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Background and AimsHistone deacetylase inhibitors [HDACi] exert potent anti-inflammatory effects. Because of the ubiquitous expression of HDACs, clinical utility of HDACi is limited by off-target effects. Esterase-sensitive motif [ESM] technology aims to deliver ESM-conjugated compounds to human mononuclear myeloid cells, based on their expression of carboxylesterase 1 [CES1]. This study aims to investigate utility of an ESM-tagged HDACi in inflammatory bowel disease [IBD].MethodsCES1 expression was assessed in human blood, in vitro differentiated macrophage and dendritic cells, and Crohn’s disease [CD] colon mucosa, by mass cytometry, quantitative polymerase chain reaction [PCR], and immunofluorescence staining, respectively. ESM-HDAC528 intracellular retention was evaluated by mass spectrometry. Clinical efficacy of ESM-HDAC528 was tested in dextran sulphate sodium [DSS]-induced colitis and T cell transfer colitis models using transgenic mice expressing human CES1 under the CD68 promoter.ResultsCES1 mRNA was highly expressed in human blood CD14+ monocytes, in vitro differentiated and lipopolysaccharide [LPS]-stimulated macrophages, and dendritic cells. Specific hydrolysis and intracellular retention of ESM-HDAC528 in CES1+ cells was demonstrated. ESM-HDAC528 inhibited LPS-stimulated IL-6 and TNF-α production 1000 times more potently than its control, HDAC800, in CES1high monocytes. In healthy donor peripheral blood, CES1 expression was significantly higher in CD14++CD16- monocytes compared with CD14+CD16++ monocytes. In CD-inflamed colon, a higher number of mucosal CD68+ macrophages expressed CES1 compared with non-inflamed mucosa. In vivo, ESM-HDAC528 reduced monocyte differentiation in the colon and significantly improved colitis in a T cell transfer model, while having limited potential in ameliorating DSS-induced colitis.ConclusionsWe demonstrate that monocytes and inflammatory macrophages specifically express CES1, and can be preferentially targeted by ESM-HDAC528 to achieve therapeutic benefit in IBD.

Background and Aims Tyrosine kinase 2 [TYK2] is required for the signalling of key cytokines in the pathogenesis of inflammatory bowel disease [IBD]. We assessed the efficacy of a novel selective TYK2 inhibitor [TYK2i] in experimental colitis, using pharmacological and genetic tools. Methods At onset of T cell transfer colitis, RAG1-/- mice received vehicle or TYK2i daily by oral gavage. T cells lacking TYK2 kinase activity [TYK2KE] were used to confirm selectivity of the inhibitor. To this end, RAG1-/- or RAG1-/-TYK2KE animals were transferred with either wild type [WT] or TYK2KE-CD45RBhigh colitogenic T cells. Loss of body weight, endoscopic disease, the disease activity index [DAI], and histopathology scores were recorded. Tissues were analysed ex vivo for lymphocyte populations by flow cytometry. The impact of TYK2 inhibition on human DC-T cell interactions were studied using autologous Revaxis specific T cell assays. Results TYK2i [70 mg/kg] prevented weight loss and limited endoscopic activity during T cell transfer colitis. TYK2i [70 mg/kg] decreased DAI. Whereas transfer of WT T cells into RAG-/-TYK2KE hosts induced colitis, TYK2KE T cells transferred into RAG1-/-TYK2KErecipients failed to do so. Ex vivo analysis showed a decrease in colon tissue Th1 cells and an increase in Th17 cells upon transfer of TYK2KE-CD45RBhigh cells. In human antigen-triggered T cells, TYK2i displayed reduced Th1 differentiation, similar to murine Th1 cells. Conclusions Oral administration of TYK2i, as well as transfer of T cells lacking TYK2 activity, reduced human Th1 differentiation and ameliorated the course of murine T cell transfer colitis. We conclude that TYK2 is a promising drug target for the treatment of IBD.

Irritable bowel syndrome (IBS) is a heterogenic, functional gastrointestinal disorder of the gut-brain axis characterized by altered bowel habit and abdominal pain. Preclinical and clinical results suggested that, in part of these patients, pain may result from fungal induced release of mast cell derived histamine, subsequent activation of sensory afferent expressed histamine-1 receptors and related sensitization of the nociceptive transient reporter potential channel V1 (TRPV1)-ion channel. TRPV1 gating properties are regulated in lipid rafts. Miltefosine, an approved drug for the treatment of visceral Leishmaniasis, has fungicidal effects and is a known lipid raft modulator. We anticipated that miltefosine may act on different mechanistic levels of fungal-induced abdominal pain and may be repurposed to IBS. In the IBS-like rat model of maternal separation we assessed the visceromotor response to colonic distension as indirect readout for abdominal pain. Miltefosine reversed post-stress hypersensitivity to distension (i.e. visceral hypersensitivity) and this was associated with differences in the fungal microbiome (i.e. mycobiome). In vitro investigations confirmed fungicidal effects of miltefosine. In addition, miltefosine reduced the effect of TRPV1 activation in TRPV1-transfected cells and prevented TRPV1-dependent visceral hypersensitivity induced by intracolonic-capsaicin in rat. Miltefosine may be an attractive drug to treat abdominal pain in IBS.

β-glucan consumption is known for its beneficial health effects, but the mode of action is unclear. While humans and mice lack the required enzymes to digest β-glucans, certain intestinal microbes can digest β-glucans, triggering gut microbial changes. Curdlan, a particulate β-glucan isolated from Alcaligenes faecalis, is used as a food additive. In this study we determined the effect of curdlan intake in mice on the intestinal microbiota and dextran sodium sulfate (DSS)-induced intestinal inflammation. The effect of curdlan on the human intestinal microbiota was assessed using i-screen, an assay for studying anaerobic microbial interactions. Mice received oral gavage with vehicle or curdlan for 14 days followed by DSS for 7 days. The curdlan-fed group showed reduced weight loss and colonic inflammation compared to the vehicle-fed group. Curdlan intake did not induce general microbiota community changes, although a specific Bifidobacterium, closely related to Bifidobacterium choerinum, was observed to be 10- to 100-fold more prevalent in the curdlan-fed group under control and colitis conditions, respectively. When tested in i-screen, curdlan induced a global change in the microbial composition of the healthy intestinal microbiota from a human. Overall, these results suggest that dietary curdlan induces microbiota changes that could reduce intestinal inflammation.

Background Recent evidence demonstrated that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) propagates in intestinal epithelial cells expressing Angiotensin-Converting Enzyme 2 (ACE2), implying that these cells represent an important entry site for the viral infection. Nicotinic receptors (nAChRs) have been put forward as potential regulators of inflammation and of ACE2 expression. As vagus nerve stimulation (VNS) activates nAChRs, we aimed to investigate whether VNS can be instrumental in affecting intestinal epithelial ACE2 expression. Methods By using publicly available datasets we qualified epithelial ACE2 expression in human intestine, and assessed gene co-expression of ACE2 and SARS-CoV-2 priming Transmembrane Serine Protease 2 (TMPRSS2) with nAChRs in intestinal epithelial cells. Next, we investigated mouse and human ACE2 expression in intestinal tissues after chronic VNS via implanted devices. Results We show co-expression of ACE2 and TMPRSS2 with nAChRs and α7 nAChR in particular in intestinal stem cells, goblet cells, and enterocytes. However, VNS did not affect ACE2 expression in murine or human intestinal tissue, albeit in colitis setting. Conclusions ACE2 and TMPRSS2 are specifically expressed in epithelial cells of human intestine, and both are co-expressed with nAChRs. However, no evidence for regulation of ACE2 expression through VNS could be found. Hence, a therapeutic value of VNS with respect to SARS-CoV-2 infection risk through ACE2 receptor modulation in intestinal epithelia could not be established.

Clinical trials suggest that vagus nerve stimulation presents an alternative approach to classical immune suppression in Crohn's disease. T cells capable of producing acetylcholine (ChAT+ T cells) in the spleen are essential mediators of the anti-inflammatory effect of vagus nerve stimulation. Besides the spleen, ChAT+ T cells are found abundantly in Peyer’s patches of the small intestine. However, the role of ChAT+ T cells in colitis pathogenesis is unknown. Here, we made use of CD4creChATfl/fl mice (CD4ChAT−/− mice) lacking ChAT expression specifically in CD4+ T cells. Littermates (ChATfl/fl mice) served as controls. In acute dextran sulfate sodium (DSS)-induced colitis (7 days of 2% DSS in drinking water), CD4ChAT−/− mice showed attenuated colitis and lower intestinal inflammatory cytokine levels compared with ChATfl/fl mice. In contrast, in a resolution model of DSS-induced colitis (5 days of 2% DSS followed by 7 days without DSS), CD4ChAT−/− mice demonstrated a worsened colitis recovery and augmented colonic histological inflammation scores and inflammatory cytokine levels as compared with ChATfl/fl mice. In a transfer colitis model using CD4+CD45RBhigh T cells, T cells from CD4ChAT−/− mice induced a similar level of colitis compared with ChATfl/fl T cells. Together, our results indicate that ChAT+ T cells aggravate the acute innate immune response upon mucosal barrier disruption in an acute DSS-induced colitis model, whereas they are supporting the later resolution process of this innate immune-driven colitis. Surprisingly, ChAT expression in T cells seems redundant in the context of T cell-driven colitis. NEW & NOTEWORTHY By using different mouse models of experimental colitis, we provide evidence that in dextran sulfate sodium-induced colitis, ChAT+ T cells capable of producing acetylcholine worsen the acute immune response, whereas they support the later healing phase of this innate immune-driven colitis.

Background & Aims 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. Methods 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 (ex vivo) and the human mast cell line HMC-1. Results 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. Conclusions 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. © 2017 AGA Institute. Chemicals/CAS: protein kinase Syk, 138674-26-7; Antifungal Agents; beta-Glucans; Protein Kinase Inhibitors; Syk Kinase; Syk protein, rat

Background MiR-511 is embedded in intron region 5 of the CD206/MRC1 gene, expressed by macrophage and dendritic cell populations. In this study, we aimed to investigate the effect of MiR-511 deficiency on intestinal inflammation in a murine T cell transfer colitis model. Methods A double MiR-511- and Rag-1 (knockout) KO mouse was generated and a T cell transfer colitis was induced by intraperitoneal injection of naïve T cells from donor WT mice. Since these mice lack mature T and B cells, first signs of inflammation appeared at week 3 after T cell injection. An endoscopy score was obtained to determine inflammation at week 3 and 5, respectively. The experiment was terminated at week 5 and severity of inflammation was assessed on the basis of weight loss, colon weight/length ratio, histology score, spleen weight and disease activity index. In addition, flow cytometry was performed for analysing immune cell populations (monocyte, macrophages, dendritic cells, neutrophils) in the colons of both control and colitis groups and T cells in the spleens of colitis group, respectively. Results Following the induction of T cell transfer colitis, colon weight/length ratio, spleen weight and endoscopic score were significantly increased in the double KO mice compared to Rag-1 KO control mice. A higher histology score and disease activity index in the double KO with no change in weight loss compared to Rag-1 KO control mice was observed. A significant increase in monocyte population in the colons of double KO was seen and increased numbers of monocytes was also observed in the double KO control group with no inflammation. Also, a higher influx of T cells in the double KO mice with a significant increase in Foxp3 and IL4 population was observed in the group with colitis. Conclusion MiR-511 deficiency aggravates intestinal inflammation compared to Rag-1 KO control mice. Also, a higher presence of monocyte as well as T cell populations were observed in these mice. Together these data show that MiR-511 is involved in the regulation of intestinal health. Future research will focus on underlying mechanisms.

Background Janus kinases (JAKs) mediate cytokine signaling involved in inflammatory bowel disease. The pan-JAK inhibitor tofacitinib has shown efficacy in the treatment of ulcerative colitis. However, concerns regarding adverse events due to their wide spectrum inhibition fueled efforts to develop selective JAK inhibitors. Given the crucial role of myeloid cells in intestinal immune homeostasis, we evaluated the effect of pan-JAK and selective JAK inhibitors on pro- and anti-inflammatory macrophage polarization and function (M1/M2) and in experimental colitis. Methods Murine bone marrow–derived macrophages or human monocytes were treated using JAK1 and JAK3 selective inhibitors (JAK1i;JAK3i) and tofacitinib and were evaluated by transcriptional, functional, and metabolic analyses. In vivo, oral administration of JAK1i and tofacitinib (10 or 30 mg/kg) was tested in both acute and acute rescue dextran sodium sulfate (DSS) colitis. Results Both tofacitinib and JAK1i but not JAK3i effectively inhibited STAT1 phosphorylation and interferon gamma–induced transcripts in M1 polarized macrophages. Strikingly, transcriptional profiling suggested a switch from M1 to M2 type macrophages, which was supported by increased protein expression of M2-associated markers. In addition, both inhibitors enhanced oxidative phosphorylation rates. In vivo, JAK1i and tofacitinib did not protect mice from acute DSS-induced colitis but ameliorated recovery from weight loss and disease activity during acute rescue DSS-induced colitis at the highest dose. Conclusion JAK1i and tofacitinib but not JAK3i induce phenotypical and functional characteristics of anti-inflammatory macrophages, suggesting JAK1 as the main effector pathway for tofacitinib in these cells. In vivo, JAK1i and tofacitinib modestly affect acute rescue DSS-induced colitis.

β-Glucans have beneficial health effects due to their immune modulatory properties. Oral administration of β-glucans affects tumour growth, microbial infection, sepsis, and wound healing. We hypothesized that pre-treatment with orally delivered soluble and particulate β-glucans could ameliorate the development of aggravate dextran sulfate sodium (DSS) induced intestinal inflammation. To study this, mice were orally pre-treated with β-glucans for 14 days. We tested curdlan (a particulate β-(1,3)-glucan), glucan phosphate (a soluble β-(1,3)-glucan), and zymosan (a particle made from Saccharomyces cerevisiae, which contains around 55% β-glucans). Weight loss, colon weight, and feces score did not differ between β-glucan and vehicle treated groups. However, histology scores indicated that β-glucan–treated mice had increased inflammation at a microscopic level suggesting that β-glucan treatment worsened intestinal inflammation. Furthermore, curdlan and zymosan treatment led to increased colonic levels of inflammatory cytokines and chemokines, compared to vehicle. Glucan phosphate treatment did not significantly affect cytokine and chemokine levels. These data suggest that particulate and soluble β-glucans differentially affect the intestinal immune responses. However, no significant differences in other clinical colitis scores between soluble and particulate β-glucans were found in this study. In summary, β-glucans aggravate the course of dextran sulfate sodium (DSS)-induced intestinal inflammation at the level of the mucosa.

Background: Irritable bowel syndrome (IBS) is a common gastrointestinal disorder associated with altered gastrointestinal microflora and increased nociception to colonic distension. This visceral hypersensitivity can be reversed in our rat maternal separation model by fungicides. Menthacarin® is a proprietary combination of essential oils from Mentha x piperita L. and Carum carvi. Because these oils exhibit antifungal and antibacterial properties, we investigated whether Menthacarin® can reverse existing visceral hypersensitivity in maternally separated rats. Methods: In non-handled and maternally separated rats, we used the visceromotor responses to colorectal distension as measure for visceral sensitivity. We evaluated this response before and 24 hours after water-avoidance stress and after 7 days treatment with Menthacarin® or control. The pre- and post-treatment mycobiome and microbiome were characterized by sequencing of fungal internal transcribed spacer 1 (ITS-1) and bacterial 16s rDNA regions. In vitro antifungal and antimicrobial properties of Menthacarin® were studied with radial diffusion assay. Key Results: Menthacarin® inhibited in vitro growth of yeast and bacteria. Water-avoidance caused visceral hypersensitivity in maternally separated rats, and this was reversed by treatment. Multivariate analyses of ITS-1 and 16S high throughput data showed that maternal separation, induced changes in the myco- and microbiome. Menthacarin® treatment of non-handled and maternally separated rats shifted the mycobiomes to more similar compositions. Conclusions & Inferences: The development of visceral hypersensitivity in maternally separated rats and the Menthacarin®-mediated reversal of hypersensitivity is associated with changes in the mycobiome. Therefore, Menthacarin® may be a safe and effective treatment option that should be tested for IBS. © 2018 The Authors. Neurogastroenterology & Motility Published by John Wiley & Sons Ltd. Chemicals / CAS: decanoic acid, 334-48-5, 3398-75-2; fluconazole, 86386-73-4; octanoic acid, 124-07-2, 1984-06-1, 74-81-7; peppermint oil, 8006-90-4; streptomycin, 57-92-1

Background Vagus nerve stimulation is currently clinically evaluated as a treatment for inflammatory bowel disease. However, the mechanism by which this therapeutic intervention can have an immune-regulatory effect in colitis remains unclear. We determined the effect of intestine-specific vagotomy or intestine-specific sympathectomy of the superior mesenteric nerve (SMN) on dextran sodium sulfate (DSS)-induced colitis in mice. Furthermore, we tested the efficacy of therapeutic SMN stimulation to treat DSS-induced colitis in rats. Methods Vagal and SMN fibers were surgically dissected to achieve intestine-specific vagotomy and sympathectomy. Chronic SMN stimulation was achieved by implantation of a cuff electrode. Stimulation was done twice daily for 5 minutes using a biphasic pulse (10 Hz, 200 μA, 2 ms). Disease activity index (DAI) was used as a clinical parameter for colitis severity. Colonic cytokine expression was measured by quantitative PCR and ELISA. Key results Intestine-specific vagotomy had no effect on DSS-induced colitis in mice. However, SMN sympathectomy caused a significantly higher DAI compared to sham-operated mice. Conversely, SMN stimulation led to a significantly improved DAI compared to sham stimulation, although no other parameters of colitis were affected significantly. Conclusions & inferences Our results indicate that sympathetic innervation regulates the intestinal immune system as SMN denervation augments, and SMN stimulation ameliorates DSS-induced colitis. Surprisingly, intestine-specific vagal nerve denervation had no effect in DSS-induced colitis.

Background In irritable bowel syndrome (IBS), familial clustering and transfer across generations may largely depend on environmental factors but this is difficult to establish in the human setting. Therefore, we aimed to set up a relevant animal model. We investigated whether susceptibility to stress induced visceral hypersensitivity in maternally separated (MS) Long Evans rats can be transferred across generations without further separation protocols and, if so, whether this depends on maternal care. Methods At adult age, we evaluated pre- vs post water avoidance (WA) changes in visceromotor response to distension in non-handled second filial generation offspring (NH-F2) of previously separated MS-F1 dams. Furthermore, the role of maternal care was evaluated by cross-fostering F2 offspring of NH-F1 and MS-F1 dams and subsequent sensitivity measurements at adult age. Involvement of mast cells in post stress hypersensitivity of NH-F2 rats was evaluated by mast cell stabilization. Key Results In adult NH-F2 offspring of MS-F1 dams, post-WA hypersensitivity to colorectal distension was observed in 80% of rats compared with 19% in offspring of NH-F1 dams. Cross-fostered pups adapted to the phenotype of the foster mother: pups of NH-F1 dams nursed by MS-F1 dams showed post-WA hypersensitivity to distension at adult age and vice versa (100% and 20% respectively). In NH-F2 rats, post-WA hypersensitivity was reversed by mast cell stabilizer doxantrazole. Conclusions & Inferences Maternal separated-induced susceptibility to stress-triggered visceral hypersensitivity is transferred across generations and this transfer depends on maternal care. Thus, MS is a suitable model to evaluate environmental triggers relevant to IBS clustering in families.

Background Mast cell activation is thought to be involved in visceral hypersensitivity, one of the main characteristics of the irritable bowel syndrome (IBS). A study was therefore undertaken to investigate the effect of the mast cell stabiliser ketotifen on rectal sensitivity and symptoms in patients with IBS. Methods 60 patients with IBS underwent a barostat study to assess rectal sensitivity before and after 8 weeks of treatment. After the initial barostat, patients were randomised to receive ketotifen or placebo. IBS symptoms and health-related quality of life were scored. In addition, mast cells were quantified and spontaneous release of tryptase and histamine was determined in rectal biopsies and compared with biopsies from 22 ageand gender-matched healthy volunteers. Results Ketotifen but not placebo increased the threshold for discomfort in patients with IBS with visceral hypersensitivity. This effect was not observed in normosensitive patients with IBS. Ketotifen significantly decreased abdominal pain and other IBS symptoms and improved quality of life. The number of mast cells in rectal biopsies and spontaneous release of tryptase were lower in patients with IBS than in healthy volunteers. Spontaneous release of histamine was mostly undetectable but was slightly increased in patients with IBS compared with healthy volunteers. Histamine and tryptase release were not altered by ketotifen. Conclusions This study shows that ketotifen increases the threshold for discomfort in patients with IBS with visceral hypersensitivity, reduces IBS symptoms and improves health-related quality of life. Whether this effect is secondary to the mast cell stabilising properties of ketotifen or H1 receptor antagonism remains to be further investigated. Trial Registration Number NTR39, ISRCTN22504486.

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.

Stress plays an important role in the development of visceral hypersensitivity, a key mechanism underlying the pathophysiology of the irritable bowel syndrome. Visceral sensitivity in rats is generally assessed under restrain conditions. To avoid this potential stress factor, we developed a model using implanted radio telemetry for remote measurement of the visceromotor response (VMR) to colorectal distention (CRD). Ten days after implantation of a radio telemetry transmitter and EMG electrodes, visceral sensitivity was evaluated by applying a standardized distension protocol (1, 1.5 and 2 mL) on three different days. In a second series, visceral sensitivity was assessed in maternally separated rats before, directly after and at 6 and 24 h after water avoidance (WA) stress. CRD resulted in a reproducible VMR response on the three different study days. In separated but not in non-handled rats, WA significantly increased visceral sensitivity at 6 h (P=0.006) and 24 h (P=0.004) after WA. Our results show that radio telemetry is a reliable and well tolerated new tool for evaluating visceral sensitivity in rats. These data further confirm that maternal separation is a good model for evaluating the mechanisms underlying visceral hypersensitivity.