Noradrenergic and cholinergic neural pathways mediate stress-induced reactivation of colitis in the rat

https://doi.org/10.1016/j.autneu.2005.12.002Get rights and content

Abstract

Evidence to date suggests that stress-induced exacerbation or relapse of intestinal inflammation in inflammatory bowel disease requires both activation of the autonomic nervous system and the activation of the immune system by the presence of previously encountered luminal antigens. The aim of the present study was to further explore these associations and to determine the role of the autonomic nervous in modulating the intestinal inflammatory response to stress. Rats healed from an initial dinitrobenzene sulfonic acid-induced colitis were given a non-colitic dose of dinitrobenzene sulfonic acid (dissolved in saline) or 0.9% saline intra-rectally and then subjected to restraint stress. Cardiac sympathovagal balance was assessed by power spectral analysis of heart rate variability data collected from telemetric electrocardiogram recordings before, during and post stress. Only rats that were stressed and received dinitrobenzene sulfonic acid showed an inflammatory relapse characterized by significant macroscopic damage and elevated myeloperoxidase activity associated with a significant infiltration of mucosal and submucosal T lymphocytes. No difference in inflammatory markers was observed in animals that received intra-rectal saline and restraint stress. Rats subjected to stress and intra-rectal dinitrobenzene sulfonic acid demonstrated an increase in sympathetic activity with a nearly four fold increase in LF : HF ratio during stress and a significant increase in heart rate. Shortly after cessation of stress, the LF : HF ratio decreased significantly, returning to baseline levels, however the heart rate remained significantly elevated over baseline levels following stress, but decreased to a level that was significantly lower than during stress. The stress/dinitrobenzene sulfonic acid-induced relapses were preventable by pre-treating rats with hexamethonium (a nicotinic cholinergic ganglion blocking agent) or the co-administration of atropine (a muscarinic cholinoceptor antagonist) and bretylium (a noradrenergic ganglion blocking agent), but was not prevented when either atropine or bretylium were administered alone. This study utilizes an established model of chemically induced colitis that when integrated with stress results in relapsing inflammatory bowel disease. Moreover, this study demonstrates that noradrenergic and cholinergic neural pathways mediate the stress response critical for the relapse of colitis.

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

References (60)

  • T. Abo et al.

    Immunomodulation by the autonomic nervous system: therapeutic approach for cancer, collagen diseases, and inflammatory bowel diseases

    Ther. Apher.

    (2002)
  • C.B. Appleyard et al.

    Reactivation of hapten-induced colitis and its prevention by anti-inflammatory drugs

    Am. J. Phsyiol.

    (1995)
  • S. Asfaha et al.

    Prolonged colonic epithelial hyporesponsiveness after colitis: role of inducible nitric oxide synthase

    Am. J. Physiol.

    (1999)
  • G.G. Berntson et al.

    Autonomic determinism: the modes of autonomic control, the doctrine of autonomic space and laws of autonomic constraint

    Psychol. Rev.

    (1991)
  • S. Bjorck et al.

    Topical treatment of ulcerative colitis with lidocaine

    Scand. J. Gastroenterol.

    (1989)
  • N.K. Boughton-Smith et al.

    Relationship between arachidonic acid metabolism, myeloperoxidase activity and leukocyte infiltration in a rat model of inflammatory bowel disease

    Agents Actions

    (1998)
  • A. Bozkurt et al.

    Anti-inflammatory effects of leptin and cholecystokinin on acetic acid-induced colitis in rats: role of capsaicin-sensitive vagal afferent fibres

    Regul. Pept.

    (2003)
  • E. Bronzetti et al.

    Muscarinic cholinergic receptor subtypes in human peripheral blood lymphocytes

    Neurosci. Lett.

    (1996)
  • J.H. Brown et al.

    Chapter 7. Muscarinic receptor agonists and antagonists

  • J. Cabarrocas et al.

    Role of enteric glial cells in inflammatory bowel disease

    Glia

    (2003)
  • I. Castagliuolo et al.

    Acute stress causes mucin release from rat colon: role of corticotropin releasing factor and mast cells

    Am. J. Physiol.

    (1996)
  • C. Cerutti et al.

    Autonomic nervous system and cardiovascular variability in rats: a spectral analysis approach

    Am. J. Physiol.

    (1991)
  • S.M. Collins et al.

    Previous inflammation alters the response of the rat colon to stress

    Gastroenterology

    (1996)
  • A.L. Colon et al.

    Stress increases susceptibility to oxidative/nitrosative mucosal damage in an experimental model of colitis in rats

    Dig. Dis. Sci.

    (2004)
  • C. Dennis et al.

    The response to vagotomy in idiopathic ulcerative colitis and regional enteritis

    Ann. Surg.

    (1946)
  • L. Dominguez-Gerpe et al.

    Lymphocyte protein synthesis: evidence that murine T cells are more affected by stress than B cells

    Immunol. Lett.

    (1996)
  • R. Duchmann et al.

    Tolerance exists towards resident intestinal flora but is broken in active inflammatory bowel disease

    Clin. Exp. Immunol.

    (1995)
  • L.C. Duffy et al.

    Relevance of major stress events as an indicator of disease activity prevalence in inflammatory bowel disease

    Behav. Med.

    (1991)
  • K. Endoh et al.

    Intragastric nicotine protection against 40% ethanol injury in the stomach. Role of ganglionic stimulation or blockade

    Dig. Dis. Sci.

    (1992)
  • K. Fujino et al.

    Inhibition of the vanilloid receptor subtype-1 attenuates TNBS-colitis

    J. Gastrointest. Surg.

    (2004)
  • F. Galeazzi et al.

    Cigarette smoke aggravates experimental colitis in rats

    Gastroenterology

    (1999)
  • M. Gue et al.

    Stress-induced enhancement of colitis in rats: CRF and arginine vasopressin are not involved

    Am. J. Physiol.

    (1997)
  • M.A. Gulpinar et al.

    Anti-inflammatory effect of acute stress on experimental colitis is mediated by cholecystokinin-B receptors

    Life Sci.

    (2004)
  • U. Haglund et al.

    Effects of adrenergic neuron and ganglion blockers on hemodynamics and plasma catecholamine levels after corticosteroids during hemorrhagic shock in the dog

    Acta Anaesthesiol.

    (1980)
  • H. Hassani et al.

    Attenuation of acute experimental colitis by preventing NPY Y1 receptor signaling

    Am. J. Physiol. Gastrointest. Liver. Physiol.

    (2005)
  • K. Jacobson et al.

    The mechanism of altered neural function in a rat model of acute colitis

    Gastroenterology

    (1997)
  • M.V. Kamath et al.

    Power spectral analysis of heart rate variability: a noninvasive signature of cardiac autonomic function

    Crit. Rev. Biomed. Eng.

    (1993)
  • N. Kihara et al.

    Vanilloid receptor-1 containing primary sensory neurones mediate dextran sulphate sodium induced colitis in rats

    Gut

    (2003)
  • K. Kyosola et al.

    Rectal mucosal adrenergic innervation and enterochromaffin cells in ulcerative colitis and irritable colon

    Scand. J. Gastroenterol.

    (1977)
  • M. Kuwahara et al.

    Power spectral analysis of heart rate variability as a new method for assessing autonomic activity in the rat

    J. Electrocardiol.

    (1994)
  • Cited by (29)

    • Everything You Always Wanted to Know About Organoid-Based Models (and Never Dared to Ask)

      2022, Cellular and Molecular Gastroenterology and Hepatology
    • Optogenetic activation of local colonic sympathetic innervations attenuates colitis by limiting immune cell extravasation

      2021, Immunity
      Citation Excerpt :

      Many inflammatory conditions are affected by the psychological state of the patient, specifically stress (Godbout and Glaser, 2006; Liu et al., 2017), which induces SNS activation. Such a connection between the mental state and inflammation is especially evident in the context of the highly innervated and immunologically active organ, the gastrointestinal tract (GIT), as seen for example, in inflammatory bowel disease (IBD; Mawdsley and Rampton, 2005; Saunders et al., 2006; Taylor and Keely, 2007). The GIT contains an internal nervous system, the enteric nervous system (ENS), and, as part of the gut-brain axis (Bellono et al., 2017; Cryan and Dinan, 2012; Foster and McVey Neufeld, 2013; Han et al., 2018; Kaelberer et al., 2018), receives inputs from the brain via the PSNS and SNS (Altschuler et al., 1993; Gabanyi et al., 2016; Straub et al., 2006; Veiga-Fernandes and Mucida, 2016).

    • Stress burden and neuroendocrine regulation of cytokine production in patients with ulcerative colitis in remission

      2018, Psychoneuroendocrinology
      Citation Excerpt :

      Furthermore, exposure of IBD patients to experimental acute stress resulted in heightened stress responses, more pronounced mast cell activation, and greater epithelial damage compared to healthy controls (Farhadi et al., 2005). These findings in humans are supported by studies of experimental stress in animal models of colitis (Reber, 2012; Saunders et al., 2006). The mechanism(s) through which psychological factors influence IBD pathophysiology and disease course remain unsettled, but likely involve interactions between neuroendocrine mediators and peripheral immune cells.

    • Stress and food allergy: Mechanistic considerations

      2014, Annals of Allergy, Asthma and Immunology
      Citation Excerpt :

      Animal research has shown that electrical stimulation of the vagus nerve results in decreased intestinal inflammation, whereas surgical removal of the vagus nerve leads to increased inflammation.35,36 Using a rat model of induced colitis, Saunders et al37 found that cholinergic neural pathways are likely involved in inflammatory relapse after a non-colitic dinitrobenzene sulfonic acid dose and restraint stress. However, they assessed only cardiac sympathovagal balance in this study.

    • Stress and visceral pain: From animal models to clinical therapies

      2012, Experimental Neurology
      Citation Excerpt :

      While it is difficult to make any assumptions on the reasons for these differences, these results suggest that inflammation alone may not always lead to visceral hypersensitivity and that the type of inflammatory insult and severity determine whether this will result in the development of postinflammatory hypersensitivity (Adam et al., 2006). In most, but not all the studies (Larsson et al., 2009), previous exposure of rats and mice to psychological or psycho-social stress was shown to enhance their susceptibility to colitis and to aggravate their colonic inflammatory response (Reber et al., 2006, 2008; Veenema et al., 2008) as well as to precipitate the reactivation of colonic inflammation in animals in which colitis had healed (Melgar et al., 2008; Saunders et al., 2006). Likewise, previous colitis was found to render the colon more susceptible to the effects of stress on enteric nerve function and to increase some parameters of inflammation in response to stress (Collins et al., 1996).

    View all citing articles on Scopus
    View full text