Noradrenergic and cholinergic neural pathways mediate stress-induced reactivation of colitis in the rat
Introduction
Inflammatory bowel disease (IBD) is an intestinal inflammatory disorder of unknown etiology thought to be precipitated by interactions between the genetically susceptible host, the mucosal immune system and enteric flora. However, the relapsing and remitting nature of the disease underlies the importance of disease modifiers that include psychological stress. While clinical observations have provided strong anecdotal evidence, few prospective studies have examined whether stress is involved in the exacerbation or precipitation of inflammatory relapses. One such study has indicated that stressful life events often precede relapses in Crohn's patients (Duffy et al., 1991), while another reported that short-term stress in ulcerative colitis patients failed to provoke relapses, whereas long-term stress increased risk of exacerbation (Levenstein et al., 2000). Moreover, in animal models of IBD, stress has been shown to augment hapten-induced colitis (Gue et al., 1997, Million et al., 1999, Pfeiffer et al., 2001, Colon et al., 2004) and dextran sulfate sodium-induced colitis (Milde and Murison, 2002) and lower the threshold for reactivation of mucosal inflammation in hapten-induced colitis (Qiu et al., 1999).
The mechanisms underlying stress-induced exacerbation or relapse of intestinal inflammation are largely unknown however brain-gut interactions via neural, hormonal and immune systems are involved. Several studies have shown that corticotrophin-releasing factor (CRF) and more recently cholecystokinin (CCK) and the urocortin (Ucn) family of neuropeptides are important mediators of the intestinal neuroendocrine stress response (Castagliuolo et al., 1996, Million et al., 1999, Santos et al., 1999, Gulpinar et al., 2004, Martinez et al., 2004). In addition, animal models of environmental stress have elucidated intestinal responses demonstrating activation of mast cells, barrier dysfunction (increased macromolecular permeability and mucus depletion) and associated bacterial adhesion and penetration into enterocytes (Wilson and Baldwin, 1999, Pfeiffer et al., 2001, Soderholm et al., 2002).
During stress, hypothalamic CRF stimulates pituitary adrenocorticotrophic hormone (ACTH) secretion, which in turn stimulates glucocorticoid release from the adrenal gland (HPA axis). However, the intestinal responses to stress are likely not entirely dependent on activation of the HPA axis, but are mediated in part by activation of the autonomic and enteric nervous systems (Saunders et al., 1997).
In acute animal models of intestinal inflammation and in patients with IBD, increasing evidence suggests a role for the sympathetic, parasympathetic and enteric nervous systems in modulating the intestinal inflammatory process (Dennis et al., 1946, Shafiroff and Hinton, 1950, Thorek, 1951, Kyosola et al., 1977, Lechin et al., 1985, Bjorck et al., 1989, Lashner et al., 1990, Pullan et al., 1994, McCafferty et al., 1997, Mazelin et al., 1998, Galeazzi et al., 1999, Cabarrocas et al., 2003, Miceli and Jacobson, 2003, Kihara et al., 2003, Nguyen et al., 2003, Bozkurt et al., 2003, Fujino et al., 2004, Hassani et al., 2005). Furthermore, the animal models of stress associated augmentation of acute colitis (Gue et al., 1997, Million et al., 1999, Pfeiffer et al., 2001, Milde and Murison, 2002, Colon et al., 2004) and stress-induced reactivation of previous colitis (Qiu et al., 1999) provide further support for neural modulation of the intestinal inflammatory response.
Interestingly, functional studies in patients with inflammatory bowel disease demonstrating autonomic nervous system dysfunction (Lindgren et al., 1991, Lindgren et al., 1993) or autonomic nervous system hyperreflexia (to various physiologic stimuli) that was more consistently associated with more severe disease and extra-intestinal manifestations (Straub et al., 1997) suggest the possibility of differing neural responses to stress. Taken together, these data suggest an important role for the nervous system in modulating intestinal inflammatory conditions and the intestinal mucosal response to stress.
Consequently, the aim of the present study was to explore the role of stress and to asses the role of noradrenergic and cholinergic neural pathways in modulating stress-induced reactivation of hapten-induced colitis. Models of stress-induced reactivation of colitis with restraint stress were developed. The models differed from the model previously described (Qiu et al., 1999) whereby saline replaced ethanol as the vehicle for delivery of the hapten, thus allowing assessment of the role of stress without the confounding effects of ethanol induced mucosal barrier disruption. Telemetric recordings of heart rate were recorded prior to, during, and following stress. Power spectral analysis of heart rate variability was utilized to provide accurate quantitative information about the interactions between sympathetic and parasympathetic nervous systems' modulation of heart rate and to determine the relative contributions of each system in this model (Kamath and Fallen, 1993). To further explore the role of cholinergic and noradrenergic neural pathways modulating the stress response, we examined the effect of subcutaneous hexamethonium, bretylium tosylate and atropine methyl nitrate. Hexamethonium, the prototypical non-depolarizing peripheral nicotinic cholinoceptor antagonist that inhibits nicotinic neurotransmission in sympathetic, parasympathetic and enteric ganglia was used alone to examine the contribution of cholinergic neural pathways, (Taylor, 2001). Bretylium that selectively accumulates in sympathetic ganglia and their postganglionic adrenergic neurons to inhibit nerve stimulated release of noradrenaline from adrenergic nerve endings (Haglund et al., 1980) or atropine that blocks binding of acetylcholine to muscarinic cholinoceptors at neuro-effector sites (Brown and Taylor, 2001) were used alone to assess contributions of noradrenergic and cholinergic neural pathways or in combination to assess contributions of both noradrenergic and cholinergic pathways.
In the present study, we present an animal model of relapsing inflammatory bowel disease that utilizes an established model of chemically induced colitis integrated with stress and further demonstrate involvement of both noradrenergic and cholinergic neural pathways in mediating the stress response.
Section snippets
Animals
Sprague Dawley rats weighing approximately 180–200 g were purchased from Charles River Laboratories (St. Constant, Que., Canada) and were maintained on standard laboratory chow and tap water ad libitum. All protocols were approved by the Animal Research Ethics Boards at McMaster University and the University of British Columbia.
Induction of acute colitis
One week after arrival, animals were anaesthetized (Enflurane–Abbott Laboratories; St. Laurent, Que., Canada) and a polyethylene (PE)-90 catheter was inserted 8 cm
Statistics
A total of 236 rats were used in the study (50 animals for the acute colitis experiments; 10 animals/group and 186 animals for the reactivation colitis experiment; 78 for the 3 day stress/reactivation and hexamethonium treatment experiments; 6–10 animals/group and 108 for the one day stress reactivation experiments and treatments with atropine and bretyllium; 6–12 animals/group). The data are expressed as mean ± SEM. One-way ANOVA with a Neuman–Keuls post hoc test for multiple comparisons was
Acute colitis
As previously described, (Jacobson et al., 1997) acute colitis was evident in rats on day five post intra-rectal DNB/EtOH accompanied by diarrhea, bloody diarrhea and weight loss. This was associated with a significant increase in macroscopic damage scores (P < 0.001) compared to saline control, a significant rise in myeloperoxidase activity (P < 0.01) and significantly more mucosal and submucosal CD3 + cells (P < 0.001) were evident in tissue sections from the distal colon (Table 1). Microscopic
Discussion
The findings from this study indicate that stress can alter host defense to luminal antigen and reactivate mucosal inflammation. In addition, this study demonstrates involvement of neural pathways in the reactivation of the colitis and provides evidence for involvement of noradrenergic and muscarinic cholinergic and nicotinic cholinergic neural pathways in the response.
Our results demonstrated that restraint stress together with exposure to the luminal hapten dinitrobenzene sulfonic acid in
Acknowledgements
We thank Ms. M-J. Smith and Mr. B. Hewlett of the Astra Laboratory, at McMaster University, for their kind and expert assistance with the Immunohistochemistry staining, Ms. P. Blennerhassett for her support and expertise with the animals and MPO tissues, and Ms. V. Mearns for her technical assistance with the model at U.B.C. We would also like to thank Dr. S. Collins for his overall support and guidance. Dr. P.R. Saunders was supported for a portion of this study by a CIHR Fellowship and a
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