Shadea Salim Rasoel

Psychological stress and corticotropin-releasing hormone increase intestinal permeability in humans by a mast cell-dependent mechanism

Objective Intestinal permeability and psychological stress have been implicated in the pathophysiology of IBD and IBS. Studies in animals suggest that stress increases permeability via corticotropin-releasing hormone (CRH)-mediated mast cell activation. Our aim was to investigate the effect of stress on intestinal permeability in humans and its underlying mechanisms. Design Small intestinal permeability was quantified by a 2 h lactulose–mannitol urinary excretion test. In a first study, 23 healthy volunteers were subjected to four different conditions: control; indomethacin; public speech and anticipation of electroshocks. In a second study, five test conditions were investigated in 13 volunteers: control; after pretreatment with disodium cromoglycate (DSCG); administration of CRH; DSCG+CRH and DSCG+public speech. Results Indomethacin, as a positive comparator (0.071±0.040 vs 0.030±0.022; p<0.0001), and public speech (0.059±0.040; p<0.01), but not the shock protocol increased intestinal permeability. Similarly, salivary cortisol was only increased after public speech. Subgroup analysis demonstrated that the effect of public speech on permeability was only present in subjects with a significant elevation of cortisol. CRH increased the lactulose–mannitol ratio (0.042±0.021 vs 0.028±0.009; p=0.02), which was inhibited by the mast cell stabiliser DSCG. Finally, intestinal permeability was unaltered by public speech with DSCG pretreatment. Conclusions Acute psychological stress increases small intestinal permeability in humans. Peripheral CRH reproduces the effect of stress and DSCG blocks the effect of both stress and CRH, suggesting the involvement of mast cells. These findings provide new insight into the complex interplay between the central nervous system and GI function in man.

Subacute stress and chronic stress interact to decrease intestinal barrier function in rats

Abstract Psychological stress increases intestinal permeability, potentially leading to low-grade inflammation and symptoms in functional gastrointestinal disorders. We assessed the effect of subacute, chronic and combined stress on intestinal barrier function and mast cell density. Male Wistar rats were allocated to four experimental groups (n = 8/group): 1/sham; 2/subacute stress (isolation and limited movement for 24 h); 3/chronic crowding stress for 14 days and 4/combined subacute and chronic stress. Jejunum and colon were collected to measure: transepithelial electrical resistance (TEER; a measure of epithelial barrier function); gene expression of tight junction molecules; mast cell density. Plasma corticosterone concentration was increased in all three stress conditions versus sham, with highest concentrations in the combined stress condition. TEER in the jejunum was decreased in all stress conditions, but was significantly lower in the combined stress condition than in the other groups. TEER in the jejunum correlated negatively with corticosterone concentration. Increased expression of claudin 1, 5 and 8, occludin and zonula occludens 1 mRNAs was detected after subacute stress in the jejunum. In contrast, colonic TEER was decreased only after combined stress, and the expression of tight junction molecules was unaltered. Increased mast cell density was observed in the chronic and combined stress condition in the colon only. In conclusion, our data show that chronic stress sensitizes the gastrointestinal tract to the effects of subacute stress on intestinal barrier function; different underlying cellular and molecular alterations are indicated in the small intestine versus the colon.

From Intestinal Permeability to Dysmotility: The Biobreeding Rat as a Model for Functional Gastrointestinal Disorders

Background Impaired intestinal barrier function, low-grade inflammation and altered neuronal control are reported in functional gastrointestinal disorders. However, the sequence of and causal relation between these events is unclear, necessitating a spontaneous animal model. The aim of this study was to describe the natural history of intestinal permeability, mucosal and neuromuscular inflammation and nitrergic motor neuron function during the lifetime of the BioBreeding (BB) rat. Methods Normoglycemic BB-diabetes prone (DP) and control rats were sacrificed at different ages and jejunum was harvested to characterize intestinal permeability, inflammation and neuromuscular function. Results Both structural and functional evidence of increased intestinal permeability was found in young BB-DP rats from the age of 50 days. In older animals, starting in the mucosa from 70 days and in half of the animals also in the muscularis propria from 110 days, an inflammatory reaction, characterized by an influx of polymorphonuclear cells and higher myeloperoxidase activity, was observed. Finally, in animals older than 110 days, coinciding with a myenteric ganglionitis, a loss of nitrergic neurons and motor function was demonstrated. Conclusion In the BB-rat, mucosal inflammatory cell infiltration is preceded by intestinal barrier dysfunction and followed by myenteric ganglionitis and loss of nitrergic function. This sequence supports a primary role for impaired barrier function and provides an insightful model for the pathogenesis of functional gastrointestinal disorders.

A Spontaneous Animal Model of Intestinal Dysmotility Evoked by Inflammatory Nitrergic Dysfunction

Background and Aims Recent reports indicate the presence of low grade inflammation in functional gastrointestinal disorders (FGID), in these cases often called “post-inflammatory” FGIDs. However, suitable animal models to study these disorders are not available. The Biobreeding (BB) rat consists of a diabetes-resistant (BBDR) and a diabetes-prone (BBDP) strain. In the diabetes-prone strain, 40–60% of the animals develop diabetes and concomitant nitrergic dysfunction. Our aim was to investigate the occurrence of intestinal inflammation, nitrergic dysfunction and intestinal dysmotility in non-diabetic animals. Methods Jejunal inflammation (MPO assay, Hematoxylin&Eosin staining and inducible nitric oxide synthase (iNOS) mRNA expression), in vitro jejunal motility (video analysis) and myenteric neuronal numbers (immunohistochemistry) were assessed in control, normoglycaemic BBDP and diabetic BBDP rats. To study the impact of iNOS inhibition on these parameters, normoglycaemic BBDP rats were treated with aminoguanidine. Results Compared to control, significant polymorphonuclear (PMN) cell infiltration, enhanced MPO activity, increased iNOS mRNA expression and a decreased ratio of nNOS to Hu-C/D positive neurons were observed in both normoglycaemic and diabetic BBDP rats. Aminoguanidine treatment decreased PMN infiltration, iNOS mRNA expression and MPO activity. Moreover, it restored the ratio of nNOS to Hu-C/D positive nerves in the myenteric plexus and decreased the abnormal jejunal elongation and dilation observed in normoglycaemic BBDP rats. Conclusions Aminoguanidine treatment counteracts the inflammation-induced nitrergic dysfunction and prevents dysmotility, both of which are independent of hyperglycaemia in BB rats. Nitrergic dysfunction may contribute to the pathophysiology of “low-grade inflammatory” FGIDs. Normoglycaemic BBDP rats may be considered a suitable animal model to study the pathogenesis of FGIDs.

269 Acute Psychological Stress Increases Small Intestinal Permeability in Healthy Volunteers via CRH-Mediated Activation of Mast Cells

G A A b st ra ct s region, MRP4 was significantly down-regulated compared with expression in biopsies from healthy subjects (P,0.001, n=7-11). Conclusions: Key components of the GC-C/cGMP signalling pathway are expressed within human colonic mucosa. However, expression of the cGMP transporter MRP4 was significantly reduced in rectosigmoid biopsies from patients with IBS-C compared to biopsies from healthy subjects. Reduced MRP4 expression may result in reduced basal release of cGMP from intestinal epithelial cells in these patients and it may underlie some aspects of the pathophysiology of IBS. 1.Gastroenterology,2011,140(5),S1,S-538. Supported by NHMRC Australia, Ironwood Pharmaceuticals Inc.

274 Antioxidant Supplementation Improves Inflammatory Nitrergic Dysfunction in Diabetic Biobreeding Prone Rats

Introduction: Loss of intestinal nitrergic motor control has been reported in Biobreeding diabetes-prone (BBDP) rats (Zandecki 2008). We previously demonstrated that loss of jejunal neuronal nitric oxide synthase (nNOS) mRNA expression occurs in the BBDP, which is related to transient intestinal inflammation (myeloperoxidase: MPO activity) and inducible nitric oxide synthase (iNOS) expression, and which is independent from the development of hyperglycemia (Kindt DDW 2009). Studies in enteric neuron cultures suggest that iNOS overexpression downregulates nNOS expression through oxidative stress (Zandecki 2006). Moreover, aminoguanidine an iNOS inhibitor, counteracts the inflammation-induced nitrergic dysfunction and prevents intestinal elongation and dilatation caused by a 3cm H2O pressure stimulus in normoglycemic BBDP (Masaoka DDW 2011). Aim: To investigate the pathophysiological role of oxidative stress in inflammatory nitrergic dysfunction, we designed a controlled antioxidant supplementation study. Methods: BBDP were fed chow supplemented with 2% vitamin E (α-tocopherol) and 2g/l vitamin C (ascorbic acid) in the drinking water from 21days onwards. After onset of diabetes, 1% vitamin E and 1g/l vitamin C were supplemented to compensate for polyphagia and polydypsia. All rats were sacrificed at 220days. Jejunal inflammation, spontaneous intestinal motility, nitrergic neuron number and nNOS mRNA expression were analyzed respectively by MPO measurements, organ bath (37°C) video analysis, NADPH-diaphorase staining and realtime PCR, both in antioxidant supplemented animals and in a control group of age-matched non-supplemented diabetic animals. Results: Compared with controls (n=7), MPO activity decreased significantly in the antioxidant supplemented group (n=6) (9.9±3.9 vs. 2.1±1.4 U/mg, p 0.05) of a 5-cm jejunum segment in response to intraluminal pressure rise In Vitro. Conclusion: Antioxidant supplementation improves inflammation-induced nitrergic dysfunction and partially prevents intestinal dysmotility in diabetic BBDP.

Tu1994 Nitrergic Neurotransmission is Impaired in Non-Diabetic BB-Rats Gastric Fundus

G A A b st ra ct s CCh were not affected by TS infection (Table). However, in TS-infected rats receiving ketotifen contractile responses to CCh were increased by 60%; an effect completely prevented by pre-incubation with NGF, while the peptide did not affect contractility by itself (Table). Overall, ketotiken had also a tendency to increase cholinergic responses in non-infected animals (Table). L-NNA responses were unaltered by TS infection or ketotifen. Conclusions: During TS infection a significant MMC infiltrate appears in the colon without other structural or functional (as contractility relates) alterations. Altered colonic contractile responses during ketotifen treatment and prevention by NGF suggests that MCs play a protective role normalizing altered motility, likely through a NGF-related mechanism. Overall, these observations suggest that the MMC infiltrate that characterize functional gastrointestinal disorders might represent a protective mechanism aiming the normalization of colonic motility. Contractility (g)

Partial Restraint Stress Induces Increased Permeability and Low Grade Inflammation in the Rat Ileum, but Not in the Jejunum

Mixed-lineage kinase-3 (MLK3) activates multiple MAPK pathways and has been shown to initiate apoptosis, proliferation, migration or differentiation in several cell types. MLK3 signaling is also required for breast cancer cell invasion. However, the role of MLK3 signaling in regulation of intestinal epithelial cell sheet migration In Vivo and In Vitro is not known. Given its role in cancer cell migration, we sought to investigate whether MLK3 signaling is important in intestinal mucosal healing and epithelial cell motility. We compared the healing of jejunal mucosal ulcers induced by topical serosal acetic acid in different age groups of MLK3 null mice with that in age-matched wild type mice and assessed the role of MLK3 signaling in human Caco-2 intestinal epithelial migration across type I collagen substrates. In older mice (30-42 weeks of age), ulcer healing at 5 days after the initial insult was reduced by 47% (p<0.05) in MLK3 null and by 29% in MLK3 heterozygous mice when compared with wild-type littermate controls. In younger animals (8-12 weeks), ulcer healing in MLK3 null mice was 25% less at day 3 (n=7, p<0.05) and 23% less at day 5 (n=7, p<0.05) than in wild-type mice. In Vitro, an MLK3 inhibitor significantly decreased closure of circular wounds in Caco-2 cell monolayers after 24 hours compared with to vehicle-treated control by 16.3% and 20.1 % respectively (p<0.05). Proliferative blockade using hydroxyurea (30mM) did not abolish the effect of MLK3 inhibitor on Caco-2 wound closure, suggesting a true effect on cell motility rather than an effect on cell proliferation. The reduction in cell motility by MLK3 inhibitor reduced level of pERK by 79% (p<0.05) and pJNK by 12% (p<0.05) revealing suppression of ERK and JNK signaling. Total MLK3 was also compared in confluent Caco-2 cells to cell that have been migrating for 96 hours. MLK3 was 53% (p<0.05) higher in migrating vs confluent cells further indicating the importance of MLK3 signaling in migrating intestinal epithelial cells. These studies reveal a novel role for MLK3 signaling in the regulation of intestinal epithelial migration and suggest that MLK3 may be an important target to influence intestinal mucosal healing.

Inflammatory Nitrergic Dysfunction Evokes Intestinal Dysmotility

COX-2 expressing vector, and HCA-7, a COX-2 expressing human colon cancer cells were used. Stemness was estimated by ability to form spheroid known to enrich in stem cells and oct-4 expression. DNA methyltransferase activity, demethylase activity, and DNA global hypomethylation were measured. Promoter methylation of tumor suppressor genes (p16, E-cadherin, and hMLH1), and an oncogene, S100A, was determined by MSP or COBRA. Expressions of DNMT-1, -3A, and -3B and demethylase genes (AID and glycosylase) were determined by RT-PCR. [Results] RIE-COX showed significantly elevated potential for spheroid formation, oct-4 expression, DNA demethylase activity and expression, promoter hypermethylation of tumor suppressors and hypomethylation of S100A, and DNMT-3a expression and activity, and it also showed significantly downregulated global DNA methylation, compared with RIE. siRNA for COX-2 or celecoxib, a COX-2 specific inhibitor, significantly inhibited spheroid formation, global hypomethylation, hypermethylation of tumor suppressor genes, and DNA demethylase activity/expression in HCA-7. siRNA for DNMT3a or AID partly suppressed spheroid formation of HCA-7 and oct-4 expression. [Conclusion] These results indicate that COX-2 is involved in intricate epigenetic regulation; simultaneous global hypomethylation and gene-specific hypermethylation, through discrete modulation of DNMT/demethylase expression, and it is also suggested that COX-2 inhibitors have a potential to prevent epigenetic dysregulation and aberrant biology of cancer stem cell in carcinogenesis.