Voltage-gated sodium channel Na 1.7 is required for acute and inflammatory pain in mice and humans but its significance for visceral pain is unknown. Here we examine the role of Na 1.7 in visceral pain processing and the development of referred hyperalgesia using a conditional nociceptor-specific Na 1.7 knockout mouse (Na 1.7 ) and selective small-molecule Na 1.7 antagonist PF-5198007. Na 1.7 mice showed normal nociceptive behaviours in response to intracolonic application of either capsaicin or mustard oil, stimuli known to evoke sustained nociceptor activity and sensitization following tissue damage, respectively. Normal responses following induction of cystitis by cyclophosphamide were also observed in both Na 1.7 and littermate controls. Loss, or blockade, of Na 1.7 did not affect afferent responses to noxious mechanical and chemical stimuli in nerve-gut preparations in mouse, or following antagonism of Na 1.7 in resected human appendix stimulated by noxious distending pressures. However, expression analysis of voltage-gated sodium channel α subunits revealed Na 1.7 mRNA transcripts in nearly all retrogradely labelled colonic neurons, suggesting redundancy in function. By contrast, using comparative somatic behavioural models we identify that genetic deletion of Na 1.7 (in Na 1.8-expressing neurons) regulates noxious heat pain threshold and that this can be recapitulated by the selective Na 1.7 antagonist PF-5198007. Our data demonstrate that Na 1.7 (in Na 1.8-expressing neurons) contributes to defined pain pathways in a modality-dependent manner, modulating somatic noxious heat pain, but is not required for visceral pain processing, and advocate that pharmacological block of Na 1.7 alone in the viscera may be insufficient in targeting chronic visceral pain.

SummaryNaV1.9 regulates normal colonic afferent mechanosensation and is required for hypersensitivity to noxious inflammatory mediators and those derived from inflammatory bowel disease tissues.

ObjectiveThe development of effective visceral analgesics free of deleterious gut-specific side effects is a priority. We aimed to develop a reproducible methodology to study visceral nociception in human tissue that could aid future target identification and drug evaluation.DesignElectrophysiological (single unit) responses of visceral afferents to mechanical (von Frey hair (VFH) and stretch) and chemical (bradykinin and ATP) stimuli were examined. Thus, serosal afferents (putative nociceptors) were used to investigate the effect of tegaserod, and transient receptor potential channel, vanilloid 4 (TRPV4) modulation on mechanical responses.ResultsTwo distinct afferent fibre populations, serosal (n=23) and muscular (n=21), were distinguished based on their differences in sensitivity to VFH probing and tissue stretch. Serosal units displayed sensitivity to key algesic mediators, bradykinin (6/14 units tested) and ATP (4/10), consistent with a role as polymodal nociceptors, while muscular afferents are largely insensitive to bradykinin (0/11) and ATP (1/10). Serosal nociceptor mechanosensitivity was attenuated by tegaserod (−20.8±6.9%, n=6, p<0.05), a treatment for IBS, or application of HC067047 (−34.9±10.0%, n=7, p<0.05), a TRPV4 antagonist, highlighting the utility of the preparation to examine the mechanistic action of existing drugs or novel analgesics. Repeated application of bradykinin or ATP produced consistent afferent responses following desensitisation to the first application, demonstrating their utility as test stimuli to evaluate analgesic activity.ConclusionsFunctionally distinct subpopulations of human visceral afferents can be demonstrated and could provide a platform technology to further study nociception in human tissue.

Activation of visceral nociceptors by inflammatory mediators contributes to visceral hypersensitivity and abdominal pain associated with many gastrointestinal disorders. Purine and pyrimidine nucleotides (e.g., ATP and UTP) are strongly implicated in this process following their release from epithelial cells during mechanical stimulation of the gut, and from immune cells during inflammation. Actions of ATP are mediated through both ionotropic P2X receptors and metabotropic P2Y receptors. P2X receptor activation causes excitation of visceral afferents; however, the impact of P2Y receptor activation on visceral afferents innervating the gut is unclear. Here we investigate the effects of stimulating P2Y receptors in isolated mouse colonic sensory neurons, and visceral nociceptor fibers in mouse and human nerve-gut preparations. Additionally, we investigate the role of Na v 1.9 in mediating murine responses. The application of UTP (P2Y 2 and P2Y 4 agonist) sensitized colonic sensory neurons by increasing action potential firing to current injection and depolarizing the membrane potential. The application of ADP (P2Y 1 , P2Y 12 , and P2Y 13 agonist) also increased action potential firing, an effect blocked by the selective P2Y 1 receptor antagonist MRS2500. UTP or ADP stimulated afferents, including mouse and human visceral nociceptors, in nerve-gut preparations. P2Y 1 and P2Y 2 transcripts were detected in 80% and 56% of retrogradely labeled colonic neurons, respectively. Na v 1.9 transcripts colocalized in 86% of P2Y 1 -positive and 100% of P2Y 2 -positive colonic neurons, consistent with reduced afferent fiber responses to UTP and ADP in Na v 1.9 Ϫ/Ϫ mice. These data demonstrate that P2Y receptor activation stimulates mouse and human visceral nociceptors, highlighting P2Y-dependent mechanisms in the generation of visceral pain during gastrointestinal disease.

The ability to sense visceral pain during appendicitis is diminished with age leading to delay in seeking healthcare and poorer clinical outcomes. To understand the mechanistic basis of this phenomenon, we examined visceral nociception in aged mouse and human tissue. Inflamed and non-inflamed appendix was collected from consenting patients undergoing surgery for the treatment of appendicitis or bowel cancer. Supernatants were generated by incubating samples in buffer and used to stimulate multiunit activity in intestinal preparations, or single unit activity from teased fibres in colonic preparations, of young and old mice. Changes in afferent innervation with age were determined by measuring the density of CGRP positive afferent fibres and by counting dorsal root ganglia back-labelled by injection of tracer dye into the wall of the colon. Finally, the effect of age on nociceptor function was studied in mouse and human colon. Afferent responses to appendicitis supernatants were greatly impaired in old mice. Further investigation revealed this was due to a marked reduction in the afferent innervation of the bowel, and a substantial impairment in the ability of the remaining afferent fibres to transduce noxious stimuli. Translational studies in human tissue demonstrated a significant reduction in the multiunit but not the single unit colonic mesenteric nerve response to capsaicin with age, indicative of a loss of nociceptor innervation. Our data demonstrates that anatomical and functional deficits in nociception occur with age, underpinning the atypical or silent presentation of appendicitis in the elderly.