 or TRPA1-stimulation was found in patients with cold injury-induced cold allodynia. The lack of TRPM8 induced axon-reflex indicates that also de-novo expression of TRPM8 on mechano-insensitive C-nociceptors does not underlie cold allodynia in these patients. We conclude from these data that the mechanisms for the induction of cold allodynia in the patients with cold injury are independent of TRPM8 or TRPA1 and differ therefore from neuropathic pain patients.

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 (75 g) 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 (>15 min). 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.

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 μl, 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 μl) 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.

Photosensitization, an exaggerated sensitivity to harmless light, occurs genetically in rare diseases, such as porphyrias, and in photodynamic therapy where short-term toxicity is intended. A common feature is the experience of pain from bright light. In human subjects, skin exposure to 405 nm light induced moderate pain, which was intensified by pretreatment with aminolevulinic acid. In heterologous expression systems and cultured sensory neurons, exposure to blue light activated TRPA1 and, to a lesser extent, TRPV1 channels in the absence of additional photosensitization. Pretreatment with aminolevulinic acid or with protoporphyrin IX dramatically increased the light sensitivity of both TRPA1 and TRPV1 via generation of reactive oxygen species. Artificial lipid bilayers equipped with purified human TRPA1 showed substantial single-channel activity only in the presence of protoporphyrin IX and blue light. Photosensitivity and photosensitization could be demonstrated in freshly isolated mouse tissues and led to TRP channel-dependent release of proinflammatory neuropeptides upon illumination. With antagonists in clinical development, these findings may help to alleviate pain during photodynamic therapy and also allow for disease modification in porphyria patients.SIGNIFICANCE STATEMENTCutaneous porphyria patients suffer from burning pain upon exposure to sunlight and other patients undergoing photodynamic therapy experience similar pain, which can limit the therapeutic efforts. This study elucidates the underlying molecular transduction mechanism and identifies potential targets of therapy. Ultraviolet and blue light generates singlet oxygen, which oxidizes and activates the ion channels TRPA1 and TRPV1. The disease and the therapeutic options could be reproduced in models ranging from isolated ion channels to human subjects, applying protoporphyrin IX or its precursor aminolevulinic acid. There is an unmet medical need, and our results suggest a therapeutic use of the pertinent antagonists in clinical development.

Background The marking technique in microneurography uses stimulus-induced changes in neural conduction velocity to characterize human C-fibers. Changes in conduction velocity are manifested as variations in the temporal latency between periodic electrical stimuli and the resulting APs. When successive recorded sweeps are displayed vertically in a “waterfall” format, APs correlated with the stimulus form visible vertical tracks. Automated detection of these latency tracks is made difficult by sometimes poor signal-to-noise ratio in recordings, spontaneous neural firings uncorrelated with the stimuli, and multi-unit recordings with crossing or closely parallel tracks. New method We developed an automated track-detection technique based on a local linearization of the latency tracks of stimulus-correlated APs. This technique enhances latency tracks, eliminates transient noise spikes and spontaneous neural activity not correlated with the stimulus, and automatically detects latency tracks across successive sweeps in a recording. Results We evaluated our method on microneurography recordings showing varying signal quality, spontaneous firing, and multiple tracks that run closely parallel and cross. The method showed excellent detection of latency tracks in all of our recordings. Comparison with existing method(s) We compare our method to the commonly used track detection method of Hammarberg as implemented in the Drever program. Conclusions Our method is a robust means of automatically detecting latency tracks in typical microneurography recordings.

Kidney disease is associated with increased cardiovascular morbidity, but underlying mechanisms are poorly understood. We tested the hypothesis that chronic renal insufficiency impairs angioadaptation in a rat model of hindlimb ischemia. Twenty male Sprague-Dawley rats (8 weeks old) underwent subtotal nephrectomy (5/6SNX) or sham surgery (each n=10). Ten weeks later, unilateral hindlimb ischemia was induced in all animals. Hindlimb perfusion was assessed by laser Doppler perfusion imaging and fluorescent microsphere injection studies 2 weeks after surgery. Ischemia-induced angiogenesis was measured by analyzing capillary density using CD31 immunofluorescence. Expression of vascular endothelial growth factor (VEGF), its receptors (VEGFRs) and inducible as well as endothelial nitric oxide (NO) synthase was measured by real-time reverse transcription-polymerase chain reaction. Laser Doppler hindpaw perfusion was significantly reduced in 5/6SNX compared to sham-operated animals. Impaired hindlimb re-perfusion in 5/6SNX vs control rats was confirmed by fluorescent microsphere injection studies (relative perfusion of ischemic vs non-ischemic limb: 68.9+/-6.4 vs 92.4+/-3.6%, P=0.005). Ischemic skeletal muscle neovascularization increased to a greater extent in sham-operated compared to 5/6SNX rats (69+/-8 vs 29+/-7%, P0.05). VEGF and VEGFR-1/2 mRNA expression increased in ischemic hindlimbs of control rats, whereas no change or a decrease was observed in 5/6SNX. In contrast, inducible and endothelial NO synthase expression did not significantly differ between sham and 5/6SNX rats. Chronic renal insufficiency impairs angiogenesis and limb perfusion in a rat hindlimb ischemia model. Impaired angioadaptation may contribute to the poor prognosis of patients with renal failure suffering from peripheral arterial disease.

Both thin afferent (nociceptors) and efferent (sympathetic sudomotor) nerve fibers can be activated electrically and chemically, resulting in neurogenic erythema and sweating. These reactions have been used before to assess the impairment of sympathetic and nociceptor fibers in humans. In this study, electrically induced sweating and erythema were assessed simultaneously in the foot dorsum and thigh, and were compared to chemically induced activation. Reproducible intensity-response relations (stimulation intensities 0-30 mA, 1 Hz) were obtained from 32 subjects. The steepest increase of the sweat response was induced at lower intensities as compared to that of the erythema (18.3 mA vs. 25.7 mA, p0.01) and reached a plateau for intensities above 25 mA, suggesting lower electrical thresholds for sudomotor fibers. Maximum flare areas induced electrically with 30 mA were smaller than those evoked chemically (flare size: 4.5 cm2 vs. 10.6 cm2). In contrast, the electrically evoked sweating rate was higher than that evoked chemically (acetylcholine, or ACh; sweating rate 0.31 vs. 0.21 microl/cm2/min, p0.01), which might be attributed to an increased effectiveness of synchronized discharge in sympathetic fibers upon electrical stimulation.

Following each action potential, C-fiber nociceptors undergo cyclical changes in excitability, including a period of superexcitability, before recovering their basal excitability state. The increase in superexcitability during this recovery cycle depends upon their immediate firing history of the axon, but also determines the instantaneous firing frequency that encodes pain intensity. To explore the mechanistic underpinnings of the recovery cycle phenomenon a biophysical model of a C-fiber has been developed. The model represents the spatial extent of the axon including its passive properties as well as ion channels and the Na/K-ATPase ion pump. Ionic concentrations were represented inside and outside the membrane. The model was able to replicate the typical transitions in excitability from subnormal to supernormal observed empirically following a conducted action potential. In the model, supernormality depended on the degree of conduction slowing which in turn depends upon the frequency of stimulation, in accordance with experimental findings. In particular, we show that activity-dependent conduction slowing is produced by the accumulation of intraaxonal sodium. We further show that the supernormal phase results from a reduced potassium current Kdr as a result of accumulation of periaxonal potassium in concert with a reduced influx of sodium through Nav1.7 relative to Nav1.8 current. This theoretical prediction was supported by data from an in vitro preparation of small rat dorsal root ganglion somata showing a reduction in the magnitude of tetrodotoxin-sensitive relative to tetrodotoxin -resistant whole cell current. Furthermore, our studies provide support for the role of depolarization in supernormality, as previously suggested, but we suggest that the basic mechanism depends on changes in ionic concentrations inside and outside the axon. The understanding of the mechanisms underlying repetitive discharges in recovery cycles may provide insight into mechanisms of spontaneous activity, which recently has been shown to correlate to a perceived level of pain.

Background and aims: This study on healthy test subjects intends to show whether one-off Low-Level Laser Therapy (LLLT) has an instant effect on the perfusion or the oxygenation of the skin tissue. These possible instant effects may have an influence on the accelerated wound healing which is often observed after application of LLLT, in addition to the usual postulated effects of LLLT which occur with a time delay normally. Study design/materials and methods: The study was carried out double-blind and placebo-controlled in two batches of testing. The test subjects received one-off LLLT on a defined area of the arch of the foot. Simultaneously a placebo treatment was carried out on the corresponding contralateral area. In the first batch of tests, the blood flow was measured immediately before and after treatment using thermography and LDI. In the second batch of tests, the blood flow and the oxygen saturation were determined immediately before and after the treatment using an O2C device. Results: No evidence that the LLLT has a significant instant effect on the circulation or the oxygen saturation could be found. Conclusion: No immediate effect of an LLLT on the perfusion or oxygenation situation is to be expected with physiologically normal starting conditions. An additional investigation should be carried out in which either the radiation dose is varied or the starting conditions are pathological (e.g. chronic wounds) in order to rule out immediate effects on circulation or oxygen saturation as the cause of the improved wound healing which is often observed.

Ciguatera, the most common form of nonbacterial ichthyosarcotoxism, is caused by consumption of fish that have bioaccumulated the polyether sodium channel activator ciguatoxin. The neurological symptoms of ciguatera include distressing, often persistent sensory disturbances such as paraesthesias and the pathognomonic symptom of cold allodynia. We show that intracutaneous administration of ciguatoxin in humans elicits a pronounced axon-reflex flare and replicates cold allodynia. To identify compounds able to inhibit ciguatoxin-induced Nav responses, we developed a novel in vitro ciguatoxin assay using the human neuroblastoma cell line SH-SY5Y. Pharmacological characterisation of this assay demonstrated a major contribution of Nav1.2 and Nav1.3, but not Nav1.7, to ciguatoxin-induced Ca2+ responses. Clinically available Nav inhibitors, as well as the Kv7 agonist flupirtine, inhibited tetrodotoxin-sensitive ciguatoxin-evoked responses. To establish their in vivo efficacy, we used a novel animal model of ciguatoxin-induced cold allodynia. However, differences in the efficacy of these compounds to reverse ciguatoxin-induced cold allodynia did not correlate with their potency to inhibit ciguatoxin-induced responses in SH-SY5Y cells or at heterologously expressed Nav1.3, Nav1.6, Nav1.7, or Nav1.8, indicating cold allodynia might be more complex than simple activation of Nav channels. These findings highlight the need for suitable animal models to guide the empiric choice of analgesics, and suggest that lamotrigine and flupirtine could be potentially useful for the treatment of ciguatera.

Objective diagnosis of small fiber impairment is difficult.We used the quantitative sudomotor axon reflex test (QSART) and axon-reflex-flare-test in the foot and thigh of 46 patients with peripheral neuropathy to assess C-fiber function in addition to conventional neurography and thermal threshold testing.In all patients, small fiber impairment was suspected because of abnormal warmth detection thresholds (76% of all tested) and/or pain in the feet. A total of 83% had reduced axon-reflex flare areas and 17% lower QSART scores. Patients with pure small fiber neuropathy had higher rates of reduced flare areas (87.5%) and sweating rates (25.5%). There was no difference between patients with and without pain regarding thermotesting and axon-reflex testing.Both axon-reflex tests are helpful to identify objectively patients with small fiber impairment. Afferent and efferent C-fiber classes can be impaired differently. These tests detect small fiber impairment, but they cannot differentiate between painful and nonpainful neuropathy.

Topical application of lidocaine is an effective approach for treatment of post-herpetic neuralgia and other painful neuropathies. Lidocaine inhibits voltage-gated Na(+) channels and it most likely reduces excitability of cutaneous sensory neurons which can be hyperexcitable or spontaneously active in states of neuropathic pain. However, lidocaine and other local anesthetics also exert a pronounced neurotoxicity and they activate the irritant receptors TRPV1 and TRPA1. In this randomized and double-blinded study, we explored the ability of lidocaine patches (5%) to alter sensory function and epidermal nerve fiber density in skin of healthy volunteers. As assessed by quantitative sensory testing, significantly elevated thresholds for touch, pin prick pain and mechanically induced wind-up were observed in skin treated with lidocaine patches. These effects reversed to baseline values within 2days after termination of the treatment. Pressure pain and thresholds for heat and cold-induced pain were not affected by the lidocaine patch. A moderate but significant decrease in epidermal nerve fiber density was observed in skin blister roofs obtained after 42days of treatment with lidocaine patches. The placebo patch did not induce any changes in sensory thresholds or nerve fiber density. In conclusion, lidocaine patches seem to have differential effects on sensory modalities in healthy skin. A degeneration of epidermal nerve fibers has previously been demonstrated for patches containing the TRPV1-agonist capsaicin and our findings suggest that this effect might also be relevant for lidocaine patches. These data warrant further studies on molecular mechanisms mediating a relief of neuropathic pain by topical lidocaine.