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Chronic pelvic pain (CPP) is the most debilitating symptom of gynaecological disorders such as endometriosis. However, it remains unclear how sensory neurons from pelvic organs affected by endometriosis, such as the female reproductive tract, detect and transmit nociceptive events and how these signals are processed within the central nervous system (CNS). Using a previously characterized mouse model of endometriosis, we investigated whether the increased pain sensitivity occurring in endometriosis could be attributed to (i) changes in mechanosensory properties of sensory afferents innervating the reproductive tract, (ii) alterations in sensory input from reproductive organs to the spinal cord or (iii) neuroinflammation and sensitization of spinal neural circuits. Mechanosensitivity of vagina‐innervating primary afferents was examined using an ex vivo single‐unit extracellular recording preparation. Nociceptive signalling from the vagina to the spinal cord was quantified by phosphorylated MAP kinase ERK1/2 immunoreactivity. Immunohistochemistry was used to determine glial and neuronal circuit alterations within the spinal cord. We found that sensory afferents innervating the rostral, but not caudal portions of the mouse vagina, developed mechanical hypersensitivity in endometriosis. Nociceptive signalling from the vagina to the spinal cord was significantly enhanced in mice with endometriosis. Moreover, mice with endometriosis developed microgliosis, astrogliosis and enhanced substance P neurokinin‐1 receptor immunoreactivity within the spinal cord, suggesting the development of neuroinflammation and sensitization of spinal circuitry in endometriosis. These results demonstrate endometriosis‐induced neuroplasticity occurring at both peripheral and central sites of sensory afferent pathways. These findings may help to explain the altered sensitivity to pain in endometriosis and provide a novel platform for targeted pain relief treatments for this debilitating disorder. [ABSTRACT FROM AUTHOR]

Chronic pelvic pain (CPP) is the primary symptom of endometriosis patients, but adequate treatments are lacking. Modulation of ion channels expressed by sensory nerves innervating the viscera has shown promise for the treatment of irritable bowel syndrome and overactive bladder. However, similar approaches for endometriosis‐associated CPP remain underdeveloped. Here, we examined the role of the voltage‐gated sodium (NaV) channel NaV1.7 in (i) the sensitivity of vagina‐innervating sensory afferents and investigated whether (ii) NaV1.7 inhibition reduces nociceptive signals from the vagina and (iii) ameliorates endometriosis‐associated CPP. The mechanical responsiveness of vagina‐innervating sensory afferents was assessed with ex vivo single‐unit recording preparations. Pain evoked by vaginal distension (VD) was quantified by the visceromotor response (VMR) in vivo. In control mice, pharmacological activation of NaV1.7 with OD1 sensitised vagina‐innervating pelvic afferents to mechanical stimuli. Using a syngeneic mouse model of endometriosis, we established that endometriosis sensitised vagina‐innervating pelvic afferents to mechanical stimuli. The highly selective NaV1.7 inhibitor Tsp1a revealed that this afferent hypersensitivity occurred in a NaV1.7‐dependent manner. Moreover, in vivo intra‐vaginal treatment with Tsp1a reduced the exaggerated VMRs to VD which is characteristic of mice with endometriosis. Conversely, Tsp1a did not alter ex vivo afferent mechanosensitivity nor in vivo VMRs to VD in Sham control mice. Collectively, these findings suggest that NaV1.7 plays a crucial role in endometriosis‐induced vaginal hyperalgesia. Importantly, NaV1.7 inhibition selectively alleviated endometriosis‐associated CPP without the loss of normal sensation, suggesting that selective targeting of NaV1.7 could improve the quality of life of women with endometriosis. [ABSTRACT FROM AUTHOR]

The distal colon and rectum (colorectum) are innervated by spinal and vagal afferent pathways. The central circuits into which vagal and spinal afferents relay colorectal nociceptive information remain to be comparatively assessed. To address this, regional colorectal retrograde tracing and colorectal distension (CRD)‐evoked neuronal activation were used to compare the circuits within the dorsal vagal complex (DVC) and dorsal horn (thoracolumbar [TL] and lumbosacral [LS] spinal levels) into which vagal and spinal colorectal afferents project. Vagal afferent projections were observed in the nucleus tractus solitarius (NTS), area postrema (AP), and dorsal motor nucleus of the vagus (DMV), labeled from the rostral colorectum. In the NTS, projections were opposed to catecholamine and pontine parabrachial nuclei (PbN)‐projecting neurons. Spinal afferent projections were labeled from rostral through to caudal aspects of the colorectum. In the dorsal horn, the number of neurons activated by CRD was linked to pressure intensity, unlike in the DVC. In the NTS, 13% ± 0.6% of CRD‐activated neurons projected to the PbN. In the dorsal horn, at the TL spinal level, afferent input was associated with PbN‐projecting neurons in lamina I (LI), with 63% ± 3.15% of CRD‐activated neurons in LI projecting to the PbN. On the other hand, at the LS spinal level, only 18% ± 0.6% of CRD‐activated neurons in LI projected to the PbN. The collective data identify differences in the central neuroanatomy that support the disparate roles of vagal and spinal afferent signaling in the facilitation and modulation of colorectal nociceptive responses. [ABSTRACT FROM AUTHOR]

Voltage-gated sodium (Na[sub.v]) channels initiate action potentials in most neurons, including primary afferent nerve fibres of the pain pathway. Local anaesthetics block pain through non-specific actions at all Na[sub.v] channels, but the discovery of selective modulators would facilitate the analysis of individual subtypes of these channels and their contributions to chemical, mechanical, or thermal pain. Here we identify and characterize spider (Heteroscodra maculata) toxins that selectively activate the Na[sub.v]1.1 subtype, the role of which in nociception and pain has not been elucidated. We use these probes to show that Na[sub.v]1.1-expressing fibres are modality-specific nociceptors: their activation elicits robust pain behaviours without neurogenic inflammation and produces profound hypersensitivity to mechanical, but not thermal, stimuli. In the gut, high-threshold mechanosensitive fibres also express Na[sub.v]1.1 and show enhanced toxin sensitivity in a mouse model of irritable bowel syndrome. Together, these findings establish an unexpected role for Na[sub.v]1.1 channels in regulating the excitability of sensory nerve fibres that mediate mechanical pain., Author(s): Jeremiah D. Osteen [1]; Volker Herzig [2]; John Gilchrist [3]; Joshua J. Emrick [1]; Chuchu Zhang [1]; Xidao Wang [4]; Joel Castro [5, 6]; Sonia Garcia-Caraballo [5, 6]; Luke [...]

Protease-activated receptor ([PAR.sub.2]) is expressed by nociceptive neurons and activated during inflammation by proteases from mast cells, the intestinal lumen, and the circulation. Agonists of [PAR.sub.2] cause hyperexcitability of intestinal sensory neurons and hyperalgesia to distensive stimuli by unknown mechanisms. We evaluated the role of the transient receptor potential vanilloid 4 (TRPV4) in [PAR.sub.2]-induced mechanical hyperalgesia of the mouse colon. Colonic sensory neurons, identified by retrograde tracing, expressed immunoreactive TRPV4, [PAR.sub.2], and calcitonin generelated peptide and are thus implicated in nociception. To assess nociception, visceromotor responses (VMR) to colorectal distension (CRD) were measured by electromyography of abdominal muscles. In [TRPV4.sup.+/+] mice, intraluminal [PAR.sub.2] activating peptide ([PAR.sub.2]-AP) exacerbated VMR to graded CRD from 6-24 h, indicative of mechanical hyperalgesia. [PAR.sub.2]-induced hyperalgesia was not observed in [TRPV4.sup.-/-] mice. [PAR.sub.2]-AP evoked discharge of action potentials from colonic afferent neurons in [TRPV4.sup.+/+] mice, but not from [TRPV4.sup.-/-] mice. The TRPV4 agonists 5',6'-epoxyeicosatrienoic acid and 4[alpha]-phorbol 12,13-didecanoate stimulated discharge of action potentials in colonic afferent fibers and enhanced current responses recorded from retrogradely labeled colonic dorsal root ganglia neurons, confirming expression of functional TRPV4. [PAR.sub.2]-AP enhanced these responses, indicating sensitization of TRPV4. Thus TRPV4 is expressed by primary spinal afferent neurons innervating the colon. Activation of [PAR.sub.2] increases currents in these neurons, evokes discharge of action potentials from colonic afferent fibers, and induces mechanical hyperalgesia. These responses require the presence of functional TRPV4. Therefore, TRPV4 is required for [PAR.sub.2]-induced mechanical hyperalgesia and excitation of colonic afferent neurons. visceral pain; proteases; protease-activated receptors; transient receptor potential channels

Ghrelin is a peptide released from gastric endocrine cells that has an orexigenic effect via a vagal pathway. Here we determine the effect of ghrelin on mechanosensitivity of upper-intestinal vagal afferent fibers in ferret and mouse. The responses of gastroesophageal vagal afferents to graded mechanical stimulation were determined in vitro before and during application of ghrelin to their peripheral endings. Three types of vagal afferent were tested: tension receptors responding to circumferential tension, mucosal receptors responding only to mucosal stroking, and tension/mucosal (TM) receptors in ferret esophagus that responded to both stimuli. In the mouse, ghrelin did not significantly affect the response of mucosal receptors to mucosal stroking with calibrated von Frey hairs. However, it significantly reduced responses of tension receptors to circumferential tension (P < 0.005; two-way ANOVA) by up to 40%. This inhibition was reversed by the ghrelin receptor antagonist [D-Lys-3]-growth hormone-releasing peptide (GHRP)-6. In the ferret, ghrelin significantly reduced the response of mucosal and TM receptors to mucosal stroking with calibrated von Frey hairs. Surprisingly, ghrelin did not significantly alter the response to circumferential tension in either tension or TM receptors. RT-PCR analysis indicated that both ghrelin and its receptor are expressed in vagal afferent cell bodies in mouse nodose ganglia. In conclusion, ghrelin selectively inhibits subpopulations of mechanically sensitive gastroesophageal vagal afferents; there is also potential for ghrelin release from vagal afferents. However, the subpopulation of afferents inhibited differs between species. These data have broad implications for ghrelin's role in food intake regulation and reflex control of gastrointestinal function. in vitro gastroesophageal preparation; neuromodulation; mechanoreceptor doi:10.1152/ajpgi.00536.2006.

Irritable bowel syndrome (IBS) is a chronic gastrointestinal disorder characterized by abdominal pain and altered bowel habit that affects ~11% of the global population. Over the past decade, preclinical and clinical studies have revealed a variety of novel mechanisms relating to the visceral analgesic effects of guanylate cyclase-C (GC-C) agonists. Here we discuss the mechanisms by which GC-C agonists target the GC-C/cyclic guanosine-3′,5′-monophosphate (cGMP) pathway, resulting in visceral analgesia as well as clinically relevant relief of abdominal pain and other sensations in IBS patients. Due to the preponderance of evidence we focus on linaclotide, a 14-amino acid GC-C agonist with very low oral bioavailability that acts within the gut. Collectively, the weight of experimental and clinical evidence supports the concept that GC-C agonists act as peripherally acting visceral analgesics. Linaclotide and plecanatide are synthetic peptide agonists of guanylate cyclase-C (GC-C), a transmembrane receptor that is predominantly located on intestinal epithelial cells. These medications have very low oral bioavailability, which, when combined with the expression profile of GC-C, provides a mechanism of action 'targeted to the gut'. Linaclotide has been shown to inhibit colonic nociceptors and reduce peripheral drive from the colon, resulting in reduced numbers of activated dorsal horn neurons within the spinal cord and reduced pain responses to noxious colorectal distension. These effects are greatest in animal models of chronic visceral hypersensitivity that drive the 'bottom-up' and/or 'top-down' sensitization relevant to irritable bowel syndrome (IBS). Activation of GC-C results in the generation and release of cyclic guanosine-3′,5′-monophosphate (cGMP) from intestinal epithelial cells. When released into the submucosal space through the basolateral membrane, extracellular cGMP acts as a neuromodulator to inhibit pain-sensing nerve fibers innervating the colon. cGMP has also been demonstrated to inhibit pain-sensing nerves from human donors. In all human Phase III clinical studies conducted to date in patients with irritable bowel syndrome with constipation (IBS-C) or chronic idiopathic constipation (CIC), linaclotide significantly improved abdominal pain, discomfort, and bloating. Recent preclinical studies also provide the prospect that linaclotide may offer a novel therapeutic option, not only for the treatment of chronic abdominal pain in IBS-C, but also comorbid bladder dysfunction and endometriosis-associated pain. [ABSTRACT FROM AUTHOR]

Three Orai (Orai1, Orai2, and Orai3) and two stromal interaction molecule (STIM1 and STIM2) mammalian protein homologues constitute major components of the store‐operated Ca2+ entry mechanism. When co‐expressed with STIM1, Orai1, Orai2 and Orai3 form highly selective Ca2+ channels with properties of Ca2+ release‐activated Ca2+ (CRAC) channels. Despite the high level of homology between Orai proteins, CRAC channels formed by different Orai isoforms have distinctive properties, particularly with regards to Ca2+‐dependent inactivation, inhibition/potentiation by 2‐aminoethyl diphenylborinate and sensitivity to reactive oxygen species. This study characterises and compares the regulation of Orai1, Orai2‐ and Orai3‐mediated CRAC current (ICRAC) by intracellular pH (pHi). Using whole‐cell patch clamping of HEK293T cells heterologously expressing Orai and STIM1, we show that ICRAC formed by each Orai homologue has a unique sensitivity to changes in pHi. Orai1‐mediated ICRAC exhibits a strong dependence on pHi of both current amplitude and the kinetics of Ca2+‐dependent inactivation. In contrast, Orai2 amplitude, but not kinetics, depends on pHi, whereas Orai3 shows no dependence on pHi at all. Investigation of different Orai1–Orai3 chimeras suggests that pHi dependence of Orai1 resides in both the N‐terminus and intracellular loop 2, and may also involve pH‐dependent interactions with STIM1. Key points: It has been shown previously that Orai1/stromal interaction molecule 1 (STIM1)‐mediated Ca2+ release‐activated Ca2+ current (ICRAC) is inhibited by intracellular acidification and potentiated by intracellular alkalinisation.The present study reveals that CRAC channels formed by each of the Orai homologues Orai1, Orai2 and Orai3 has a unique sensitivity to changes in intracellular pH (pHi). The amplitude of Orai2 current is affected by the changes in pHi similarly to the amplitude of Orai1. However, unlike Orai1, fast Ca2+‐dependent inactivation of Orai2 is unaffected by acidic pHi. In contrast to both Orai1 and Orai2, Orai3 is not sensitive to pHi changes.Domain swapping between Orai1 and Orai3 identified the N‐terminus and intracellular loop 2 as the molecular structures responsible for Orai1 regulation by pHi.Reduction of ICRAC dependence on pHi seen in a STIM1‐independent Orai1 mutant suggested that some parts of STIM1 are also involved in ICRAC modulation by pHi. [ABSTRACT FROM AUTHOR]

Introduction: Improved understanding of vestibulodynia pathophysiology is required to develop appropriately targeted treatments. Established features include vulvovaginal hyperinnervation, increased nociceptive signalling and hypersensitivity. Emerging evidence indicates macrophage-neuron signalling contributes to chronic pain pathophysiology. Macrophages are broadly classified as M1 or M2, demonstrating pro-nociceptive or anti-nociceptive effects respectively. This study investigates the impact of clodronate liposomes, a macrophage depleting agent, on nociceptive signalling in a mouse model of vestibulodynia. Methods: Microinjection of complete Freund's adjuvant (CFA) at the vaginal introitus induced mild chronic inflammation in C57Bl/6J mice. A subgroup was treated with the macrophage depleting agent clodronate. Control mice received saline. After 7 days, immunolabelling for PGP9.5, F4/80+CD11c+ and F4/80+CD206+ was used to compare innervation density and presence of M1 and M2 macrophages respectively in experimental groups. Nociceptive signalling evoked by vaginal distension was assessed using immunolabelling for phosphorylated MAP extracellular signal-related kinase (pERK) in spinal cord sections. Hyperalgesia was assessed by visceromotor response to graded vaginal distension. Results: CFA led to increased vaginal innervation (p < 0.05), increased pERK-immunoreactive spinal cord dorsal horn neurons evoked by vaginal-distension (p < 0.01) and enhanced visceromotor responses compared control mice (p < 0.01). Clodronate did not reduce vaginal hyperinnervation but significantly reduced the abundance of M1 and M2 vaginal macrophages and restored vaginal nociceptive signalling and vaginal sensitivity to that of healthy control animals. Conclusions: We have developed a robust mouse model of vestibulodynia that demonstrates vaginal hyperinnervation, enhanced nociceptive signalling, hyperalgesia and allodynia. Macrophages contribute to hypersensitivity in this model. Macrophage-sensory neuron signalling pathways may present useful pathophysiological targets. [ABSTRACT FROM AUTHOR]