Rat Model Of Visceral Hypersensitivity Research Articles

P114 Role of NLRX1 Agonist NX-13 in Reducing Visceral Hypersensitivity in Preclinical Gastrointestinal Inflammation

Abstract Background NLRX1 activation reduces inflammation by decreasing oxidative stress and altering cellular metabolism within multiple cell types implicated in ulcerative colitis (UC). Colitis animal models demonstrated reduced disease severity and a phase 1b clinical trial showed signs of rapid symptom and endoscopic improvement in patients with active UC.1, 2 Abdominal pain driven by visceral hypersensitivity may persist in patients even after inflammation has resolved, negatively affecting their quality of life.3, 4 Therapeutic agents which address inflammation, epithelial healing, and visceral hypersensitivity concurrently may provide greater symptomatic relief and improved quality of life as many patients, even in remission, still complain of abdominal pain. We here describe the effects of oral NX-13 in a rat model of visceral hypersensitivity. Methods Rats (n=8) were dosed daily for a period of 3 days with NX-13 or vehicle. Under anesthesia, electrodes were positioned to monitor oblique abdominal muscle contraction and a colonic balloon catheter was inserted intra-anally. Visceral pain was assessed at baseline and 3 h post lipopolysaccharide injection (1 mg/kg subcutaneous) through measuring of visceromotor response (VMR) via electromyogram (EMG) recording and visual assessment of abdominal withdrawal reflex (AWR). Data are represented as median (IQR) and statistical significance determined by non-parametric Mann-Whitney U test. Results Compared to the vehicle control, oral NX-13 delayed the onset of muscle contraction in response to colonic distension in LPS-treated rats. Further, NX-13 treated rats experienced reduced contraction intensity and reduced sustained abdominal muscle contraction period compared to the vehicle control. Specifically, NX-13 decreased the number of AWR during colonic expansion compared to the control group (p=0.01, Fig1A). Moreover, NX-13 desensitized the VMR response by numerically increasing the minimum volume of the colonic distension balloon required to induce significant VMR. The mean minimum volume of water injected required to induce significant VMR increased 23%, from 650 μL in the vehicle group to 800 μL in the NX-13 treated rats (p=0.16, Fig1B). Lastly, NX-13 visually reduced the maximum abdominal EMG amplitude during colonic distention (p=0.19, Fig1C). Conclusion Adequate management of persistent pain in IBD patients with or without active bowel inflammation remains an unmet need in the treatment of IBD. The potential for NX-13 to specifically reduce visceral hypersensitivity and abdominal pain will be evaluated further in the ongoing phase 2 human NEXUS trial in patients with UC. 1Leber et al. J Immunol 203(12) 2Peyrin-Biroulet et al. JCC (17)Supp 3Abreu et al. JCC (14)Supp 4Wils et al. J Clin Med 11(15)

Melatonin attenuated chronic visceral pain by reducing Nav1.8 expression and nociceptive neuronal sensitization.

Irritable bowel syndrome (IBS) is a common functional gastrointestinal disorder, and its specific pathogenesis is still unclear. We have previously reported that TTX-resistant (TTX-R) sodium channels in colon-specific dorsal root ganglion (DRG) neurons were sensitized in a rat model of visceral hypersensitivity induced by neonatal colonic inflammation (NCI). However, the detailed molecular mechanism for activation of sodium channels remains unknown. This study was designed to examine roles for melatonin (MT) in sensitization of sodium channels in NCI rats. Colorectal distention (CRD) in adult male rats as a measure of visceral hypersensitivity. Colon-specific dorsal root ganglion (DRG) neurons were labeled with DiI and acutely dissociated for measuring excitability and sodium channel current under whole-cell patch clamp configurations. Western blot and Immunofluorescence were employed to detect changes in expression of Nav1.8 and MT2. The results showed that rats exhibited visceral hypersensitivity after NCI treatment. Intrathecal application of melatonin significantly increased the threshold of CRD in NCI rats with a dose-dependent manner, but has no role in the control group. Whole-cell patch clamp recording showed that melatonin remarkably decreased the excitability and the density of TTX-R sodium channel in DRG neurons from NCI rats. The expression of MT2 receptor at the protein level was markedly lower in NCI rats. 8MP, an agonist of MT2 receptor, enhanced the distention threshold in NCI rats. Application of 8MP reversed the enhanced hypersensitivity of DRG neurons from NCI rats. 8MP also reduced TTX-R sodium current density and modulated dynamics of TTX-R sodium current activation. These data suggest that sensitization of sodium channels of colon DRG neurons in NCI rats is most likely mediated by MT2 receptor, thus identifying a potential target for treatment for chronic visceral pain in patients with IBS.

Tandospirone prevents stress-induced anxiety-like behavior and visceral hypersensitivity by suppressing theta oscillation enhancement via 5-HT1A receptors in the anterior cingulate cortex in rats.

Tandospirone, a third-generation of antianxiety agent with fewer side effects, has been widely used in the treatment of anxiety. Moreover, it is interesting that tandospirone has been found to relieve gastrointestinal symptoms in patients with refractory irritable bowel syndrome who also have psychological dysfunctions. However, the underlying mechanism remains unclear. In this study, using a visceral hypersensitivity rat model induced by chronic water avoidance stress to mimic the symptoms of irritable bowel syndrome, we found that tandospirone relieved anxiety-like behavior and visceral hypersensitivity induced by stress. Meanwhile, stressed rats had increased 5-HT concentration, less 5-HT1A receptor expression, and enhanced theta oscillations in the anterior cingulate cortex (ACC). Furthermore, the power of the theta band in ACC is positively correlated with the level of visceral sensitivity. Activation of 5-HT1A receptors by its agonist, 8-OH-DPAT, to compensate for their effect in ACC reduced the enhancement of theta oscillations in ACC slices in stressed rats, whereas 5-HT1A receptor antagonist, WAY100135, facilitates theta oscillations in slices of normal rats. Tandospirone reduced the enhancement of theta band power in ACC in vitro and in vivo, thus alleviating anxiety-like behavior and visceral hypersensitivity through 5-HT1A receptors in stressed rats. These results suggest a novel mechanism by which tandospirone activates 5-HT1A receptors to relieve stress-induced anxiety and visceral hypersensitivity by suppressing theta oscillation enhancement in ACC.

Changes in intestinal barrier protein expression and intestinal flora in a rat model of visceral hypersensitivity.

Destruction of the intestinal mucosal barrier and visceral hypersensitivity are main pathogenesis of irritable bowel syndrome (IBS). The study aimed to establish a rat model of visceral hypersensitivity and explore mechanisms involved the changes of the intestinal barrier protein expression and intestinal flora. A rat model of visceral hypersensitivity was established and evaluated using abdominal withdrawal reflex (AWR) scores, colonic paracellular permeability, and gastrointestinal motility. The expression of tight junction proteins, aquaporin proteins (AQPs), phosphorylated ERK, and proteinase-activated receptor-2 (PAR-2) was determined. The intestinal microflora was evaluated by high-throughput sequencing of the 16S rRNA gene. In model rats, AWR score and fecal water content were significantly increased, gastrointestinal motilities were disorder and characterized by an inhibition of gastric motility and an enhancement of small intestinal and colonic movement. The expressions of colonic occludin, ZO-1, AQP3, and AQP8 were decreased but claudin-2 and claudin-4 were markedly increased. Imbalance of intestinal flora appeared and showed an obvious decrease of Lactobacillus and an increase of Clostridiales_bacterium. Additionally, the total serine protease activity in feces, the expressions of PAR2 and phosphorylated ERK in the colon tissues were increased significantly. The model rats of visceral hypersensitivity possess the decreased expression of occludin, ZO-1, AQP3, AQP8, and the increased expression of claudin-2 and claudin-4, meanwhile develop an imbalance of intestinal flora which probably increase serine protease activity, thereby activating the PAR2/ERK signaling and causing the intestinal barrier disorder.

The Role of Intestinal Microbiota and Mast Cell in a Rat Model of Visceral Hypersensitivity.

Background/AimsTo explore the role of intestinal flora and mast cells in visceral hypersensitivity (VH).MethodsThe experimental animals were divided into 4 groups control group, VH group, VH + VSL#3 group, and VH + ketotifen group. Stool samples were collected from each group (n = 3) for a further analysis using 16S ribosomal DNA gene sequence. Visceral sensitivity was evaluated by abdominal withdrawal reflex (AWR) score. Colon tissues of rats were obtained from each group. Mast cells were detected by toluidine blue staining. The degranulation of mast cells was assessed by transmission electron microscopy.ResultsVH rat model could successfully be induced by acetic acid enema combined with partial limb restraint method. Compared with rats in the control group, AWR score, number of mast cells, and degranulation of mast cells were increased in the VH rats, which could be reduced by administration of ketotifen or probiotic VSL#3. Clostridium sensu stricto 1 abundance was higher in the VH group compared to the control group, which could be restored by application of probiotic VSL#3.ConclusionsProbiotic VSL#3 decreases visceral sensitivity in VH rats. The mechanism may be related to mast cell and intestinal flora. Change of Clostridium sensu stricto 1 abundance may be a basis for VH observed in irritable bowel syndrome and may be prevented by specific probiotic administration.

Electroacupuncture Relieves Irritable Bowel Syndrome by Regulating IL-18 and Gut Microbial Dysbiosis in a Trinitrobenzene Sulfonic Acid-Induced Post-Inflammatory Animal Model.

Post inflammatory irritable bowel syndrome (PI-IBS), a subset of IBS, is characterized by symptoms of visceral pain, bloating, and changed bowel habits that occur post initial episode of intestinal infection. Gut microbial dysbiosis or inflammation plays a key role in the pathogenesis of abdominal hypersensitivity of PI-IBS. Electroacupuncture (EA) stimulation results in an alleviated PI-IBS-associated symptom. This study investigated the effect of EA on IL-18 and gut microbial dysbiosis in one visceral hypersensitive rat models with PI-IBS. A trinitrobenzene sulfonic acid (TNBS)-induced visceral hypersensitivity rat model was developed. EA stimulation was applied to the ST25 and ST36 acupoints. Animals were assessed using abdominal withdrawal reflex (AWR) scores to determine the development of colonic visceral hypersensitivity. The 16S rRNA was used to correlate microbial diversity. IL-18 expression in colon was quantified by quantitative real-time PCR and western blotting. We identified that model rats had an increased visceral hypersensitivity to colorectal distention at different distention pressures compared with the normal group. Sensitivity to colorectal distention decreased after EA stimulation. The composition of the fecal microbiota was different between groups. Specifically, in the model group Empedobacter, Psychrobacter, Enterococcus, Butyricimonas, Vampirovibrio, Kurthia, Intestinimonas, Neisseria, Falsiporphyromonas, Bilophila, Fusobacterium, Alistipes, Veillonella, Flavonifractor, Clostridium XlVa weremore abundantaffected genera, whereas Lactobacillus was enriched in normal rats. EA stimulation was correlated with significant decrease in the phyla of Fusobacteria. The mRNA and protein levels of IL-18 were higher in the model group. Meanwhile, EA stimulation attenuated this response. In a word, our findings suggest that PI-IBS is associated with significant increase in IL-18 levels as well as an alteration in microbiome diversity. These changes can be reversed with EA treatment. EA stimulation has a positive effect in alleviating symptoms of visceral hypersensitivity and protecting the gastrointestinal tract.

The relationship between adherens junction and tight junction and clinical symptoms in patients with diarrhea predominant irritable bowel syndrome

Objective: To investigate the association between adherens junction proteins E-cadherin and β-catenin and tight junction protein claudin-2 and clinical symptoms in patients with diarrhea predominant irritable bowel syndrome (IBS-D). Methods: Cecal biopsy tissues were collected from IBS-D patients (n=26) according to Rome Ⅲ criterion and healthy controls (n=26). The duration of symptoms, abdominal pain score and mean weekly bowel movements were recorded. Colorectal dilatation combined with restraint stress were applied to establish visceral hypersensitivity rat model. Abdominal contraction reflex (AWR) was applied to assess the visceral sensitivity in rats. The stool frequency within 1 hour was recorded after establishing the rat model. The expression of E-cadherin、β-catenin and claudin-2 were assessed by Western blot and immunofluorescence microscopy. Intercellular ultrastructure was observed by transmission electron microscopy. Results: Compared with the healthy controls, the protein expression of E-cadherin and β-catenin in cecal epithelium in IBS-D patients were significantly lower (P=0.015 and P=0.005, respectively), while claudin-2 was significantly higher (P=0.000). Reduced E-cadherin and β-catenin expression was associated high abdominal pain score (r=-0.463, P=0.017 and r=-0.407, P=0.039). The lower expression of β-catenin was associated with longer duration of symptoms (r=-0.458, P=0.019). The protein expression of E-cadherin and ß-catenin in the cecal epithelium of the visceral hypersensitivity rats were significantly lower (P=0.004 and P=0.003, respectively), while claudin-2 was significantly higher (P=0.008). Reduced E-cadherin and ß-catenin expression was associated high visceral sensitivity in IBS-D rats (r=-0.639, P=0.047 and r=-0.888, P=0.001). Conclusions: Intercellular ultrastructure alterations well as cecal β-catenin and E-cadherin protein expression decrease and are associated with high abdominal pain score in IBS-D patients and hypersensitivity rats. β-catenin is further associated with prolonged duration of symptoms in IBS-D patients. The expression of E-cadherin and β-catenin may play a vital role in visceral sensitivity and intestinal barrier dysfunction in IBS-D.

Effects of Shugan Hewei Granule on Depressive Behavior and Protein Expression Related to Visceral Sensitivity in a Rat Model of Nonerosive Reflux Disease.

Objective To explore the effect of Shugan Hewei Granule (SGHWG) and to provide the experimental basis for its clinical application. Methods 40 healthy male Wistar rats were divided into 5 groups, with 8 rats in each group, including control group, model group, normal saline (NS) group, SGHWG group, and Rabeprazole group. The control group was not treated. The model group was treated with fructose intake and mental stress to be the model of NERD. The other groups were treated as the model group and then gavaged with the corresponding drugs. The pH value of lower third of esophagus, immobile time in tail suspension test, CRF protein expression in both hypothalamus and anterior cingulate cortex (ACC), and SP protein in esophageal mucosa in lower third of esophagus detected by immunofluorescence and NMDAR1 protein expression in spinal cord detected by immunohistochemistry of each group were compared. Results The pH values of both the SGHWG group and the Rabeprazole group were higher than that of the model group (P<0.01), but the Rabeprazole group increased more obviously. The immobile time of the SGHWG group was shorter than that of the model group (P<0.01) and the Rabeprazole group (P<0.05). The expression of the CRF in the hypothalamus and ACC, NMDAR1 in the spinal cord, and SP in the esophageal mucosa in lower third of esophagus of the SGHWG group decreased significantly, compared with the model group (P<0.01), and was obviously lower than that in the Rabeprazole group (P<0.05). Conclusions This study provided an evidence that SGHW formula was inferior to Rabeprazole in acid inhibition, but it might reduce the expression of CRF protein of hypothalamus and ACC, lower the levels of NMDAR1 in spinal dorsal horn and SP in esophageal mucosa in lower third of esophagus, and regulate depressive behavior simultaneously, related to the improvement of visceral hypersensitivity in rat model of NERD.

Mechanisms of Probiotic VSL#3 in a Rat Model of Visceral Hypersensitivity Involves the Mast Cell-PAR2-TRPV1 Pathway.

Mast cells (MCs), PAR2 and TRPV1, play a key role in the regulation of visceral pain. Several studies have found that probiotics regulate visceral sensitivity. The purpose of the current study was to explore the role of MC-PAR2-TRPV1 in VH and the mechanism of VSL#3 in a rat model of VH. A total of 64 rats were randomly divided into eight groups: Control VH, VH + ketotifen, VH + FSLLRY-NH2, VH + SB366791, VH + VSL#3, VH + VSL#3 + capsaicin, and VH + VSL#3 + SLIGRL-NH2. The rat model of VH was induced by acetic acid enema and the partial limb restraint method. VH was assessed by the abdominal withdrawal reflex score. MCs in colonic tissue were detected by the toluidine blue staining assay. The expression of PAR2 and TRPV1 in DRGs (L6-S1) was measured by immunohistochemistry and Western blotting. The established VH was abolished by treatment with ketotifen, a mast cell stabilizer FSLLRY-NH2, a PAR2 antagonist SB366791 a TRPV1 antagonist, and probiotic VSL#3 in rats. The administration of ketotifen or probiotic VSL#3 caused a decrease in mast cell number in the colon and decreased PAR2 and TRPV1 expression in DRGs. Intrathecal injection of FSLLRY-NH2 or SB366791 caused decreased expression of PAR2 and/or TRPV1 in DRGs in VH rats. SLIGRL-NH2, a PAR2 agonist, and capsaicin, a TRPV1 agonist, blocked the effects of probiotic VSL#3. The probiotic VSL#3 decreases VH in rat model of VH. The mechanism may be related with the mast cell-PAR2-TRPV1 signaling pathway.

Regulation of Colonic Mucosal MicroRNA Expression via Multiple Targets in Visceral Hypersensitivity Rats by Tongxieyaofang.

PurposeThis study aimed to screen for differentially expressed microRNAs (miRNAs) in the colons of rats with visceral hypersensitivity to build the expression profiles of miRNAs therein and to determine the mechanism of Tongxieyaofang use in the treatment of irritable bowel syndrome (IBS).Materials and MethodsForty Sprague-Dawley rats were divided randomly into four groups: control group, model control group (induced by rectum stimulus and evaluated by abdominal withdraw reaction), treatment control group (normal saline), and Tongxieyaofang group (treated with Tongxieyaofang). We screened for differential expression of colonic mucosal miRNAs using liquid chip technology and verified the expression thereof using reverse transcription-PCR.ResultsThe visceral hypersensitivity rat model was successfully established. We found the expression of let-7f, let-7i, miR-130b, miR-29a, miR-132, miR-21, and miR-375 to be up-regulated (p<0.05), while the expression of miR-24, miR-31a, miR-192, miR-221, and miR-223 was down-regulated (p<0.05) in the visceral hypersensitivity rats. After treatment with Tongxieyaofang, the expression of let-7f, let-7i, miR-130b, miR-29a, miR-132, miR-21, and miR-375 was reduced (p<0.05), whereas the expression of miR-24, miR-31a, miR-192, miR-221, miR-223 was increased, compared to the treatment control group (p<0.05).ConclusionMiRNAs play a pivotal role in visceral hypersensitivity and might be targets in the treatment of IBS by Tongxieyaofang.

Intestinal Fungal Dysbiosis Is Associated With Visceral Hypersensitivity in Patients With Irritable Bowel Syndrome and Rats

Visceral hypersensitivity is one feature of irritable bowel syndrome (IBS). Bacterial dysbiosis might be involved in the activation of nociceptive sensory pathways, but there have been few studies of the role of the mycobiome (the fungal microbiome) in the development of IBS. We analyzed intestinal mycobiomes of patients with IBS and a rat model of visceral hypersensitivity. We used internal transcribed spacer 1-based metabarcoding to compare fecal mycobiomes of 18 healthy volunteers with those of 39 patients with IBS (with visceral hypersensitivity or normal levels of sensitivity). We also compared the mycobiomes of Long-Evans rats separated from their mothers (hypersensitive) with non-handled (normally sensitive) rats. We investigated whether fungi can cause visceral hypersensitivity using rats exposed to fungicide (fluconazole and nystatin). The functional relevance of the gut mycobiome was confirmed in fecal transplantation experiments: adult maternally separated rats were subjected to water avoidance stress (to induce visceral hypersensitivity), then given fungicide and donor cecum content via oral gavage. Other rats subjected to water avoidance stress were given soluble β-glucans, which antagonize C-type lectin domain family 7 member A (CLEC7A or DECTIN1) signaling via spleen-associated tyrosine kinase (SYK), a SYK inhibitor to reduce visceral hypersensitivity, or vehicle (control). The sensitivity of mast cells to fungi was tested with mesenteric windows (exvivo) and the human mast cell line HMC-1. α diversity (Shannon index) and mycobiome signature (stability selection) of both groups of IBS patients differed from healthy volunteers, and the mycobiome signature of hypersensitive patients differed from that of normally sensitive patients. We observed mycobiome dysbiosis in rats that had been separated from their mothers compared with non-handled rats. Administration of fungicide to hypersensitive rats reduced their visceral hypersensitivity to normal levels of sensitivity. Administration of cecal mycobiomes from rats that had been separated from their mothers (but not non-handled mycobiome) restored hypersensitivity to distension. Administration of soluble β-glucans or a SYK inhibitor reduced visceral hypersensitivity, compared with controls. Particulate β-glucan (a DECTIN-1 agonist) induced mast cell degranulation in mesenteric windows and HMC-1 cells responded to fungal antigens by release of histamine. In an analysis of patients with IBS and controls, we associated fungal dysbiosis with IBS. In studies of rats, we found fungi to promote visceral hypersensitivity, which could be reduced by administration of fungicides, soluble β-glucans, or a SYK inhibitor. The intestinal fungi might therefore be manipulated for treatment of IBS-related visceral hypersensitivity.

Neonatal Maternal Deprivation Enhances Presynaptic P2X7 Receptor Transmission in Insular Cortex in an Adult Rat Model of Visceral Hypersensitivity.

Insular cortex (IC) is involved in processing the information of pain. The aim of this study was to investigate roles and mechanisms of P2X7 receptors (P2X7Rs) in IC in development of visceral hypersensitivity of adult rats with neonatal maternal deprivation (NMD). Visceral hypersensitivity was quantified by abdominal withdrawal reflex threshold to colorectal distension (CRD). Expression of P2X7Rs was determined by qPCR and Western blot. Synaptic transmission in IC was recorded by patch-clamp recording. The expression of P2X7Rs and glutamatergic neurotransmission in IC was significantly increased in NMD rats when compared with age-matched controls. Application of BzATP (P2X7R agonist) enhanced the frequency of spontaneous excitatory postsynaptic currents (sEPSC) and miniature excitatory postsynaptic currents (mEPSC) in IC slices of control rats. Application of BBG (P2X7R antagonist) suppressed the frequencies of sEPSC and mEPSC in IC slices of NMD rats. Microinjection of BzATP into right IC significantly decreased CRD threshold in control rats while microinjection of BBG or A438079 into right IC greatly increased CRD threshold in NMD rats. Data suggested that the enhanced activities of P2X7Rs in IC, likely through a presynaptic mechanism, contributed to visceral hypersensitivity of adult rats with NMD.

Role of 5-HT1A receptor in insular cortex mediating stress - induced visceral sensory dysfunction.

5-HT1A receptors (HTR1As) in the insular cortex are thought to be related with the generation of stress-induced functional gastrointestinal disorders (FGIDs), but its mechanism is not clear. Visceral hypersensitivity is one important pathophysiological mechanism of FGIDs. This study aimed to explore the role of HTR1As in mediating stress-induced visceral hypersensitivity and its mechanism in the insular cortex. Visceral hypersensitivity rat model was established by water avoidance stress (WAS) and the visceral sensitivity was measured by electromyogram. The activities of HTR1As were regulated by microinjecting the HTR1A agonist and antagonist into the insular cortex. The expression levels of 5-HT, HTR1A, N-methyl-d-aspartic acid receptor subtype 2B (NR2B) and c-fos were observed by RT-PCR, Western Blot and immunohistochemical staining. In WAS rats, the expression levels of 5-HT and HTR1As in the insular cortex were significantly lower (p < 0.05) than that in sham WAS and normal rats, but the levels of c-fos and NR2B were significantly higher (p < 0.05). After microinjecting HTR1As agonist into the insular cortex of WAS rats, the visceral sensitivity and the expression levels of NR2B and c-fos in insular cortex significantly decreased (p < 0.05). The HTR1As-NR2B signal pathway of insular cortex plays an important role in regulating stress-induced visceral hypersensitivity.

Adrenergic stimulation sensitizes TRPV1 through upregulation of cystathionine β-synthetase in a rat model of visceral hypersensitivity.

The pathogenesis of pain in irritable bowel syndrome (IBS) is poorly understood and treatment remains difficult. The present study was designed to investigate roles of adrenergic signaling and the endogenous hydrogen sulfide producing enzyme cystathionine β-synthetase (CBS) in a previously validated rat model of IBS induced by neonatal colonic inflammation (NCI). Here we showed that NCI-induced visceral hypersensitivity (VH) was significantly attenuated by β2 subunit inhibitor but not by β1 or β3 or α subunit inhibitor. NCI markedly elevated plasma norepinephrine (NE) concentration without alteration in expression of β2 subunit receptors in dorsal root ganglion (DRGs) innervating the colon. In addition, NCI markedly enhanced TRPV1 and CBS expression in the colon DRGs. CBS inhibitor AOAA reversed the upregulation of TRPV1 in NCI rats. In vitro experiments showed that incubation of DRG cells with NE markedly enhanced expression of TRPV1, which was reversed by application of AOAA. Incubation of DRG cells with the H2S donor NaHS greatly enhanced TRPV1 expression. Collectively, these data suggest that activation of adrenergic signaling by NCI sensitizes TRPV1 channel activity, which is likely mediated by upregulation of CBS expression in peripheral sensory neurons, thus contributing to chronic visceral hypersensitivity.

Role of the potassium chloride cotransporter isoform 2-mediated spinal chloride homeostasis in a rat model of visceral hypersensitivity.

Visceral hypersensitivity represents an important hallmark in the pathophysiology of irritable bowel syndrome (IBS), of which the mechanisms remain elusive. The present study was designed to examine whether cation-chloride cotransporter (CCC)-mediated chloride (Cl(-)) homeostasis of the spinal cord is involved in chronic stress-induced visceral hypersensitivity. Chronic visceral hypersensitivity was induced by exposing male Wistar rats to water avoidance stress (WAS). RT-PCR, Western blotting, and immunohistochemistry were used to assess the expression of CCCs in the spinal cord. Patch-clamp recordings were performed on adult spinal cord slices to evaluate Cl(-) homeostasis and Cl(-) extrusion capacity of lamina I neurons. Visceral sensitivity was estimated by measuring the abdominal withdrawal reflex in response to colorectal distension (CRD). After 10 days of WAS exposure, levels of both total protein and the oligomeric form of the K(+)-Cl(-) cotransporter isoform 2 (KCC2), but not Na(+)-K(+)-2Cl(-) transporter isoform 1 (NKCC1), were significantly decreased in the dorsal horn of the lumbosacral spinal cord. The downregulation of KCC2 resulted in a depolarizing shifted equilibrium potential of GABAergic inhibitory postsynaptic current and impaired Cl(-) extrusion capacity in lamina I neurons of the lumbosacral spinal cord from WAS rats. Acute noxious CRD disrupted spinal KCC2 expression and function 2 h after the final distention in sham rats, but not in WAS rats. Pharmacological blockade of KCC2 activity by intrathecal injection of a KCC2 inhibitor [(dihydroindenyl)oxy] alkanoic acid enhanced visceral nociceptive sensitivity in sham rats, but not in WAS rats. These results suggest that KCC2 downregulation-mediated impairment of spinal cord Cl(-) homeostasis may play an important role in chronic stress-induced visceral hypersensitivity.

Involvement of protein kinase ζ in the maintenance of hippocampal long-term potentiation in rats with chronic visceral hypersensitivity.

The hippocampal long-term potentiation (LTP) was implicated in the formation of visceral hypersensitivity in rats with irritable bowel syndrome in our previous study. Recent studies have shown that protein kinase M ζ (PKMζ) may be responsible for the maintenance of LTP in memory formation. However, it remains unclear whether PKMζ is involved in the visceral hypersensitivity. In this study, a rat model of visceral hypersensitivity was generated by neonatal maternal separation (NMS). The visceral hypersensitivity was assessed by recording responses of the external oblique abdominal muscle to colorectal distension. Our results demonstrated that hippocampal LTP and visceral hypersensitivity were enhanced significantly in rats of NMS. ζ-Pseudosubstrate inhibitory peptide (ZIP) could dose dependently inhibit the maintenance of Cornu Ammonis area 1 LTP in rats of NMS. Furthermore, Western blot data showed that the expression of hippocampal phosphorylated PKMζ (p-PKMζ) significantly increased in rats of NMS. In addition, bilateral intrahippocampal injections of ZIP attenuated the visceral hypersensitivity dose dependently in rats of NMS. The maximal inhibition was observed at 30 min, and significant inhibition lasted for 1.5–2 h after ZIP application. Besides, data from the open-field test and Morris water maze showed that ZIP did not influence the movement and spatial procedural memory in rats of NMS. In conclusion, p-PKMζ might be a critical protein in the maintenance of hippocampal LTP, which could result in visceral hypersensitivity.

Decreased miR-199 augments visceral pain in patients with IBS through translational upregulation of TRPV1

ObjectiveMany patients with irritable bowel syndrome IBS not only have abdominal pain but also may suffer from visceral hypersensitivity and heighted visceral nociception. Moreover, IBS has few effective therapeutic agents...

887 Analgesic Action of Resolvin D1 to Combat Visceral Pain in a Rat Model of Visceral Hypersensitivity

G A A b st ra ct s root ganglia (DRG) neurons. The aim of this study was to examine the downstream signaling pathways mediating these effects, with the focus on Smad 3 and TRPV1. Methods: In vitro patch clamp studies were performed on dissociated DRG neurons. For in vivo studies, CP was induced by intraductal injection of trinitrobenzene sulfonic acid (TNBS) in adult male rats, with controls receiving the same volume of vehicle, followed by intrapancreatic injection of DiI, a retrograde neuronal tracer, to label the DRG neurons that project to pancreas. Three weeks later, SIS3 (2.5mg/kg i.p. daily) was administered for 1 week. Subsequently, thoracic DRGs were harvested and stained for pSmad3 using immunohistochemistry. Phosphorylated Smad3 (pSmad3), Smad3 and TRPV1 protein levels in the thoracic DRGs (T8T12) were measured using immunoblot assay. Results: DRG cells were cultured with vehicle (1μg/ml DMSO-solvent for SIS3), TGF β1(10ng/ml), SIS3-TGF β1(10ng/ml-0.3μM), respectively for 48 hours and then TRPV1 currents were determined by the response to 1μM of capsaicin. TGFβ1 significantly enhanced TRPV1 currents in DRG neurons in culture (99.06±24 pA/pF; n=6) compared to controls (44.77±14 pA/pF; n=8), an effect that was blocked by SIS3 (26.23±4.4 pA/pF; n=6) (p<0.01 by ANOVA). However, sis3 was unable to block TRPA1 currents induced by mustard oil, attesting to the relative specificity of this effect. In vivo, as shown previously, SIS3 significantly attenuates both somatic referred hypersensitivity (measured by VFF) and pancreatic hyperalgesia (measured by behavioral response to pancreatic electrical stimulation) in rats with CP. pSmad3 positive neurons in the DiI-labeled thoracic neurons were significantly increased in CP rats (79.06±4.72% compare with control 67.15±1.53%, p<0.05), which can be reduced by treatment with SIS3 (63.64±2.21%, P<0.05). Immunoblot confirmed that pSmad3 levels, but not total Smad3, were significantly reduced in the DRGs (T8-T12) of rats treated with SIS3 relative to vehicle treated rats (P<0.05). Finally, compared with vehicle treatment we found that SIS3 significantly reduced TRPV1 protein levels in rats with CP (106.11±9.44 vs 70.32±6.17, P<0.05). Conclusions: The present results demonstrate a novel link between TGFβ1 signaling and TRPV1 function and expression, an effect that appears to be mediated by Smad3. These results provide the fundamental basis for developing novel therapeutic approaches for CPinduced pain.

Abnormal dendritic cells mediated immune response in a rat model of visceral hypersensitivity

To explore the role of altered characteristics of intestinal dendritic cell (DC) in the induction of visceral hyperalgesia through the activation of mast cells in a rat model of irritable bowel syndrome (IBS). A total of 20 Sprague-Dawley rats were divided into modeling and control groups (n = 10 each). The IBS rat model was established by combining colorectal distention with restraint stress. Abdominal withdrawal reflex (AWR) was employed to evaluate visceral sensitivity. The surface marker of intestinal DC was analyzed by immunohistochemistry. Toluidine blue staining was used to determine the number of mast cells (MC). The expressions of interleukin (IL) -4 and IL-9 in colonic mucosa were measured by enzyme-linked immunosorbent assay (ELISA) and the level of proteinase-activated receptor-2 (PAR-2) was measured by Western blot. Mesenteric lymph node (MLN) DC and splenic CD4(+)/CD8(+)T cells were isolated and purified by magnetic label-based technique. Cytokine production of MLN DC co-culturing with CD4(+) or CD8(+)T cells was determined. The number of colonic MC in modeling group was more than that in control group ((2.73 ± 0.21) vs (1.13 ± 0.10), P = 0.000). The expressions of PAR-2, IL-4 and IL-9 in colonic mucosa were all higher than those in control group (2.13 ± 0.81 vs 0.42 ± 0.29, (7.2 ± 1.2) vs (3.3 ± 1.0) pg/ml, (7.3 ± 1.3) vs (5.2 ± 0.6) pg/ml, P = 0.026, 0.000, 0.001). Co-cultured MLN DC with CD4(+) T cells showed a predominant IL-4 secretion in the modeling group ((1.22 ± 0.33) vs (0.80 ± 0.48) pg/ml, P = 0.000). Increased colonic DC may stimulate CD4(+) T cells to secrete a high level of IL-4 to cause the degranulation of mast cells and the generation of visceral hypersensitivity in IBS rats.

Sensitization of sodium channels by cystathionine β-synthetase activation in colon sensory neurons in adult rats with neonatal maternal deprivation

BackgroundThe pathogenesis of pain in irritable bowel syndrome (IBS) is poorly understood and treatment remains difficult. We have previously reported that TTX-resistant (TTX-R) sodium channels in colon-specific dorsal root ganglion (DRG) neurons were sensitized and the expression of the endogenous hydrogen sulfide producing enzyme cystathionine β-synthetase (CBS) was upregulated in a rat model of visceral hypersensitivity induced by neonatal maternal deprivation (NMD). However, the detailed molecular mechanism for activation of sodium channels remains unknown. This study was designed to examine roles for CBS–H2S signaling in sensitization of sodium channels in a previously validated rat model of IBS. MethodsNeonatal male rats (postnatal days 2–15) were exposed to a 3hour period of daily maternal separation with temperature maintained at ~33°C. Colon-specific dorsal root ganglion (DRG) neurons were labeled with DiI and acutely dissociated for measuring excitability and sodium channel current under whole-cell patch clamp configurations. The expression of NaV1.8 was analyzed by Western blot and Immunofluorescence study. The endogenous H2S producing enzyme CBS antagonist was injected intraperitoneally. ResultsWe showed that CBS was colocalized with NaV1.8 in colon-specific DRG neurons pre-labeled with DiI. Pretreatment of O-(Carboxymethyl) hydroxylamine hemihydrochloride (AOAA), an inhibitor of CBS, significantly reduced expression of NaV1.8 in NMD rats. AOAA treatment also inhibited the TTX-R sodium current density, right-shifted the V1/2 of activation curve, and reversed hyperexcitability of colon-specific DRG neurons in NMD rats. Conversely, addition of NaHS, a donor of H2S, greatly enhanced TTX-R sodium current density, left shifted the activation curve and enhanced excitability of colon DRG neurons in age-matched healthy rats. Furthermore, application of H-89, an inhibitor of protein kinase A, markedly attenuated the potentiation of TTX-R sodium current density by NaHS. ConclusionThese data suggest that sensitization of sodium channels of colon DRG neurons in NMD rats is most likely mediated by CBS–H2S signaling, thus identifying a potential target for treatment for chronic visceral pain in patients with IBS.