Peripheral signaling pathways contributing to non-histaminergic itch in humans.

BACKGROUND: Chronic itch (chronic pruritus) is a major therapeutic challenge that remains poorly understood despite the extensive recent analysis of human pruriceptors. It is unclear how the peripheral nervous system differentiates the signaling of non-histaminergic itch and pain. METHODS: Here we used psychophysical analysis and microneurography (single nerve fiber recordings) in healthy human volunteers to explore the distinct signaling mechanisms of itch, using the pruritogens ß-alanine, BAM 8-22 and cowhage extract. RESULTS: The mode of application (injection or focal application using inactivated cowhage spicules) influenced the itch/pain ratio in sensations induced by BAM 8-22 and cowhage but not ß-alanine. We found that sensitizing pre-injections of prostaglandin E2 increased the pain component of BAM 8-22 but not the other pruritogens. A-fibers contributed only to itch induced by ß-alanine. TRPV1 and TRPA1 were necessary for itch signaling induced by all three pruritogens. In single-fiber recordings, we found that BAM 8-22 and ß-alanine injection activated nearly all CM-fibers (to different extents) but not CMi-fibers, whereas cowhage extract injection activated only 56% of CM-fibers but also 25% of CMi-fibers. A "slow bursting discharge pattern" was evoked in 25% of CM-fibers by ß-alanine, in 35% by BAM 8-22, but in only 10% by cowhage extract. CONCLUSION: Our results indicate that no labeled line exists for these pruritogens in humans. A combination of different mechanisms, specific for each pruritogen, leads to itching sensations rather than pain. Notably, non-receptor-based mechanisms such as spatial contrast or discharge pattern coding seem to be important processes. These findings will facilitate the discovery of therapeutic targets for chronic pruritus, which are unlikely to be treated effectively by single receptor blockade.

A novel role for follistatin in hypersensitivity following cystitis.

AIMS: Previous studies have shown that the activin-binding protein follistatin reduces inflammation in several mouse models of colitis. To determine whether follistatin also has a beneficial effect following bladder inflammation, we induced cystitis in mice using cyclophosphamide (CYP) and examined the relationship between bladder hypersensitivity and bladder follistatin expression. METHODS: Adult female C57BL/6 mice were treated with CYP (100 mg/kg) or vehicle (saline) three times over 5 days. Bladder hypersensitivity was assessed by recording the visceromotor response (VMR) to urinary bladder distension and in vitro single-fiber bladder afferent recording. Follistatin gene expression was measured using qRT-PCR. Immunohistochemistry was employed for further characterization. RESULTS: Bladder hypersensitivity was established by day 6 and persisted to day 14 in CYP-treated mice. On day 14, hypersensitivity was accompanied by increases in follistatin gene expression in the bladder. Follistatin-like immunoreactivity colocalized with laminin, and the percentage of structures in the lamina propria that were follistatin-positive was increased in CYP-treated mice. Exogenous follistatin increased VMR and afferent responses to bladder distension in CYP- but not vehicle-treated mice. CONCLUSIONS: Chronic bladder pain following CYP treatment is associated with increased follistatin expression in the bladder. These results suggest a novel, pro-nociceptive role for follistatin in cystitis, in contrast with its proposed therapeutic role in colitis. This protein has exciting potential as a biomarker and therapeutic target for bladder hypersensitivity. Neurourol. Urodynam. 36:286-292, 2017. © 2015 Wiley Periodicals, Inc.

Temperature increase significantly enhances nociceptive responses of C-fibers to ATP, high K+, and acidic pH in mice.

It is well established that temperature affects the functioning of almost all biomolecules and, consequently, all cellular functions. Here, we show how temperature variations within a physiological range affect primary afferents' spontaneous activity in response to chemical nociceptive stimulation. An ex vivo mouse hind limb skin-saphenous nerve preparation was used to study the temperature dependence of single C-mechanoheat (C-MH) fibers' spontaneous activity. Nociceptive fibers showed a basal spike frequency of 0.097 ± 0.013 Hz in control conditions (30°C). Non-surprisingly, this activity decreased at 20°C and increased at 40°C, showing moderate temperature dependence with Q10∼2.01. The fibers' conduction velocity was also temperature-dependent, with an apparent Q10 of 1.38. Both Q10 for spike frequency and conduction velocity were found to be in good correspondence with an apparent Q10 for ion channels gating. Then we examined the temperature dependence of nociceptor responses to high K+, ATP, and H+. Receptive fields of nociceptors were superfused with solutions containing 10.8 mM K+, 200 µM ATP, and H+ (pH 6.7) at three different temperatures: 20, 30, and 40°C. We found that at 30 and 20°C, all the examined fibers were sensitive to K+, but not to ATP or H+. At 20°C, only 53% of fibers were responsible for ATP; increasing the temperature to 40°C resulted in 100% of sensitive fibers. Moreover, at 20°C, all observed fibers were silent to pH, but at 40°C, this number was gradually increased to 87.9%. We have found that the temperature increase from 20 to 30°C significantly facilitated responses to ATP (Q10∼3.11) and H+ (Q10∼3.25), leaving high K+ virtually untouched (Q10∼1.88 vs. 2.01 in control conditions). These data suggest a possible role of P2X receptors in coding the intensity of non-noxious thermal stimuli.

Properties of Nav1.8ChR2-positive and Nav1.8ChR2-negative afferent mechanoreceptors in the hindpaw glabrous skin of mice.

Nav1.8-positive afferent fibers are mostly nociceptors playing a role in mediating thermal and mechanical pain, but mechanoreceptors within these afferents have not been fully investigated. In this study, we generated mice expressing channel rhodopsin 2 (ChR2) in Nav1.8-positive afferents (Nav1.8ChR2), which showed avoidance responses to mechanical stimulation and nocifensive responses to blue light stimulation applied to hindpaws. Using ex vivo hindpaw skin-tibial nerve preparations made from these mice, we characterized properties of mechanoreceptors on Nav1.8ChR2-positive and Nav1.8ChR2-negative afferent fibers that innervate the hindpaw glabrous skin. Of all Aß-fiber mechanoreceptors, small portion was Nav1.8ChR2-positive. Of all Aδ-fiber mechanoreceptors, more than half was Nav1.8ChR2-positive. Of all C-fiber mechanoreceptors, almost all were Nav1.8ChR2-positive. Most Nav1.8ChR2-positive Aß-, Aδ-, and C-fiber mechanoreceptors displayed slowly adapting (SA) impulses in response to sustained mechanical stimulation, and their mechanical thresholds were high in the range of high threshold mechanoreceptors (HTMRs). In contrast, sustained mechanical stimulation applied to Nav1.8ChR2-negative Aß- and Aδ-fiber mechanoreceptors evoked both SA and rapidly adapting (RA) impulses, and their mechanical thresholds were in the range of low threshold mechanoreceptors (LTMRs). Our results provide direct evidence that in the mouse glabrous skin, most Nav1.8ChR2-negative Aß-, Aδ-fiber mechanoreceptors are LTMRs involving in the sense of touch, whereas Nav1.8ChR2-positive Aß-, Aδ-, and C-fiber mechanoreceptors are mainly HTMRs involving in mechanical pain.

Responses of cutaneous C-fiber afferents and spinal microglia after hindlimb cast immobilization in rats.

Previous studies have shown that persistent limb immobilization using a cast increases nociceptive behavior to somatic stimuli in rats. However, the peripheral neural mechanisms of nociception remain unclear. Using single-fiber electrophysiological recordings in vitro, we examined the general characteristics of cutaneous C-fiber afferents in the saphenous nerve and their responsiveness to mechanical and heat stimuli in a rat model of immobilization-induced pain by subjecting the rats to hindlimb cast immobilization for 4 weeks. The mechanical response of C-fibers appeared to increase in the model; however, statistical analysis revealed that neither the response threshold nor the response magnitude was altered. The general characteristics and heat responses of the C-fibers were not altered. The number of microglia and cell diameters significantly increased in the superficial dorsal horn of the lumbar spinal cord. Thus, activated microglia-mediated spinal mechanisms are associated with the induction of nociceptive hypersensitivity in rats after persistent cast immobilization.

Targeting Two-Pore-Domain Potassium Channels by Mechanical Stretch Instantaneously Modulates Action Potential Transmission in Mouse Sciatic Nerves.

Recent reports indicate dominant roles of TRAAK and TREK-1 channels, i.e., mechanosensitive two-pore-domain potassium channels (K2P) at the nodes of Ranvier for action potential repolarization in mammalian peripheral nerves. Functional changes in mammalian peripheral nerve conduction by mechanical stretch studied by recording compound action potentials lack the necessary resolution to detect subtle neuromodulatory effects on conduction velocity. In this study, we developed a novel in vitro approach that enables single-fiber recordings from individual mouse sciatic nerve axons while delivering computer-controlled stepped stretch to the sciatic nerve trunk. Axial stretch instantaneously increased the conduction delay in both myelinated A-fibers and unmyelinated C-fibers. Increases in conduction delay linearly correlated with increases in axial stretch ratio for both A- and C-fibers. The slope of the increase in conduction delay versus stretch ratio was steeper in C-fibers than in A-fibers. Moderate axial stretch (14-19% of in vitro length) reversibly blocked 37.5% of unmyelinated C-fibers but none of the eight myelinated A-fibers tested. Application of arachidonic acid, an agonist to TRAAK and TREK-1 to sciatic nerve trunk, blocks axonal transmission in both A- and C-fibers with delayed onset and prolonged block. Also, the application of an antagonist ruthenium red showed a tendency of suppressing the stretch-evoked increase in conduction delay. These results could draw focused research on pharmacological and mechanical activation of K2P channels as a novel neuromodulatory strategy to achieve peripheral nerve block.

Peripheral nociceptive mechanisms in an experimental rat model of fibromyalgia induced by repeated cold stress.

Fibromyalgia (FM) is a debilitating disease characterized by generalized and persistent musculoskeletal pain. Although central mechanisms are strongly implicated in the pathogenesis of FM, the involvement of peripheral mechanisms is poorly understood. To understand the peripheral nociceptive mechanisms, we examined muscular nociceptors in an FM model, which was made by exposing rats to repeated cold stress (RCS). A single muscle C-fiber nociceptors were identified through the teased fiber technique using ex vivo muscle-nerve preparations. Response properties of C-fibers to noxious stimuli were systematically analyzed. Messenger RNA expression of neurotrophic factors and inflammatory mediators were also studied in the muscle. In the RCS group, the mechanical response threshold of C-fibers, measured using a ramp mechanical stimulus, was significantly decreased, and the response magnitude was significantly increased in the RCS group when compared with the SHAM group, where the environmental temperature was not altered. The general characteristics of C-fibers and the responsiveness to noxious cold and heat stimuli were similar between the two groups. Messenger RNAs of neurotrophic factors and inflammatory mediators were not changed in the muscle during and after RCS. These results suggest that augmentation of the mechanical response of muscle C-fiber nociceptors contributes to hyperalgesia in the RCS model.

Vagally Mediated Gut-Brain Relationships in Appetite Control-Insights from Porcine Studies.

Signals arising from the upper part of the gut are essential for the regulation of food intake, particularly satiation. This information is supplied to the brain partly by vagal nervous afferents. The porcine model, because of its sizeable gyrencephalic brain, omnivorous regimen, and comparative anatomy of the proximal part of the gut to that of humans, has provided several important insights relating to the relevance of vagally mediated gut-brain relationships to the regulation of food intake. Furthermore, its large size combined with the capacity to become obese while overeating a western diet makes it a pivotal addition to existing murine models, especially for translational studies relating to obesity. How gastric, proximal intestinal, and portal information relating to meal arrival and transit are encoded by vagal afferents and their further processing by primary and secondary brain projections are reviewed. Their peripheral and central plasticities in the context of obesity are emphasized. We also present recent insights derived from chronic stimulation of the abdominal vagi with specific reference to the modulation of mesolimbic structures and their role in the restoration of insulin sensitivity in the obese miniature pig model.

Monocyte chemoattractant protein-1-induced excitation and sensitization to mechanical stimulation of mechanosensitive C-fiber afferents in rat skin.

It has been previously demonstrated that chemokine monocyte chemoattractant protein-1 (MCP-1/CCL2) increases the excitability of nociceptive neurons after peripheral nerve injury or inflammation. Moreover, decreased nocifensive mechanical threshold in behavioral tests and increased calcium influx in cultured dorsal root ganglion neurons by MCP-1 application have been reported. However, the effects of MCP-1 on peripheral afferent terminals have not been studied yet. The present study aimed to examine the effect of MCP-1 on the response of cutaneous unmyelinated afferents. For this purpose, single fiber recordings of mechanosensitive C-afferents were made in vitro from skin-saphenous nerve preparations excised from rats euthanized by CO2. Since IB4-positive neurons were previously implicated in MCP-1 induced mechanical hyperalgesia, sensitivity to α,ß-methylene ATP (metATP), an indicator of IB4-positive neurons, was also studied. Application of MCP-1 100 ng/ml to the receptive field elicited excitation in one half of mechanosensitive unmyelinated afferents in the skin. MCP-1 also sensitized metATP insensitive fibers to mechanical stimulation, but not metATP sensitive fibers. The incidence of heat sensitive fibers was decreased in the MCP-1 treated group with a decrease in the response threshold. These results demonstrate MCP-1 is an effective stimulant of mechanosensitive unmyelinated peripheral afferents in the rat skin.

Computational solution of spike overlapping using data-based subtraction algorithms to resolve synchronous sympathetic nerve discharge.

Sympathetic nerves conveying central commands to regulate visceral functions often display activities in synchronous bursts. To understand how individual fibers fire synchronously, we establish "oligofiber recording techniques" to record "several" nerve fiber activities simultaneously, using in vitro splanchnic sympathetic nerve-thoracic spinal cord preparations of neonatal rats as experimental models. While distinct spike potentials were easily recorded from collagenase-dissociated sympathetic fibers, a problem arising from synchronous nerve discharges is a higher incidence of complex waveforms resulted from spike overlapping. Because commercial softwares do not provide an explicit solution for spike overlapping, a series of custom-made LabVIEW programs incorporated with MATLAB scripts was therefore written for spike sorting. Spikes were represented as data points after waveform feature extraction and automatically grouped by k-means clustering followed by principal component analysis (PCA) to verify their waveform homogeneity. For dissimilar waveforms with exceeding Hotelling's T(2) distances from the cluster centroids, a unique data-based subtraction algorithm (SA) was used to determine if they were the complex waveforms resulted from superimposing a spike pattern close to the cluster centroid with the other signals that could be observed in original recordings. In comparisons with commercial software, higher accuracy was achieved by analyses using our algorithms for the synthetic data that contained synchronous spiking and complex waveforms. Moreover, both T(2)-selected and SA-retrieved spikes were combined as unit activities. Quantitative analyses were performed to evaluate if unit activities truly originated from single fibers. We conclude that applications of our programs can help to resolve synchronous sympathetic nerve discharges (SND).