Endothelin-1 Exhibiting Pro-Nociceptive and Pro-Peristaltic Activities Is Increased in Peritoneal Carcinomatosis

Background: Peritoneal carcinomatosis often results in alterations in intestinal peristalsis and recurrent abdominal pain. Pain management in these patients is often unsatisfactory. This study aimed to investigate whether endothelin-1 (EDN1) was involved in pain mediation in peritoneal carcinomatosis, and thus whether the EDN1 pathway could be a new therapeutic target for peritoneal carcinomatosis-associated pain.Methods: EDN1 plasma levels and abdominal pain severity were assessed in patients with abdominal tumors, with or without peritoneal carcinomatosis, and in healthy donors. The effects of EDN1 on the visceromotor response to colorectal distension, and on colonic contractions were then examined in mice, and the mechanism of action of EDN1 was then investigated by measuring the impact of EDN1 exposure on calcium mobilization in cultured neurons. Inhibition studies were also performed to determine if the effects of EDN1 exposure could be reversed by EDN1-specific receptor antagonists.Results: A positive correlation between EDN1 plasma levels and abdominal pain was identified in patients with peritoneal carcinomatosis. EDN1 exposure increased visceral sensitivity and the amplitude of colonic contractions in mice and induced calcium mobilization by direct binding to its receptors on sensory neurons. The effects of EDN1 were inhibited by antagonists of the EDN1 receptors.Conclusions: This preliminary study, using data from patients with peritoneal carcinomatosis combined with data from experiments performed in mice, suggests that EDN1 may play a key role mediating pain in peritoneal carcinomatosis. Our findings suggest that antagonists of the EDN1 receptors might be beneficial in the management of pain in patients with peritoneal carcinomatosis.

Eosinophil-associated micro-inflammation in the gastroduodenal tract contributes to gastric hypersensitivity in a rat model of early life adversity

Gastric hypersensitivity is a major pathophysiological feature of functional dyspepsia (FD). Recent clinical studies have shown that a large number of FD patients present with gastroduodenal micro-inflammation, which may be involved in the pathophysiology of FD. However, no animal model reflecting this clinical characteristic has been established. The underlying mechanism between micro-inflammation and FD remains unknown. In this study, using a maternal separation (MS)-induced FD model, we aimed to reproduce the gastroduodenal micro-inflammation and reveal the interaction between gastroduodenal micro-inflammation and gastric hypersensitivity. The MS model was established by separating newborn Sprague Dawley rats for 2 h a day from postnatal day 1 to day 10. At 7-8 weeks of age, electromyography was used to determine the visceromotor response to gastric distention (GD) and immunohistochemistry was performed to detect distension-associated neuronal activation as well as immunohistological changes. Our results demonstrated that MS-induced FD rats underwent gastric hypersensitivity with GD at 60 and 80 mmHg, which are related to increased p-ERK1/2 expression in the dorsal horn of T9-T10 spinal cords. Eosinophils, but not mast cells, were significantly increased in the gastroduodenal tract, and the co-expression rate of CD11b and major basic protein significantly increased in MS rats. Treatment with dexamethasone reversed gastric hypersensitivity in MS-induced FD rats by inhibiting eosinophil infiltration. These findings indicated that neonatal MS stress induces eosinophil-associated gastroduodenal micro-inflammation and gastric hypersensitivity in adulthood in rats. Micro-inflammation contributes to gastric hypersensitivity; therefore, anti-inflammatory therapy may be effective in treating FD patients with gastroduodenal micro-inflammation.

The Anti-Inflammatory and Pain-Relieving Effects of AR170, an Adenosine A3 Receptor Agonist, in a Rat Model of Colitis

The pharmacological activation of A3 receptors has shown potential usefulness in the management of bowel inflammation. However, the role of these receptors in the control of visceral hypersensitivity in the presence of intestinal inflammation has not been investigated. The effects of AR170, a potent and selective A3 receptor agonist, and dexamethasone (DEX) were tested in rats with 2,4-dinitrobenzene sulfonic acid (DNBS)-induced colitis to assess their tissue inflammatory parameters. The animals received AR170, DEX, or a vehicle intraperitoneally for 6 days, starting 1 day before the induction of colitis. Visceral pain was assessed by recording the abdominal responses to colorectal distension in animals with colitis. Colitis was associated with a decrease in body weight and an increase in spleen weight. The macroscopic damage score and tissue tumor necrosis factor (TNF), interleukin 1β (IL-1β), and myeloperoxidase (MPO) levels were also enhanced. AR170, but not DEX, improved body weight. Both drugs counteracted the increase in spleen weight, ameliorated macroscopic colonic damage, and decreased TNF, IL-1β, and MPO tissue levels. The enhanced visceromotor response (VMR) in rats with colitis was decreased via AR170 administration. In rats with colitis, AR170 counteracted colonic inflammatory cell infiltration and decreased pro-inflammatory cytokine levels, thereby relieving visceral hypersensitivity.

Optogenetic inhibition of colon epithelium reduces hypersensitivity in a mouse model of inflammatory bowel disease

ABSTRACTVisceral pain is a prevalent symptom of inflammatory bowel disease (IBD) that can be difficult to treat. Pain and hypersensitivity are mediated by extrinsic primary afferent neurons (ExPANs) that innervate the colon. Recent studies indicate that the colon epithelium contributes to initiating ExPAN firing and nociceptive responses. Based on these findings we hypothesized that the epithelium contributes to inflammation-induced hypersensitivity. A key prediction of this hypothesis is that inhibition of the epithelium would attenuate nociceptive signaling and inflammatory hypersensitivity. To test this hypothesis, the inhibitory yellow light activated protein archaerhodopsin was targeted to the intestinal epithelium (villin-Arch) or the ExPANs (TRPV1-Arch) that innervate the colon. Visceral sensitivity was assessed by measuring the visceromotor response (VMR) to colorectal distension (CRD), with and without yellow light illumination of the colon lumen. Inhibition of the colon epithelium in healthy villin-Arch mice significantly diminished the CRD-induced VMR. Direct inhibition of ExPANs during CRD using TRPV1-Arch mice showed that ExPAN and epithelial inhibition were similarly effective in reducing the VMR to CRD. We then investigated the effect of epithelial and ExPAN inhibition in the dextran sulfate sodium (DSS) model of inflammatory bowel disease (IBD). Inhibition of the colon epithelium significantly decreased DSS-induced hypersensitivity and was comparable to inhibition of ExPANS. Together these results reveal the potential of targeting the colon epithelium for treatment of pain.

Altered colonic sensory and barrier functions by CRF: roles of TLR4 and IL-1

Visceral allodynia and increased colonic permeability are considered to be crucial pathophysiology of irritable bowel syndrome (IBS). Corticotropin-releasing factor (CRF) and immune-mediated mechanisms have been proposed to contribute to these changes in IBS, but the precise roles have not been determined. We explored these issues in rats in vivo. The threshold of visceromotor response, i.e., abdominal muscle contractions induced by colonic balloon distention was electrophysiologically measured. Colonic permeability was estimated by quantifying the absorbed Evans blue in colonic tissue. Intraperitoneal injection of CRF increased the permeability, which was blocked by astressin, a non-selective CRF receptor antagonist, but astressin2-B, a selective CRF receptor subtype 2 (CRF2) antagonist did not modify it. Urocortin 2, a selective CRF2 agonist inhibited the increased permeability by CRF. Eritoran, a toll-like receptor 4 (TLR4) antagonist or anakinra, an interleukin-1 receptor antagonist blocked the visceral allodynia and the increased gut permeability induced by CRF. Subcutaneous injection of lipopolysaccharide (immune stress) or repeated water avoidance stress (WAS, psychological stress), 1 h daily for 3 days induced visceral allodynia and increased gut permeability (animal IBS models), which were also blocked by astressin, eritoran or anakinra. In conclusion, stress-induced visceral allodynia and increased colonic permeability were mediated via peripheral CRF receptors. CRF induced these visceral changes via TLR4 and cytokine system, which were CRF1 dependent, and activation of CRF2 inhibited these CRF1-triggered responses. CRF may modulate immune system to alter visceral changes, which are considered to be pivotal pathophysiology of IBS.

Optogenetic silencing of primary afferents reduces evoked and ongoing bladder pain

Patients with interstitial cystitis/bladder pain syndrome (IC/BPS) suffer from chronic pain that severely affects quality of life. Although the underlying pathophysiology is not well understood, inhibition of bladder sensory afferents temporarily relieves pain. Here, we explored the possibility that optogenetic inhibition of bladder sensory afferents could be used to modulate bladder pain. Specifically, we chose to study the role of Nav1.8+ sensory afferents before and after induction of a mouse model of bladder pain. The light-activated inhibitory proton pump Archaerhodopsin (Arch) was expressed under control of the Nav1.8+ promoter to selectively silence these neurons. Optically silencing Nav1.8+ afferents significantly blunted the evoked visceromotor response to bladder distension and led to small but significant changes in bladder function. To study of the role of these fibers in freely behaving mice, we developed a fully implantable, flexible, wirelessly powered optoelectronic system for the long-term manipulation of bladder afferent expressed opsins. We found that optogenetic inhibition of Nav1.8+ fibers reduced both ongoing pain and evoked cutaneous hypersensitivity in the context of cystitis, but had no effect in uninjured, naïve mice. These results suggest that selective optogenetic silencing of bladder afferents may represent a potential future therapeutic strategy for the treatment of bladder pain.

Absence of diurnal variation in visceromotor response to colorectal distention in normal Long Evans rats

Background: Enhanced colorectal sensitivity (i.e. visceral hypersensitivity) is thought to be a pathophysiological mechanism in irritable bowel syndrome (IBS). In healthy men a circadian variation in rectal perception to colonic distention was described. Disturbed day and night rhythms, which occur in shift work and trans meridian flights, are associated with the prevalence of IBS. This raises the question whether disruptions of circadian control are responsible for the observed pathology in IBS. Prior to investigating altered rhythmicity in relation to visceral hypersensitivity in a rat model for IBS, it is relevant to establish whether normal rats display circadian variation similar to healthy men. Methodology and findings: In rodents colorectal distension leads to reproducible contractions of abdominal musculature. We used quantification of this so called visceromotor response (VMR) by electromyography (EMG) to assess visceral sensitivity in rats. We assessed the VMR in normal male Long Evans rats at different time points of the light/dark cycle. Although a control experiment with male maternal separated rats confirmed that intentionally inflicted (i.e. stress induced) changes in VMR can be detected, normal male Long Evans rats showed no variation in VMR along the light/dark cycle in response to colorectal distension.Conclusions: In the absence of a daily rhythm of colorectal sensitivity in normal control rats it is not possible to investigate possible aberrancies in our rat model for IBS.

A Balance Theory of Peripheral Corticotropin-Releasing Factor Receptor Type 1 and Type 2 Signaling to Induce Colonic Contractions and Visceral Hyperalgesia in Rats

Several recent studies suggest that peripheral corticotropin-releasing factor (CRF) receptor type 1 (CRF1) and CRF2 have a counter regulatory action on gastrointestinal functions. We hypothesized that the activity balance of each CRF subtype signaling may determine the changes in colonic motility and visceral sensation. Colonic contractions were assessed by the perfused manometry, and contractions of colonic muscle strips were measured in vitro in rats. Visceromotor response was determined by measuring contractions of abdominal muscle in response to colorectal distensions (CRDs) (60 mm Hg for 10 min twice with a 30-min rest). All drugs were administered through ip route in in vivo studies. CRF increased colonic contractions. Pretreatment with astressin, a nonselective CRF antagonist, blocked the CRF-induced response, but astressin2-B, a selective CRF2 antagonist, enhanced the response by CRF. Cortagine, a selective CRF1 agonist, increased colonic contractions. In in vitro study, CRF increased contractions of muscle strips. Urocortin 2, a selective CRF2 agonist, itself did not alter the contractions but blocked this increased response by CRF. Visceromotor response to the second CRD was significantly higher than that of the first. Astressin blocked this CRD-induced sensitization, but astressin2-B or CRF did not affect it. Meanwhile, astressin2-B together with CRF significantly enhanced the sensitization. Urocortin 2 blocked, but cortagine significantly enhanced, the sensitization. These results indicated that peripheral CRF1 signaling enhanced colonic contractility and induced visceral sensitization, and these responses were modulated by peripheral CRF2 signaling. The activity balance of each subtype signaling may determine the colonic functions in response to stress.

The role of the anteriolateral bed nucleus of the stria terminalis in stress-induced nociception

Activation of the central amygdala (CeA) by corticosterone (CORT) induces somatic and colonic hypersensitivity through corticotrophin-releasing factor (CRF)-dependent mechanisms. However, the importance of the bed nucleus of the stria terminalis (BNST), part of the extended amygdala, on nociception remains unexplored. In the present study, we test the hypothesis that stimulation of the CeA by CORT induces somatic and colonic hypersensitivity through activation of the anteriolateral BNST (BNSTAL). Animals were implanted with micropellets of CORT or cholesterol (CHOL) onto the CeA or the BNSTAL. Mechanical sensitivity was quantified using electronic von Frey filaments, and colonic nociception was measured by quantifying a visceromotor response to graded colorectal distension. In situ hybridization was used to determine mRNA levels for CRF, CRF1, and CRF2 receptors in the BNSTAL. In a second group, animals were implanted bilaterally with 1) CORT or CHOL micropellets onto the CeA; and 2) cannulas localized to the BNSTAL to administer a CRF1 receptor antagonist (CP376395). Animals implanted with CORT onto the CeA, but not the BNSTAL, exhibited increased expression of CRF mRNA and increased CRF1-to-CRF2 receptor ratio in the BNST, as well as somatic and colonic hypersensitivity compared with CHOL controls. Infusion of CP376395 into the BNSTAL inhibited somatic and colonic hypersensitivity in response to elevated amygdala CORT. Somatic and colonic hypersensitivity induced by elevated amygdala CORT is mediated via a CRF1 receptor-dependent mechanism in the BNSTAL. The CeA through a descending pathway involving the BNSTAL plays a pivotal role in somatic and colonic nociception.

Long-term sensitization of mechanosensitive and -insensitive afferents in mice with persistent colorectal hypersensitivity

Afferent input contributes significantly to the pain and colorectal hypersensitivity that characterize irritable bowel syndrome. In the present study, we investigated the contributions of mechanically sensitive and mechanically insensitive afferents (MIAs; or silent afferents) to colorectal hypersensitivity. The visceromotor response to colorectal distension (CRD; 15–60 mmHg) was recorded in mice before and for weeks after intracolonic treatment with zymosan or saline. After CRD tests, the distal colorectum with the pelvic nerve attached was removed for single-fiber electrophysiological recordings. Colorectal afferent endings were located by electrical stimulation and characterized as mechanosensitive or not by blunt probing, mucosal stroking, and circumferential stretch. Intracolonic zymosan produced persistent colorectal hypersensitivity (>24 days) associated with brief colorectal inflammation. Pelvic nerve muscular-mucosal but not muscular mechanosensitive afferents recorded from mice with colorectal hypersensitivity exhibited persistent sensitization. In addition, the proportion of MIAs (relative to control) was significantly reduced from 27% to 13%, whereas the proportion of serosal afferents was significantly increased from 34% to 53%, suggesting that MIAs acquired mechanosensitivity. PGP9.5 immunostaining revealed no significant loss of colorectal nerve fiber density, suggesting that the reduction in MIAs is not due to peripheral fiber loss after intracolonic zymosan. These results indicate that colorectal MIAs and sensitized muscular-mucosal afferents that respond to stretch contribute significantly to the afferent input that sustains hypersensitivity to CRD, suggesting that targeted management of colorectal afferent input could significantly reduce patients' complaints of pain and hypersensitivity.