Hypersensitivity In Irritable Bowel Syndrome Research Articles

Quercetin Attenuates Visceral Hypersensitivity and 5-Hydroxytryptamine Availability in Postinflammatory Irritable Bowel Syndrome Rats: Role of Enterochromaffin Cells in the Colon

Intestinal enterochromaffin (EC) cell hyperplasia and increased 5-hydroxytryptamine (5-HT) availability play key roles in the pathogenesis of abdominal hypersensitivity of irritable bowel syndrome (IBS). This study aims to study the effect of quercetin on visceral pain and 5-HT availability in postinflammatory IBS (PI-IBS) rats. PI-IBS model rats were administered quercetin by gavage at doses of 5, 10, and 20 mg/kg for 14 days. Compared with normal rats, the visceral pain threshold of PI-IBS rats was markedly decreased and the abdominal motor response to colon distension was markedly increased. The EC cell count and 5-HT level, as well as tryptophan hydroxylase (TPH) protein, were all significantly elevated in PI-IBS rats, while the 5-HT reuptake transporter (serotonin transporter) was reduced. Genes that are responsible for enteroendocrine cell differentiation, that is, Ngn3 and pdx1, were significantly increased in the PI-IBS group. Quercetin treatment markedly elevated the pain threshold pressure and decreased the visceral motor response of PI-IBS animals; and EC cell density and 5-HT level, as well as TPH expression, in the PI-IBS group were all reduced by quercetin. Quercetin treatment also significantly reduced colonic expression of Ngn3 and pdx1 of PI-IBS. Findings from the present study indicated that the analgesic effect of quercetin on PI-IBS may result from reduction of 5-HT availability in the colon, and the regulatory role of quercetin in endocrine progenitors may contribute to reduced EC cells.

MiRNA-29a modulates visceral hyperalgesia in irritable bowel syndrome by targeting HTR7

Some patients with irritable bowel syndrome (IBS) have visceral hypersensitivity, which contributes to their abdominal pain. miRNA-29 was detected to be significantly upregulated in colonic tissues of patients with IBS. However, it is unknown whether miRNA-29a is involved in the visceral hypersensitivity pathogenesis of IBS. This study aimed to investigate whether miRNA-29a participates in visceral hypersensitivity in IBS. We investigated miRNA-29a in intestinal biopsies collected during endoscopy of patients with IBS (n = 10) and healthy volunteers (control) (n = 10). In addition, a water avoidance stress (WAS)-induced visceral hypersensitivity IBS mouse model was established. The abdominal withdrawal reflex (AWR) scores of mice in response to colorectal distention were used to assess visceral sensitivity. Reverse transcription quantitative-polymerase chain reaction (RT-qPCR) was used to measure miRNA-29a levels. Immunofluorescence, RT-qPCR and western blot were used to measure 5-HT7 receptor (HTR7) levels. Bioinformatic analysis and luciferase reporter assays were used to detect the direct relationship between miRNA-29a and HTR7. Finally, alterations in the levels of HTR7 and miRNA-29a were measured in the human intestinal epithelial cell line NCM460 after transfection with miRNA-29a inhibitor or mimic. Intestinal tissues from patients with IBS and WAS-induced IBS mice had increased levels of miRNA-29a, but reduced levels of HTR7. MiRNA-29a knockout resulted in overexpression of HTR7 and attenuated visceral hyperalgesia in WAS-induced IBS mice. HTR7 was a direct target of miRNA-29a. Based on analyses of intestinal tissue samples from patients with IBS and WAS-induced miRNA-29a–/– mice, miRNA-29a plays a role in the visceral hyperalgesia pathogenesis of IBS, probably through regulating HTR7 expression.

Effects of diet with different vitamin D concentration on visceral sensitivity in rats with irritable bowel syndrome

Objective To explore the mechanism of vitamin D in the genesis of irritable bowel syndrome (IBS). Methods A total of 50 newborn Sprague Dawley (SD) rats were divided into three model groups and two control groups (saline control group and normal control group). The model was induced by dilute acetic acid enema. After rats were weaned, the rats of three IBS model groups were fed with normal diet, vitamin D deficient diet and vitamin D sufficient diet, respectively. The visceral sensitivity of the rats was assessed by the colorectal distention experiment. The intestinal tissues of rats was taken for histological score, and the intestinal mast cell (MC) was also counted. The mRNA level of vitamin D receptor (VDR) in colon tissues of rats was determined by real-time reverse transcription-polymerase chain reaction (RT-PCR). T test or rank sum test was performed for statistical analysis. Results When the water volume of balloon was 0.5 mL and 1.0 mL, the abdominal withdrawal reflex (AWR) scores of male rats of the vitamin D deficient model group were 2.67±0.33 and 3.60±0.28, which were higher than those of normal vitamin D model group (1.93±0.15 and 3.20±0.18), and the differences were statistically significant (t=4.491 and 2.683, both P 0.05). The number of MC in the mucosal tissue of the sigmoid colon of female rats in IBS model group was 41.00±19.80, which was higher than that in normal control group (12.40±5.35), and the difference was statistically significant (t=3.118, P=0.030). The number of MC in the mucosal tissue of ileum of the female rats in vitamin D deficiency model group was 16.00±3.71, which was higher than that in normal vitamin D model group (7.30±2.66), and the difference was statistically significant (t=4.263, P=0.003). The VDR mRNA level in the colon tissues of male model rats of normal vitamin D model group was 1.48±0.33, which was higher than that of saline control group and normal control group (0.97±0.21 and 1.00±0.21; t=2.590 and 2.482, both P<0.05). The VDR mRNA levels in colon tissues of female rats of vitamin D deficient model group was 1.90±0.66, which was higher than that of normal control group (1.00±0.14), and the difference was statistically significant (t=2.649, P=0.038). Conclusions Vitamin D may affect visceral hypersensitivity in IBS, and MC may involve in vitamin D induced visceral hypersensitivity. Key words: Irritable bowel syndrome; Vitamin D; Models, animal; Mast cells; Visceral sensitivity

Activating metabotropic glutamate receptor‑7 attenuates visceral hypersensitivity in neonatal maternally separated rats.

Increasing evidence has indicated that metabotropic glutamate receptor-7 (mGluR7) is an important target for reducing anxiety and stress-associated behaviours. Notably, mood disorders exhibit high levels of comorbidity with gastrointestinal dysfunction; however, the role of mGluR7 outside of the central nervous system is currently unknown. Activating mGluR7 likely increases colonic secretory function. Therefore, the present study aimed to evaluate the possible effects of mGluR7 on the visceral hypersensitivity of irritable bowel syndrome (IBS) in rats. The expression levels of mGluR7 were assessed in the colon tissues of rats with neonatal maternal separation (NMS)-induced visceral hypersensitivity using reverse transcription-quantitative polymerase chain reaction, western blotting and immunohistochemistry. In addition, the mGluR7 agonist AMN082 (3 or 10 mg/kg; i.p.) was administered 1 h prior to the visceral hypersensitivity test, and the effects of AMN082 were then observed on the nuclear factor (NF)-κB signalling pathway. The mRNA and protein expression levels of mGluR7 were upregulated in the colon mucosa of NMS rats compared with in normal control rats. Notably, administration of AMN082 (10 mg/kg) attenuated colorectal distension (CRD)-induced visceral hypersensitivity in NMS rats. In addition, interleukin-10 and transforming growth factor-β mRNA expression levels were upregulated, whereas interferon-γ mRNA expression levels were downregulated in the NMS + AMN082 group compared with in NMS rats. The number of cluster of differentiation 3+ T cells in the intestinal mucosa and myeloperoxidase activity were decreased in NMS + AMN082 rats. Furthermore, AMN082 treatment reduced the protein expression levels of phosphorylated-NF-κB in the colon tissue of NMS rats. These results indicated that activation of mGluR7 may attenuate CRD-induced visceral hypersensitivity in experimental IBS and reduce the abnormal immune cytokine response. In addition, it was suggested that the role of AMN082 in modulating the inflammatory response may be partially associated with inhibiting NF-κB activation. These data suggested that targeting mGluR7 may be useful in the treatment of stress-associated IBS.

MicroRNA-200a Targets Cannabinoid Receptor 1 and Serotonin Transporter to Increase Visceral Hyperalgesia in Diarrhea-predominant Irritable Bowel Syndrome Rats.

Background/AimsMicroRNAs (miRNAs) were reported to be responsible for intestinal permeability in diarrhea-predominant irritable bowel syndrome (IBS-D) rats in our previous study. However, whether and how miRNAs regulate visceral hypersensitivity in IBS-D remains largely unknown.MethodsWe established the IBS-D rat model and evaluated it using the nociceptive visceral hypersensitivity test, myeloperoxidase activity assay, restraint stress-induced defecation, and electromyographic (EMG) activity. The distal colon was subjected to miRNA microarray analysis followed by isolation and culture of colonic epithelial cells (CECs). Bioinformatic analysis and further experiments, including dual luciferase assays, quantitative real-time polymerase chain reaction, western blot, and enzyme-linked immunosorbent assay, were used to detect the expression of miRNAs and how it regulates visceral hypersensitivity in IBS-D rats.ResultsThe IBS-D rat model was successfully established. A total of 24 miRNAs were differentially expressed in the distal colon of IBS-D rats; 9 were upregulated and 15 were downregulated. Among them, the most significant upregulation was miR-200a, accompanied by downregulation of cannabinoid receptor 1 (CNR1) and serotonin transporter (SERT). MiR-200a mimic markedly inhibited the expression of CNR1/SERT. Bioinformatic analysis and luciferase assay confirmed that CNR1/SERT are direct targets of miR-200a. Rescue experiments that overexpressed CNR1/SERT significantly abolished the inhibitory effect of miR-200a on the IBS-D rats CECs.ConclusionsThis study suggests that miR-200a could induce visceral hyperalgesia by targeting the downregulation of CNR1 and SERT, aggravating or leading to the development and progression of IBS-D. MiR-200a may be a regulator of visceral hypersensitivity, which provides potential targets for the treatment of IBS-D.

Tu1635 - Symptoms Profile and Ileocecal Hypersensitivity in Irritable Bowel Syndrome, Functional Constipation and Healthy Controls. A Study using the Wireless Motility Capsule (WMC)

Tu1635 - Symptoms Profile and Ileocecal Hypersensitivity in Irritable Bowel Syndrome, Functional Constipation and Healthy Controls. A Study using the Wireless Motility Capsule (WMC)

MLCK-mediated intestinal permeability promotes immune activation and visceral hypersensitivity in PI-IBS mice.

Alterations in intestinal permeability regulated by tight junctions (TJs) are associated with immune activation and visceral hypersensitivity in irritable bowel syndrome (IBS). Myosin light chain kinase (MLCK) is an important mediator of epithelial TJ. The aim of this study is to investigate the role of MLCK in the pathogenesis of IBS using a post infectious IBS (PI-IBS) mouse model. Trichinella spiralis-infected PI-IBS mouse model was used. Urine lactulose/mannitol ratio was measured to assess intestinal epithelial permeability. Western blotting was used to evaluate intestinal TJ protein (zonula occludens-1) and MLCK-associated protein expressions. Immune profile was assessed by measuring Th (T helper) 1/Th2 cytokine expression. Visceral sensitivity was determined by abdominal withdrawal reflex in response to colorectal distension. Eight weeks after inoculation with T. spiralis, PI-IBS mice developed decreased pain and volume thresholds during colorectal distention, increased urine lactulose/mannitol ratio, elevated colonic Th1/Th2 cytokine ratio, and decreased zonula occludens-1 expression compared to the control mice. MLCK expression was dramatically elevated in the colonic mucosa of PI-IBS mice compared to the control mice, alongside increased pMLC/MLC and decreased MLCP expression. Administration of MLCK inhibitor and TJ blocker both reversed the increased intestinal permeability, visceral hypersensitivity, and Th1-dominant immune profile in PI-IBS mice. MLCK is a pivotal step in inducing increased intestinal permeability promoting low-grade intestinal immune activation and visceral hypersensitivity in PI-IBS mice. MLCK inhibitor may provide a potential therapeutic option in the treatment of IBS.

Clostridium butyricum regulates visceral hypersensitivity of irritable bowel syndrome by inhibiting colonic mucous low grade inflammation through its action on NLRP6.

Visceral hypersensitivity induced by stress is quite common in irritable bowel syndrome (IBS) patients. Probiotics play an important role in reducing visceral hypersensitivity in IBS patients. However, the mechanism has not been clearly elucidated. In this study, we investigated the role of nod-like receptor pyrin domain-containing protein 6 (NLRP6) in Clostridium butyricum-regulated IBS induced by stress. Our results showed that NLRP6 was down-regulated in IBS group colon tissues. In addition, IL-18, IL-1β, myeloperoxidase (MPO), d-lactic acid (D-LA), and CD172a were up-regulated in the IBS group of colonic mucous. IL-18 and IL-1β were also increased after the NLRP6 gene was silenced. Pathological score suggested low inflammation of colonic mucous rather than terminal ileum. Water-avoidance stress (WAS) showed visceral hypersensitivity to colonic distension. However, treatment with Clostridium butyricum reversed these results, exerting a beneficial effect. In conclusion, Clostridium butyricum may exert a beneficial action on visceral hypersensitivity of IBS by inhibiting low grade inflammation of colonic mucous through its action on NLRP6.

Are characteristics of abdominal pain helpful to identify patients with visceral hypersensitivity in irritable bowel syndrome? Results of a prospective study.

Some consider that patients with visceral hypersensitivity may represent a separate entity within the IBS population not only from a pathophysiological but also from a clinical perspective. The aim of this prospective exploratory study was to assess whether characteristics of abdominal pain in IBS patients could be suggestive of hypersensitivity. This prospective study included consecutive IBS patients selected by Rome III criteria. Validated scores (IBS-SSS, Bristol stool scale, HADS) were used to phenotype patients who were also asked to describe the main location of their abdominal pain on a simple image (abdomen divided into 6 zones). Progressive isobaric rectal distensions were performed to demonstrate, with the ascending method of limits, allodynia (pain threshold lower than 24mmHg). Fifty patients (women: 72%), 42.6±15.7years old, were included. Sub-types were IBS-D, IBS-C and IBS-M in 58%, 22% and 20% of cases, respectively. Allodynia was present in 18% of cases. Neither IBS-SSS nor intensity of pain was predictive of hypersensitivity. In hypersensitive patients, pain was more often located in one of the two iliac fossa (P=0.02) and located outside these areas in only 11% of cases. The sensitivity and the specificity of this pain location to differentiate hyper from normosensitive patients were 0.89 and 0.59, respectively. The location of pain is different between hyper and normosensitive IBS patients. Pain located outside one of the two iliac fossa suggests that the patient is normosensitive.

Biomarkers for visceral hypersensitivity in patients with irritable bowel syndrome.

Increased visceral sensitivity is observed in up to 60% of patients with Irritable Bowel Syndrome (IBS). Mucosal inflammation, altered neuroendocrine activity and intraluminal metabolic processes may contribute to the development of visceral hypersensitivity. Previously, we demonstrated that biomarkers, indicative for these biological processes, were altered in IBS patients compared to healthy controls. However, how these processes relate to visceral hypersensitivity is unknown. The aim of this study was to provide insight in biological processes associated with visceral hypersensitivity. Fecal and plasma biomarkers were measured in normosensitive and hypersensitive IBS patients. A total of 167 IBS patients underwent a rectal barostat procedure to assess visceral sensitivity to pain. Based on the outcome, patients were classified into a normosensitive or hypersensitive group. Calprotectin, human β-defensin 2 (HBD2), chromogranin A (CgA), and short chain fatty acids (SCFAs) were measured in feces, citrulline in plasma, and serotonin and its main metabolite 5-hydroxyindoleacetic acid (5-HIAA) in platelet-poor plasma. Fecal markers and plasma citrulline were measured in 83 hypersensitive and 84 normosensitive patients, while platelet-poor plasma for the assessment of serotonin and 5-HIAA was available for a subgroup, i.e. 53 hypersensitive and 42 normosensitive patients. No statistically significant differences were found in concentrations of biomarkers between groups. Adjustment of the analyses for potential confounders, such as medication use, did not alter this conclusion. Our findings do not support a role for the biological processes as ascertained by biomarkers in visceral hypersensitivity in IBS patients. This study is registered in the US National Library of Medicine (clinicaltrials.gov, NCT00775060).

Piezo2: A Candidate Biomarker for Visceral Hypersensitivity in Irritable Bowel Syndrome?

Background/AimsCurrently, there exists no biomarker for visceral hypersensitivity in irritable bowel syndrome (IBS). Piezo proteins have been proven to play an important role in the mechanical stimulation to induce visceral pain in other tissues and may also be a biomarker candidate. The aim of this study was to test the expressions of Piezo1 and Piezo2 proteins in the intestinal epithelial cells from different intestinal segments and to explore the correlation between Piezo proteins expression and visceral pain threshold.MethodsPost-infectious IBS was induced in mice via a Trichinella spiralis infection. Visceral sensitivity was measured with abdominal withdrawal reflex to colorectal distention. Inflammation in the small intestine and colon was scored with H&E staining. Expression location of Piezo proteins was confirmed by immunohistochemistry. Abundance of Piezo proteins were measured with real-time reverse transcriptase polymerase chain reaction.ResultsPiezo1 and Piezo2 proteins were expressed in the intestinal epithelial cells. The expression levels of Piezo1 and Piezo2 were abundant in the colon than the small intestine (P < 0.001 for Piezo1, P = 0.003 for Piezo2). Expression of Piezo2 in the colon significantly correlated to the visceral sensitivity (r = −0.718, P = 0.001) rather than the mucosal inflammation.ConclusionPiezo2 is a candidate biomarker for visceral hypersensitivity in IBS.

Colonic N-methyl-d-aspartate receptor contributes to visceral hypersensitivity in irritable bowel syndrome.

N-methyl-d-aspartate receptor (NMDAR) in brain, spinal cord, and enteric nervous system is involved in visceral hypersensitivity. This study aimed to reveal the functional expression of NMDAR on mucosal cells in colon and to investigate the downstream signal pathway from colonic NMDAR activation to visceral hypersensitivity in irritable bowel syndrome (IBS). The expression of mucosal NMDAR in IBS patients and healthy controls was assessed by immunohistochemistry and Western blot and correlated with abdominal pain/discomfort scores quantified by a validated questionnaire. Electromyography recording in response to colorectal distension was recorded to measure the colonic sensitivity of mice receiving NMDA administration intracolonically. Brain-derived neurotrophic factor (BDNF) expression and extracellular signal-regulated kinase (ERK) pathway activation were examined in human colonic epithelial HT29 cells after NMDA stimulation, with or without MK801 or U0126 pretreatment. A significant upregulation of mucosal NMDAR was observed in IBS patients compared with controls, which was significantly correlated with abdominal pain/discomfort scores. Intracolonic administration of NMDA in normal mice produced increased colonic sensitivity to colorectal distension and elevated expression of BDNF and activation of ERK. Activation of NMDAR in colonic epithelial HT29 cells in vitro induced increased BDNF secretion in cell supernatants and higher BDNF expression in cells, as well as elevated phosphorylated ERK. This study demonstrated that the activation of mucosal NMDAR in colon may contribute to the visceral hypersensitivity in IBS, by increasing production of BDNF in an ERK-dependent pathway.

Brain functional connectivity is associated with visceral sensitivity in women with Irritable Bowel Syndrome.

Increased perception of visceral stimuli is a key feature of Irritable Bowel Syndrome (IBS). While altered resting-state functional connectivity (rsFC) has been also reported in IBS, the relationship between visceral hypersensitivity and aberrant rsFC is unknown. We therefore assessed rsFC within the salience, sensorimotor and default mode networks in patients with and without visceral hypersensitivity and in healthy controls (HCs).An exploratory resting-state functional magnetic resonance imaging study was performed in 41 women with IBS and 20 HCs. Group independent component analysis was used to derive intrinsic brain networks. Rectal thresholds were determined and patients were subdivided into groups with increased (hypersensitive IBS, N = 21) or normal (normosensitive IBS, N = 20) visceral sensitivity. Between-group comparisons of rsFC were carried-out using region-of-interest analyses and peak rsFC values were extracted for correlational analyses.Relative to normosensitive IBS, hypersensitive patients showed increased positive rsFC of pregenual anterior cingulate cortex and thalamus within the salience network and of posterior insula within the sensorimotor network. When compared to both hypersensitive IBS and HCs, normosensitive IBS showed decreased positive rsFC of amygdala and decreased negative rsFC in dorsal anterior insula within the DMN. DMN and sensorimotor network rsFC were associated with rectal perception thresholds, and rsFC in posterior insula was correlated with reported symptom severity in IBS.Our exploratory findings suggest that visceral sensitivity in IBS is related to changes in FC within resting-state networks associated with interoception, salience and sensory processing. These alterations may play an important role in hypervigilance and hyperalgesia in IBS.

PDIA3 gene induces visceral hypersensitivity in rats with irritable bowel syndrome through the dendritic cell-mediated activation of T cells.

This study investigated the mechanism of protein disulfide-isomerase A3 (PDIA3)-induced visceral hypersensitivity in irritable bowel syndrome (IBS). Rats were treated with saline (control), acetic acid and restraint stress (IBS model), empty vector (RNAi control) and PDIA3-RNAi vector (PDIA3-RNAi). Mesenteric lymph node DCs (MLNDCs) and splenic CD4+/CD8+ T cells were isolated for co-cultivation. Compared with control, MLNDCs co-cultured with CD4+ or CD8+ T cells showed an increased ability to promote T cell proliferation and produced more IL-4 or IL-9 secretion. Compared with the RNAi control, MLNDCs from the PDIA3 knockdown models were less effective in promoting the proliferation of CD4+/CD8+ T cells. It is concluded that PDIA3 plays an important role in the development of IBS through the DC-mediated activation of T cells, resulting in degranulation of MCs and visceral hypersensitivity.

Ion channels, ion channel receptors, and visceral hypersensitivity in irritable bowel syndrome.

Ion channels are expressed throughout the gastrointestinal system and regulate nearly every aspect of digestion, including fluid secretion and absorption, motility, and visceral sensitivity. It is therefore not surprising that in the setting of functional bowel disorders, such as irritable bowel syndrome (IBS), ion channels are often altered in terms of expression level and function and are a target of pharmacological intervention. This is particularly true of their role in driving abdominal pain through visceral hypersensitivity (VH), which is the main reason IBS patients seek medical care. In the study by Scanzi etal., in the current issue of this journal, they provide evidence that the T-type voltage-gated calcium channel (Cav ) Cav 3.2 is upregulated in human IBS patients, and is necessary for the induction of an IBS-like disease state in mice. In this mini-review, we will discuss the contribution of specific ion channels to VH in IBS, both in human patients and rodent models. We will also discuss how Cav 3.2 may play a role as an integrator of multiple environmental stimuli contributing toward VH.

Zeta Inhibitory Peptide as a Novel Therapy to Control Chronic Visceral Hypersensitivity in a Rat Model.

BackgroundThe pathogenesis of multiple chronic visceral pain syndromes, such as irritable bowel syndrome (IBS), is not well known, and as a result current therapies are ineffective. The objective of this study was to investigate the effect of spinal protein kinase M zeta (PKMζ) on visceral pain sensitivity in rats with IBS to better understand the pathogenesis and investigate the effect of zeta inhibitory peptide (ZIP) as a therapy for chronic visceral pain.MethodsVisceral hypersensitivity rats were produced by neonatal maternal separation (NMS). Visceral pain sensitivity was assessed by electromyographic (EMG) responses of abdominal muscles to colorectal distention (CRD). Spinal PKMζ and phosphorylated PKMζ (p-PKMζ) were detected by western blot. Varying doses of ZIP were intrathecally administered to investigate the role of spinal PKMζ in chronic visceral hypersensitivity. The open field test was used to determine if ZIP therapy causes spontaneous motor activity side effects.ResultsGraded CRD pressure significantly increased EMG responses in NMS rats compared to control rats (p < 0.05). p-PKMζ expression increased in the thoracolumbar and lumbosacral spinal cord in the IBS-like rats with notable concomitant chronic visceral pain compared to control rats (p < 0.05). EMG data revealed that intrathecal ZIP injection (1, 5, and 10 μg) dose-dependently attenuated visceral pain hypersensitivity in IBS-like rats.ConclusionsPhosphorylated PKMζ may be involved in the spinal central sensitization of chronic visceral hypersensitivity in IBS, and administration of ZIP could effectively treat chronic visceral pain with good outcomes in rat models.

Colonic mucosal N‐methyl‐D‐aspartate receptor mediated visceral hypersensitivity in a mouse model of irritable bowel syndrome

The aim of this study was to investigate whether colonic mucosal N-methyl-D-aspartate receptor (NMDAR) participates in visceral hypersensitivity in irritable bowel syndrome (IBS). C57BL/6 mice were administered intrarectally with trinitrobenzenesulfonic acid (TNBS) for the establishment of an IBS-like visceral hypersensitivity model. Those received an equivalent volume of 50% ethanol were regarded as the controls. Abdominal withdrawal reflex (AWR) scores in response to colorectal distention (CRD) were used to assess visceral sensitivity. NMDAR levels in the colonic mucosa were detected by both immunohistochemistry and Western blot. The concentrations of glutamate and ammonia in the feces of the mice were measured. Changes in visceral sensitivity after the intracolonic administration of ammonia or NMDAR antagonist were recorded. The established IBS-like mouse model of visceral hypersensitivity showed no evident inflammation in the colon. NMDAR levels in the colonic mucosa of the IBS-like mice were significantly higher compared with the controls, and were positively associated with AWR scores. The glutamate level in the feces of the TNBS-treated mice was similar to that of the controls, although the ammonia level was significantly higher. Intracolonic administration of ammonia induced visceral hypersensitivity in mice, which was repressed by pretreatment with NMDAR antagonist MK801. Overexpressed NMDAR in the colonic mucosa may participate in the pathogenesis of visceral hypersensitivity in IBS. Our study identifies the effect of ammonia in the colonic lumen on NMDAR in the colonic mucosa as a potential novel targeted mechanism for IBS treatment.

Role of colonic mast cell infiltration and nerve growth factor in visceral hypersensitivity of irritable bowel syndrome

Objective To investigate the role of mucosal mast cells infiltration and degranulation with nerve growth factor (NGF) in development of visceral hypersensitivity in Sprague-Dawley (SD) rats. Methods The model of visceral hypersensitivity of irritable bowel syndrome (IBS) was established in 19 neonate SD rats with intestinal stimulation (rectalballon distention) on 8th, 10th and 12th postnatal days. The other 19 neonate SD rats without colonic distention were assigned to the control group. After rats grew up (six to eight weeks old), the visceral sensitivity was tested by abdominal withdrawal reflex (AWR) in 10 rats of each group. Mast cell infiltration and degranulation were observed with toluidine blue staining in colon tissue slides. The NGF level of intestinal tissues was detected by enzyme-linked immunosorbent assay (ELISA) methods in the left nine rats of each group. The culture system of dorsal root ganglias (DRG) from the neonatal rats was set up. The changes of electrophysilogical characters of DRG stimulated with NGF (100 ng/mL) for four days were recorded with patch-clamp.Paired t test was performed for comparison between groups. Results The results of AWR indicated that neonatal colonic stimulation could significantly increase visceral sensitivity after growing up. Under 20, 40 and 60 mmHg (1 mmHg=0.133 kPa) distention pressure, visceral sensitivity scores of visceral hypersensitivity rats and rats of control group were 1.00±0.50 vs 1.67±0.50, 1.89±0.31 vs 2.89±0.34 and 2.89±0.33 vs 3.89±0.33, the differences were statistically significant (t=-2.83, -6.00 and -6.00, all P<0.05). The results of master cells staining in tissue slides showed colonic master cells infiltration was obvious in rats with visceral hypersensitivity, and part of mast cells were degranulation. The result of ELISA demonstrated that NGF level of visceral hypersensitivity rats was significantly higher than that of control group ((11.07±3.06) pg/mg vs (2.38±1.88) pg/mg, t=-6.93, P<0.05). The results of electrophysilogical tests of primary cultured DRG indicated that compared with blank control growp, the action potential threshold of neuron in NGF 100 ng/mL group significantly decreased ((-18.0±2.1) mV vs (-29.0±2.5) mV, t=12.26, P<0.05) and discharge frequency increased ((5.0±1.4)/800 ms vs (12.0±3.2)/800 ms, t=-8.40, P<0.05). Meanwhile, neuron voltage-gated K+ current density remarkably decreased, most were sustained delayed rectifier K+ current (IK) decreasing ((279.0±48.0) pA/pF vs (203.0±39.0) pA/pF, t=6.18, P<0.05). Conclusion Colonic stimulation in neonatal rats could cause intestinal master cells infiltration and degranulation, which induced changes of neuron electrophysilogical characters and resulted in visceral hypersensitivity after growing up. Key words: Irritable bowel syndrome; Hyperesthesia; Ganglion, spinal; Mast cells; Nerve growth factor

Expression and distribution of SP and its NK1 receptor in the brain-gut axis in neonatal maternally separated rat model with visceral hypersensitivity

Neurokinin-1 receptor (NK1R) is a high affinity Substance P (SP) receptor and plays a key role in visceral hypersensitivity in irritable bowel syndrome (IBS). Early life stress is a significant risk factor in IBS. The aim of the present study was to investigate the influence of neonatal maternal separation on the expression and distribution of SP and its receptor along the brain-gut axis in a neonatal maternally separated rat model with visceral hypersensitivity. Male neonatal Sprague-Dawley rats, 2-21-day old, were randomly distributed into maternal separation groups of 3 h daily maternal separation (MS) or non-handling (NH). These rats underwent colorectal balloon distention (CRD) upon reaching adulthood. Immunofluorescence was used to examine the distal colon, lumbosacral spinal cord, and the brainstem to semi-quantitatively determine SP and NK1R expression before and after CRD. The following features were assessed: percentage SP-positive area in colonic muscle layer, the number of NK1R-positive myenteric plexus, SP-positive area and NK1-positivity score in the dorsal horn and the brainstem. Neither of these was altered in the MS and NH groups before or after CRD. These results suggest that the SP system might play little role in the development of visceral hyperalgesia in the neonatal maternal separation rat model.

Excessive coupling of the salience network with intrinsic neurocognitive brain networks during rectal distension in adolescents with irritable bowel syndrome: a preliminary report.

The neural network mechanisms underlying visceral hypersensitivity in irritable bowel syndrome (IBS) are incompletely understood. It has been proposed that an intrinsic salience network plays an important role in chronic pain and IBS symptoms. Using neuroimaging, we examined brain responses to rectal distension in adolescent IBS patients, focusing on determining the alteration of salience network integrity in IBS and its functional implications in current theoretical frameworks. We hypothesized that (i) brain responses to visceral stimulation in adolescents are similar to those in adults, and (ii) IBS is associated with an altered salience network interaction with other neurocognitive networks, particularly the default mode network (DMN) and executive control network (ECN), as predicted by the theoretical models. Irritable bowel syndrome patients and controls received subliminal and liminal rectal distension during imaging. Stimulus-induced brain activations were determined. Salience network integrity was evaluated by the functional connectivity of its seed regions activated by rectal distension in the insular and cingulate cortices. Compared with controls, IBS patients demonstrated greater activation to rectal distension in neural structures of the homeostatic afferent and emotional arousal networks, especially the anterior cingulate and insular cortices. Greater brain responses to liminal vs subliminal distension were observed in both groups. Particularly, IBS is uniquely associated with an excessive coupling of the salience network with the DMN and ECN in their key frontal and parietal node areas. Our study provided consistent evidence supporting the theoretical predictions of altered salience network functioning as a neuropathological mechanism of IBS symptoms.