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

Autonomic Neuroscience: Basic and Clinical 124 (2006) 56 – 68 www.elsevier.com/locate/autneu

Noradrenergic and cholinergic neural pathways mediate stress-induced reactivation of colitis in the rat Paul R. Saunders, Paula Miceli, Bruce A. Vallance, Lu Wang, Sarah Pinto, Gervais Tougas, Markad Kamath, Kevan Jacobson * Intestinal Disease Research Program, McMaster University, Hamilton, Ontario, Canada Departments of Medicine and Pediatrics, McMaster University, Hamilton, Ontario, Canada CURE, UCLA Los Angeles, California, USA British Columbia Research Institute, B.C. Children’s Hospital, University of British Columbia, Vancouver, B.C., Canada Department of Pediatrics, B.C. Children’s Hospital, University of British Columbia, Vancouver, B.C., Canada Received 8 June 2005; received in revised form 10 November 2005; accepted 5 December 2005

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 intrarectal 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 acidinduced relapses were preventable by pre-treating rats with hexamethonium (a nicotinic cholinergic ganglion blocking agent) or the coadministration 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. D 2005 Elsevier B.V. All rights reserved. Keywords: Inflammatory bowel disease; Restraint stress; Inflammatory relapse; Noradrenergic; Cholinergic nervous systems

1. Introduction * Corresponding author. British Columbia’s Research Institute and British Columbia’s Children’s Hospital, Division of Gastroenterology, 4480 Oak Street, Room K4-181 Vancouver, B.C. V6H 3V4, Canada. Tel.: +1 604 875 2332; fax: +1 604 875 3244. E-mail address: [email protected] (K. Jacobson). 1566-0702/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.autneu.2005.12.002

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

P.R. Saunders et al. / Autonomic Neuroscience: Basic and Clinical 124 (2006) 56 – 68

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.,

57

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, 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.

58

P.R. Saunders et al. / Autonomic Neuroscience: Basic and Clinical 124 (2006) 56 – 68

2. Materials and methods 2.1. 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. 2.2. 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 proximal to the anus, approximately positioned at the level of the splenic flexure, and 15 mg (per rat) of dinitrobenzene sulfonic acid (DNB-ICN Biomedicals; Aurora Ohio, USA) in 250 Al of 40% ethanol (DNB/EtOH) was administered. Control animals received 250 Al of 0.9% saline. Animals were returned to their home cages for six weeks, to allow the initial colitis to resolve in keeping with previous studies showing return to baseline of colitis markers by that point (Collins et al., 1996; Qiu et al., 1999). 2.3. Acute colitis Positive control animals for the acute colitis, (n = 10) were euthanized by cervical dislocation on day 5 post induction of the initial colitis (Fig. 1). To determine whether stress was capable of replacing ethanol’s role in the initial induction of colitis, one group of rats received 3 days of restraint stress (as described below) followed by intra-rectal administration of dinitrobenzene sulfonic acid (15 mg dissolved in 250 Al saline, stress and DNB/saline, n = 10)

Acute Colitis

30 min post stress (on days 0, 1, 2). Control animals received intra-rectal dinitrobenzene sulfonic acid (15 mg dissolved in 250 Al saline) without preceding stress (DNB/ saline, n = 10) or were subjected to 3 days of restraint stress followed by intra-rectal administration of 250 Al of 0.9% saline (stress/saline, n = 10). Absolute control animals received 250 Al of 0.9% saline intra-rectally (n = 10). The rats were euthanized on day 5 and their colons removed and examined. 2.4. Stress-induced reactivation of colitis Six weeks following initial induction of colitis (DNB/ EtoH), animals were either exposed to repeated stress sessions or a single session of stress (Fig. 1). The animals were randomly assigned to one of the following treatment groups: (i) administration of 250 Al of 0.9% saline by intra-rectal injection on days 42, 43 and 44 (control), (ii) restraint stress in plastic disposable rodent restrainers (Braintree Scientific, Braintree MA, USA) for 3 h followed 30 min later by administration of 250 Al of 0.9% saline by intra-rectal injection on days 42, 43 and 44 (stress and saline); (iii) administration of DNB (15 mg in 250 Al 0.9% saline) intra-rectally while under anesthesia with Enflurane on days 42, 43 and 44 (DNB/saline); (iv) restraint stress in plastic cones for 3 h, followed 30 min later by administration of DNB (15 mg in 250 Al 0.9% saline) while under anesthesia with enflurane on days 42, 43 and 44 (stress and DNB/saline) or (v) to determine whether a single session of stress was sufficient to reactivate the colitis an additional model was developed. Restraint stress was applied for 3 h only on day 42 followed 1 h later by administered of intra-rectal dinitrobenzene sulfonic acid (20 mg in 250 Al saline, single stress and DNB/saline). All rats (n = 10/ group in groups i – iv and n = 12/group in group v) were sacrificed by cervical dislocation on day 44, 1 h post treatment where applicable.

Stress-induced Reaction Colitis

Stress

Stress Day

0 1 2 3 4

5

Post induction of acute colitis animals allowed to recover for 6 weeks

42 43 44

Fig. 1. Acute colitis was induced on day 0 by intrarectal administration of dinitrobenzene sulfonic acid dissolved in ethanol, (DNB/EtOH). In separate experiments, animals were subject to restraint stress on days 0, 1 and 2 followed by intrarectal DNB in saline (stress and DND/saline). Control animals received intrarectal DNB in saline (DNB/saline) or stress followed by intrarectal saline (stress/ saline). Animals were sacrificed on day 5. For stress reactivation colitis experiments animals were allowed to recover for 42 days following induction of acute colitis. On day 42 the animals were divided into those who received intrarectal saline for 3 days (control), restraint stress followed by intrarectal saline for 3 days (stress and saline), intrarectal DNB in saline for 3 days (DNB/ saline), restraint stress followed by intrarectal DNB in saline for 3 days (stress and DNB/saline) or restraint stress followed by intrarectal DNB in saline for 1 day (stress and DNB/saline). Pharmacologic intervention consisted of subcutaneous hexamethonium administered prior to restraint stress with or without intrarectal DNB in saline for 3 days (stress/DNB + hexamethonium) or subcutaneous atropine methyl nitrate or bretylium tosylate administered either separately or in combination on day 42 prior to restraint stress (with an additional post stress dose of atropine) with or without intrarectal DNB/saline (stress/ DNB + atropine – bretylium). See Materials and methods for details.

P.R. Saunders et al. / Autonomic Neuroscience: Basic and Clinical 124 (2006) 56 – 68

2.5. Assessment of colonic inflammation The inflammatory response was assessed by determination of macroscopic damage score, tissue myeloperoxidase activity, histology and mucosal infiltration of CD3+ T-lymphocytes except in the case of the single stress model where the inflammatory response was assessed 143by macroscopic damage and myeloperoxidase activity. 2.5.1. Macroscopic damage score The colons were excised, opened along the mesenteric border, and pinned flat on a gelatin coated Petri dish. An observer blinded to the treatment assigned a macroscopic damage score using criteria previously outlined in detail (Appleyard and Wallace, 1995). Briefly, the damage score consisted of a score for severity and extent of ulceration (0– 10), summed with scores for the absence or presence of diarrhea (0 or 1; diarrhea being defined as loose or watery stool) and adhesions (0, 1, or 2), and the maximum thickness of the wall of the colon (in mm). A mean macroscopic damage score and standard error of the mean was calculated for each group of rats. The site of maximum macroscopic damage was located 5 to 7 cm proximal to the anus. Adjacent segments of colon (each ¨2 cm in length) or corresponding sites from control animals were removed for assessment of myeloperoxidase (MPO) activity, histology, and immunohistochemistry. 2.5.2. Colonic myeloperoxidase activity and histology Retrieved colonic segments (2 cm) were snap frozen in liquid nitrogen and assayed for myeloperoxidase activity within seven days using a previously described method (Boughton-Smith et al., 1998). Myeloperoxidase is an enzyme found in intracellular granules of neutrophil granulocytes and other cells of myeloid origin and its activity is widely used as a marker of inflammation (Smith and Castro, 1978). One unit of myeloperoxidase activity was defined as the quantity able to convert 1 Amol of hydrogen peroxide to water per minute at 25 -C and was expressed as units per milligram of tissue. Adjacent full-thickness sections of tissue were retrieved and submitted for routine histological processing and microscopy. The tissue sections were immediately fixed in 10% neutral buffered formalin for 24 h, then transferred to 70% ethanol, processed and embedded in a solid paraffin block. Three-micrometer sections were stained with hematoxylin and eosin and examined under light microscopy by an investigator blinded to the treatments. 2.5.3. Immunostaining of CD3+ cells on colonic sections Immunostaining for T lymphocytes was performed, as T lymphocytes are reflective of chronic inflammation and are required for reactivation of colitis by stress (Qiu et al., 1999). Colonic tissues were fixed in 10% neutral buffered formalin for 24 h followed by 70% ethanol for an additional 24 h. Paraffin-embedded colonic sections cut 3 Am thick were

59

deparaffinized, rehydrated and then placed in freshly prepared methanol H2O2 solution for 30 min to block endogenous peroxidase activity (Vallance et al., 1998; Wang et al., 2000). To assist antigen retrieval, sections were treated for 20 min at 37 -C in 0.05% trypsin (Sigma) and 0.25% CaCl2 in 0.05M tris buffered saline (TBS), pH 7.6 and non-specific binding sites were blocked with 5% normal goat serum in TBS. Tissue sections were exposed to rabbit antihuman CD3 (Dako A/S; Denmark) at a dilution of 1 : 600 for 1 h at room temperature, followed by incubation with biotinylated goat anti-rabbit antibody and ABC staining (Zymed Laboratories; San Francisco CA, USA). The slides were developed with 0.2% aminoethylcarbazole and hydrogen peroxide in 0.05M acetate buffer for 15 min, then counter stained with haematoxylin. Slides were coded to prevent bias and the total number of mucosal CD3+ cells were counted (including intra-epithelial and lamina propria lymphocytes) in at least 20 intact crypts. Lymphoid follicles were excluded. The thickness of the tissue (from the submucosal-inner circular muscle layer border to the apical epithelium) was measured along with the mean crypt width; the square area of tissue examined was calculated and the cell counts expressed per square millimeter. 2.5.4. Assessment of autonomic nervous system activity: sympathovagal balance To assess the response and role of the autonomic nervous system in the stress response, telemetric recordings of heart rate were collected from conscious rats. Prior to initial induction of colitis, the Data Sciences International (DSI) biopotential activity transmitter TA10ETA-F20 was surgically implanted subcutaneously (dorsal) and positioned between the shoulder blades of each rat while under ketamine hydrochloride (90 mg/kg) and xylazine (20 mg/kg; McMaster Animal facility) anaesthesia. The two electrocardiogram (ECG) recording electrodes were sutured to muscle in the right anterior chest wall (sternomastoid or major pectoris) and the left upper abdominal musculature (internal oblique), so that the ventral representation of the heart was located between the two electrodes. One week following recovery the rats were acclimatized to the recording room with four visits over the next week. A telemetry system (DSI, St. Paul MN) permitted the acquisition of ECG signals from rats pre-stress (baseline), during stress (at the 2 h time point of 3 h of restraint stress), and post stress (30 – 60 min after cessation of stress). The three ECG recordings were made prior to initial induction of colitis or prior to intra-rectal saline administration and then six weeks later. Additional experiments were carried out on non-stressed rats, 5 days post-induction of initial colitis to determine the effect of colitis on sympathovagal balance. The ECG signal was digitized with a 12 bit analog conversion (DATAQ Instruments, Akron OH) and recorded at a sampling rate of 1 kHz. ECG data were analyzed by software developed by the author M.K. A QRS detection algorithm located stable and

60

P.R. Saunders et al. / Autonomic Neuroscience: Basic and Clinical 124 (2006) 56 – 68

Table 1 Inflammatory activity in acute dinitrobenzene sulfonic acid-induced colitis

Saline control DNB/saline DNB/EtOH Stress/saline Stress and DNB/saline

Damage score

MPO (units/mg)

CD3+ cells (no./mm2)

0.0 T 0.0 0.19 T 0.03 2.02 T 0.22* 0.16 T 0.08 0.6 T 0.22 -

0.59 T 0.18 0.78 T 0.09 20.73 T 5.11* 0.52 T 0.12 8.39 T 1.71 .

10.47 T 2.11 n.d 37.82 T 4.49** n.d. n.d.

Macroscopic damage score, myeloperoxidase activity and number of mucosal and submucosal CD3+ cells in control rats (0.9% saline), rats that received intrarectal dinitrobenzene sulfonic acid in ethanol (DNB/EtOH) and rats that were subjected to restraint stress followed by intrarectal dinitrobenzene sulfonic acid in saline (stress and DNB/saline). Results are expressed as means T SEM for 10 rats per group except for quantitative analysis of mucosal and submucosal CD3+ cells where 6 rats per group where examined. There was an overall significant difference in the Damage Score, P < 0.0001. Post hoc analysis indicated that DNB/EtOH rats had significantly increased damage scores compared to all control groups ( P < 0.001) and Stress and DNB/saline rats ( P < 0.001), whereas stress and DNB/saline rats had significantly increased damage scores compared to saline control ( P < 0.001). There was also an overall significant difference in MPO activity, P < 0.001. DNB/EtOH rats had significantly increased MPO activity compared to all control groups ( P < 0.01) and Stress and DNB/saline rats ( P < 0.05), whereas stress and DNB/saline rats had significantly increased MPO activity compared to saline control and stress/saline ( P < 0.001 and 0.05, respectively). DNB/EtOH rats had significantly more mucosal and submucosal CD3+ cells compared to saline control, P = 0.0003. * and ** indicate groups significantly different from all controls ( P < 0.01, P < 0.001, respectively), . and - indicate groups significantly different from DNB/EtOH ( P < 0.05, P < 0.001, respectively), n.d. indicates not determined.

noise-independent points on the R wave. An R –R time interval series was then generated from the continuous ECG data. A beat-to-beat heart rate variability (HRV) signal was computed and then re-sampled at 6 Hz using linear interpolation to obtain an equally sampled time series. A record length of 768 (128 s) points from the re-sampled signal was selected for power spectral analysis. The mean value of the signal was removed and the equally sampled HRV signals were subjected to a 4th order high pass Butterworth filter with a cut-off at 0.015 Hz. A Blackman– Tukey power spectral method was then utilized on the filtered HRV data. Earlier studies (Cerutti et al., 1991; Kuwahara et al., 1994) indicated that most of the low frequency (LF: predominantly sympathetic) power was in the range of 0.015 – 1.0 Hz and the high frequency (HF: parasympathetic) power was in the range of 1.0 – 3.0 Hz. Each of the spectral band areas were calculated by integrating the power contained within each frequency range and expressing as absolute units (beats/minute)2. The areas were normalized by dividing the integrated power within each range by the total power contained in the entire spectrum. Finally, the LF : HF ratio was calculated as the ratio of the two normalized areas, to provide a measure of relative sympathovagal balance. 2.5.5. Assessment of contribution of the nervous system 2.5.5.1. In vivo treatment with hexamethonium 2.5.5.1.1. Assessment of contribution of nicotinic cholinergic pathways. Previously inflamed rats were pretreated with the nicotinic ganglionic blocking agent hexamethonium (10 mg/kg in dH2O, Sigma) or dH2O administered subcutaneously 15 min prior to 3 h of physical restraint stress followed by intra-rectal DNB/saline (n = 10) on days 42, 43 and 44 (Fig. 1). In addition, previously inflamed rats were pretreated with hexamethonium followed by intrarectal saline (control), DNB or stress plus intra-rectal saline (n = 6 animals/group) as described. This dose was chosen as

it has been shown to be effective in blocking nicotinic receptors present on autonomic ganglia (Endoh et al., 1992). On day 44 rats were sacrificed and intestinal segments were collected for determination of macroscopic damage (n = 10 stress/DNB/saline and n = 6 control, DNB and stress/ saline), myeloperoxidase activity (n = 8 stress/DNB/saline and n = 6 control, DNB and stress/saline) and immunohistochemistry (n = 6 stress/DNB/saline and n = 6 control, DNB and stress/saline). 2.5.5.1.2. In vivo treatment with atropine methyl nitrate and bretylium tosylate 2.5.5.1.2.1. Assessment of contribution of muscarinic cholinergic and noradrenergic pathways. To assess the contributions of nordrenergic and cholinergic pathways in mediating stress’ role as a modulator of the relapsing colitis (Fig. 1), previously inflamed rats (n = 12/group) were treated with either a) dH2O (1 ml/kg), b) atropine methyl nitrate (2 mg/kg), a muscarinic cholinoceptor antagonist (which inhibits the actions of acetylcholine on exocrine glands and smooth muscles), c) bretylium tosylate (16 mg/kg), a ganglionic noradrenergic blocking agent (which selectively accumulates in sympathetic ganglia and their postganglionic adrenergic neurons to inhibit noradrenaline release) or d) both atropine methyl nitrate and bretylium tosylate (same doses as given individually) injected subcutaneously on day 42, 30 min prior to beginning 3 h of restraint stress. One hour after cessation of restraint stress, rats were administered intra-rectal DNB (20 mg dissolved in saline). Rats that received atropine methyl nitrate (either alone or in conjunction with bretylium tosylate) received a second subcutaneous injection of atropine (2 mg/kg) 30 min prior to DNB/saline administration as the half-life for atropine is approximately 4 h. In addition, previously inflamed rats were treated atropine, bretylium or both as described above followed by intra-rectal saline (control), DNB or stress plus intra-rectal saline (n = 6 animals/group). On day 44 rats were sacrificed and intestinal segments were collected for determination of macroscopic damage and myeloperoxidase

P.R. Saunders et al. / Autonomic Neuroscience: Basic and Clinical 124 (2006) 56 – 68

61

Fig. 2. Histological appearance of tissue sections for saline control (A), acute dinitrobenzene sulfonic acid induced colitis (B), 6 weeks post induction of acute dinitrobenzene sulfonic acid induced colitis (C), stress-induced reactivation colitis (D), pretreatment with hexamethonium (E) or atropine and bretyllium (F) followed by Stress/DNB. (A), normal histological appearance, (B), focal ulceration with fibropurulent material overlying granulation tissue in the ulcer crater. Deep to this transmural inflammation with extension into the muscle layers of both acute and chronic inflammatory cells. (C), epithelial recovery with resolution of the increased inflammatory cell infiltrate, (D), focal ulceration, crypt destruction and transmural inflammation with extension of both acute and chronic inflammatory cells into the muscle layers, (E), intact epithelium with markedly reduced infiltration of mucosal and submucosal inflammatory cells, (F), minimal epithelial damage with reduced infiltration of mucosal and submucosal inflammatory cells. The bar represent 100 Am.

activity (n = 12 stress/DNB/saline and n = 6 control, DNB and stress/saline).

performed and when appropriate a student’s T-test was used for select comparisons. A P-value less than 0.05 was considered statistically significant.

3. Statistics 4. Results 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 T SEM. One-way ANOVA with a Neuman – Keuls post hoc test for multiple comparisons was

4.1. 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

62

P.R. Saunders et al. / Autonomic Neuroscience: Basic and Clinical 124 (2006) 56 – 68

A

**

3

‡ 2

1

Control

MPO Activity (units/mg)

40

Stress

DNB

Stress/DNB + Hexamethonium

inflammation with both acute and chronic inflammatory cells (Fig. 2B). Rats that were exposed to three consecutive days of restraint stress followed by intra-rectal dinitrobenzene sulfonic acid in saline rather than ethanol and sacrificed on day 5, demonstrated mild elevations in mean macroscopic damage score that was significantly increased compared to saline control ( P < 0.001, Table 1) and myeloperoxidase activity that was significantly elevated compared to both saline control and stress/saline ( P < 0.001 and 0.05, respectively, Table 1). These markers of inflammation were significantly lower than measures made from rats administered dinitrobenzene sulfonic acid in ethanol (Table 1). Microscopic analysis of tissue sections from these rats revealed a mild increase in lamina propria mononuclear cells.

*

B

4.2. Reactivation of colitis

30

†

20

10

Control

Stress

DNB

In animals examined 6 weeks post induction of colitis, all inflammatory markers including macroscopic damage score, MPO activity and infiltration of mucosal and submucosal CD3+ cells had returned to control levels in addition to epithelial recovery and resolution of the increased inflammatory cell infiltrate (Fig. 2C). However a mild degree of scaring and muscular hyperplasia was evident in some animals. Rats that were stressed and then administered intrarectal dinitrobenzene sulfonic acid in saline daily for three

Stress/DNB + Hexamethonium

Fig. 3. Six weeks post induction of acute colitis, rats received either intrarectal saline (control), intra-rectal dinitrobenzene sulfonic acid (DNB), restraint stress (stress), or restraint stress followed by intrarectal dinitrobenzene sulfonic acid (stress/DNB) for 3 consecutive days together with pretreatment with either subcutaneous dH2O or hexamethonium. No demonstrable differences were observed with hexamethonium pretreatment in control, DNB and stress animals (n = 6 animals/group, data not shown). A) There was an overall significant difference in mean macroscopic damage scores ( P < 0.0001). Post hoc testing indicated that Stress/DNB rats had significantly elevated damage scores ( P < 0.001) compared to control, stress, or dinitrobenzene sulfonic acid rats. Pre-treatment with hexamethonium (+Hexamethonium) in stress/DNB rats significantly attenuated the damage score ( P < 0.001). Results are expressed as mean T SEM for 10 rats in each group. B) There was an overall significant difference in tissue myeloperoxidase activity ( P = 00.0008). Stress/DNB rats had significantly higher myeloperoxidase activity than Controls ( P < 0.01) or rats exposed to either stress or dinitrobenzene sulfonic acid alone ( P < 0.001, 0.01, respectively). Pretreatment with hexamethonium (+Hexamethonium) in stress/DNB rats prevented the increase in myeloperoxidase activity ( p < 0.01). Results are expressed as mean T SEM for 8 rats in each group. *( P < 0.01) or **( P < 0.001) indicates a significant difference from controls, .( P < 0.01) or -( P < 0.001) indicates a significant difference from stress/DNB rats.

( 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 analysis of tissue sections from acute colitic animals demonstrated focal ulceration, overlying fibropurulent material and transmural

80

CD3+ cells (no./mm2)

Macroscopic Damage Score

4

*

60

† 40

20

Control

Stress

DNB

Stress/DNB + Hexamethonium

Fig. 4. Quantitative analysis of mucosal and submucosal CD3+ cells six weeks post induction of colitis in rats that received either intra-rectal saline (control), intra-rectal dinitrobenzene sulfonic acid (DNB), restraint stress (stress) or restraint stress followed by intra-rectal dinitrobenzene sulfonic acid (stress/DNB), for 3 consecutive days. There was an overall significant difference in the number of CD3+ cells/mm2 ( P = 0.0014). Post hoc analysis indicated that stress/DNB rats had significantly more mucosal and submucosal CD3+ cells compared to controls ( P < 0.001), or rats that had a previous colitis but were exposed to either stress alone ( P < 0.01) or in DNB alone ( P < 0.05). No difference in number of CD3+ cells was observed between absolute controls (rats not previously exposed to dinitrobenzene sulfonic acid) and control. Pre-treatment with hexamethonium (+Hexamethonium) in stress/DNB rats significantly attenuated the increase in CD3+ cells ( P < 0.05). Bars represent mean T SEM for n = 6 rats per group. * Indicate a significant difference from control, and . indicates the pretreatment group was significantly different from the stress/DNB group.

P.R. Saunders et al. / Autonomic Neuroscience: Basic and Clinical 124 (2006) 56 – 68 5

A

Macroscopic Damage Score

*** ***

4

** 3

2

1

Control

MPO Activity (units/mg)

50

Stress/DNB

**

B

+Atropine +Bretylium +Atropine-Bretylium

*

**

40

30

20

†

10

Control

Stress/DNB

+Atropine

+Bretylium +Atropine-Bretylium

Fig. 5. Six weeks post induction of colitis, rats received either intra-rectal saline (control) or restraint stress followed by intra-rectal dinitrobenzene sulfonic acid (stress/DNB) on only one occasion together with pretreatment with either subcutaneous dH2O, atropine or bretylium or both atropine and bretylium. The colons were examined 2 days later. No demonstrable differences were observed with pharmacologic pretreatment in control, DNB and stress animals (n = 6 animals/group, data not shown). A) There was an overall significant difference in macroscopic damage scores of rat colons, ( P < 0.0001). Post hoc analysis indicated that stress/DNB rats had significantly elevated scores ( P < 0.001) compared to control rats. Pretreatment with atropine (+Atropine) or bretylium (+Bretylium) in stress/ DNB rats failed to affect the damage scores as these groups were also significantly different from control ( P < 0.01, P < 0.001, respectively). Rats pre-treated with both atropine and bretylium demonstrated reduced damage scores but this just failed to reach significance compared to stress/DNB ( P = 0.06), however the score was also not significantly different from control. Bars represent mean T SEM for 12 rats in each group. B) There was also an overall significant difference in tissue myeloperoxidase activity ( P = 0.0012). Stress/DNB rats had significantly higher myeloperoxidase activity than controls ( P < 0.01) as did rats pretreated with either atropine or bretylium ( P < 0.05, 0.01, respectively). Stress/DNB rats pretreated with both atropine and bretylium had significantly lower myeloperoxidase activity than stress/DNB ( P < 0.05) and the + Atropine and + Bretylium rats ( P < 0.05). The myeloperoxidase activity in the atropine plus bretylium treated rats was also not significantly different from control. Bars represent mean T SEM for 12 rats in each group. *( P < 0.05), **( P < 0.01) or ***( P < 0.001) indicates a significant difference from controls, . ( P < 0.05) indicates a significant difference from stress/DNB rats.

consecutive days (stress and DNB/saline), six weeks following induction of initial colitis, developed a clinically relevant reactivation of the colitis associated with bloody

63

diarrhea. Moreover, inflammatory markers were not significantly different from that observed in animals with acute colitis. The reactivation of colitis was associated with a significant increase in the mean macroscopic damage score as compared to saline controls, stress only, and DNB/saline only treatments ( P < 0.001 for all 3 groups), (Fig. 3A). In addition, there was a 10-fold increase in myeloperoxidase activity as compared to saline controls ( P < 0.01), (Fig. 3B). Myeloperoxidase activity was also significantly elevated when compared to stress alone or DNB/saline alone ( P < 0.001; P < 0.01, respectively), (Fig. 3B), whereas rats that were only stressed or only received intra-rectal DNB/ saline had little or no inflammation at all; any inflammatory process that was evident was mild. Moreover, there were no significant differences in the damage scores or myeloperoxidase activity in either stress alone or DNB/saline alone groups when compared to saline controls (Fig. 3). Microscopic analysis of tissue sections demonstrated areas of focal ulceration, crypt destruction and transmural inflammation with extension of both acute and chronic inflammatory cells into the muscle layers (Fig. 2D). The histological appearance was similar to that observed on day 5 following induction of acute colitis. Immunohistochemistry for CD3+ cells revealed increased numbers of mucosal and submucosal CD3+ cells in tissues of stress plus DNB/saline treated rats (Fig. 4). Cell counts demonstrated a significant 6-fold increase in the number of CD3+ cells/mm2 compared to saline treated controls ( P < 0.001). Cell counts for stress plus DNB/saline treated animals were also significantly different from the stress only and DNB/saline only treatment groups ( P < 0.01; P < 0.05, respectively), (Fig. 4). Rats subjected to a single session of stress followed by intra-rectal DNB dissolved in saline 1 h after cessation of stress 6 weeks post induction of acute colitis developed a dramatic reactivation of the colitis associated with bloody diarrhea, similar to that observed in rats subjected to 3 consecutive days of restraint stress followed by intra-rectal DNB/saline. The reactivation of colitis was associated with a significant increase in mean macroscopic damage score as compared to control rats healed from their initial colitis, ( P < 0.001), (Fig. 5A). In addition, there was nearly a 15-fold increase in myeloperoxidase activity compared to control rats healed from their initial colitis ( P < 0.001), (Fig. 5B). 4.3. Power spectral analysis of HRV — autonomic nervous system activity To assess the role of the autonomic nervous system in the stress response, HRV was assessed before, during, and after stress. Cardiac sympathovagal balance was assessed by power spectral analysis of heart rate variability data collected from telemetric electrocardiogram recordings. The animals were observed to be profoundly sympathetic with enhancement in sympathetic activity to stress. At baseline the LF : HF ratio in rats not exposed to DNB was 6 which is higher than

64

P.R. Saunders et al. / Autonomic Neuroscience: Basic and Clinical 124 (2006) 56 – 68 30

A

(pre DNB)

450

** LF : HF Ratio

400

20

**

350

‡

10

300

Heart Rate (beats/min)

**

250 baseline

30

B

stress post stress

baseline

stress post stress

450

(post DNB)

* LF : HF Ratio

400 20

*†

350

10 300

‡

Heart Rate (beats/min)

**

250 baseline

stress post stress

baseline

stress post stress

Fig. 6. Telemetric recordings of heart rate were collected from conscious rats prior to stress, during stress, and post stress in naı¨ve rats (not exposed to dinitrobenzene sulfonic acid), (A) and in rats that had recovered from DNB colitis 6 weeks earlier, (B). Low frequency (LF, predominantly sympathetic activity) to high frequency (HF, parasympathetic activity) ratios were determined to provide an overall indication of cardiac autonomic tone. A) In naı¨ve rats there was an overall significant difference in the LF : HF ratios ( P = 0.0007, n = 6 – 10 recordings per group). Post hoc testing revealed that the LF : HF ratios were significantly elevated during stress ( P < 0.001) compared to baseline values, and significantly lowered to baseline levels post stress ( P < 0.001). There was also an overall significant difference in heart rate, ( P < 0.0001, n = 6 – 10 recordings per group). Heart rates were significantly elevated compared to baseline measures ( P < 0.001), but failed to decrease significantly post stress, and remained significantly elevated compared to baseline measures ( P < 0.001). B) In previously inflamed rats, there was also an overall significant difference in the LF : HF ratios (for this period as well, P = 0.0008, n = 6 – 10 recordings per group). The LF : HF ratios were significantly elevated during stress ( P < 0.01) compared to baseline values, and significantly lowered to baseline levels post stress ( P < 0.001). There was also an overall significant difference in heart rate, ( P < 0.0001, n = 6 – 10 recordings per group). The heart rates were significantly elevated compared to baseline measures ( P < 0.001) and did significantly slow down post stress ( P < 0.01), but remained significantly elevated compared to baseline measures ( P < 0.01). Black bars represent mean T SEM of the LF : HF ratios (left y axis) while gray bars represent mean T SEM of the heart rates (right y axis). *( P < 0.01) or **( P < 0.001) indicates a significant difference from baseline measures, .( P < 0.01) or -( P < 0.001) indicates significant differences in post stress measures compared to stress measures.

the ratio observed in humans (LF : HF ratio around 1) demonstrating that rats are normally more profoundly sympathetic, (Fig. 6A), (15). As anticipated, 2 h into the restraint stress session there was a significant increase in the relative sympathetic activity characterized by a significant increase in heart rate and LF : HF ratio which doubled over baseline levels ( P < 0.001). Following cessation of stress the LF : HF ratio returned to baseline levels that was significantly lower than during stress ( P < 0.001). However, the heart rate remained significantly elevated above baseline values ( P < 0.001) at a level not significantly different from the elevated heart rate observed during stress (Fig. 6A). Previously inflamed rats subjected to the same stress regimen demonstrated a similar pattern of increased sympathetic activity with a nearly four fold increase in the

LF : HF ratio during stress ( P < 0.01), and a significant increase in heart rate (Fig. 6B). Similarly, shortly after cessation of stress the LF : HF ratio decreased significantly, ( P < 0.001), returning to baseline levels. However, the heart rate remained significantly elevated over baseline levels following stress ( P < 0.01), but decreased to a level that was significantly lower than during stress ( P < 0.01), (Fig. 6B). There were no significant differences in the specific LF : HF ratios between the groups, pre and post dinitrobenzene sulfonic acid, across any of the times recordings made (baseline, during stress, or post stress). However, the magnitudes of the overall increases in LF : HF ratios during stress and the subsequent decreases post stress were greater in the rats that had recovered from the initial colitis than in rats not exposed to dinitrobenzene sulfonic acid.

P.R. Saunders et al. / Autonomic Neuroscience: Basic and Clinical 124 (2006) 56 – 68

Power spectral analysis of heart rate variability done on electrocardiogram recordings from non-stressed rats 5 days post-induction of the initial colitis, showed no significant differences for either LF : HF ratio or resting heart rate when compared to baseline recordings in non-inflamed rats (data not shown). 4.4. Hexamethonium pre-treatment Pre-treating rats that had previously received intra-rectal DNB/EtOH with subcutaneous hexamethonium prior to stress and DNB/saline administration dramatically attenuated the onset of bloody diarrhea and reactivation of the colitis. Hexamethonium significantly attenuated the mucosal ulceration, significantly diminished the mean macroscopic damage score ( P < 0.001) and reduced the myeloperoxidase activity ( P < 0.01), (Fig. 3A and B). Furthermore, hexamethonium significantly attenuated the inflammatory response, preventing epithelial ulceration and markedly reducing the infiltration of mucosal and submucosal inflammatory cells (Fig. 2E, 4). 4.5. Atropine and bretylium pre-treatment Neither pre-treatment with atropine nor bretylium alone significantly influenced the stress-induced reactivation of colitis (Fig. 5A, B). However, when atropine and bretylium were both administered to rats prior to stress (plus a second dose of atropine given post stress and prior to DNB/saline administration) the onset of bloody diarrhea and reactivation of the colitis was attenuated. This was associated with a significant reduction in myeloperoxidase activity ( P < 0.05), (Fig. 5B), evidence of minimal epithelial damage and a reduction in the infiltration of mucosal and submucosal inflammatory cells (Fig. 2F). Although this was associated with a decrease in the mean macroscopic damage score, the score was not significantly different from that observed with stress and DNB/saline treated rats given only saline or stress and DNB/saline treated rats given either atropine or bretylium alone. However, the mean damage score from stress and DNB/saline rats pretreated with both atropine and bretylium were also not significantly different from rats given only saline (Fig. 5A).

5. 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

65

acid in previously sensitized rats resulted in reactivation of a clinically relevant colitis associated with bloody diarrhea, a significant increase in macroscopic damage scores, tissue myeloperoxidase activity and an increased number of mucosal and submucosal T lymphocytes. The importance of re-exposure to luminal antigen is further supported by our previous work where stress without re-exposure to luminal trinitrobenzene sulfonic acid was associated with altered neural function (decreased noradrenaline release in vitro) and elevated tissue myeloperoxidase levels, but failed to reactivate colitis (Collins et al., 1996). Similarly, in the present study, rats exposed to stress alone failed to reactivate the colitis. In previous studies in patients with inflammatory bowel disease, local immunocytes isolated from involved portions were noted to react with autologous bacterial antigens, whereas, immunocytes from a non-involved portion of intestine and immunocytes from ‘‘control’’ resected tissue were unreactive to luminal antigen (Duchmann et al., 1995). Additionally, murine studies have shown that local immunocytes present at the site of original intestinal injury reacted to a second exposure of dinitrobenzene sulfonic acid administered systemically (Appleyard and Wallace, 1995; Palmen et al., 1995). Although the present study did not provide direct evidence that T lymphocytes mediated the reactivation of colitis, the model did show a significant increase in number of CD3+ cells recruited into the mucosa and submucosa that was associated with epithelial damage (ulceration and crypt destruction) and mucosal edema. Furthermore, previous murine studies have shown that sensitized CD4+ T cells were necessary for dinitrobenzene sulfonic acid reactivation when dinitrobenzene sulfonic acid was administered systemically (Palmen et al., 1998) or intra-rectally (Qiu et al., 1999). In addition, murine studies have demonstrated that T cells are profoundly affected by stress (Dominguez-Gerpe and Lefkovits, 1996). When examining the role of stress in the induction of the initial colitis, we observed that stress was effective at facilitating dinitrobenzene sulfonic acid-induced colonic inflammation when administered in saline however the degree of inflammation was mild and far less marked that that observed in the reactivation model. The reason why stress was capable of precipitating a clinically relevant relapse but was only capable of inducing a mild degree of inflammation in the acute setting is not yet understood. However the behavior of the colonic epithelium following stress was likely different between rats previously sensitized to dinitrobenzene sulfonic acid and those exposed for the first time. It has been shown that despite normal histological appearances and baseline electrophysiological measures, the colonic epithelium from rats healed from an initial trinitrobenzene sulfonic acid-induced colitis (6 weeks post treatment) demonstrated impaired secretory responses to various prosecretory stimuli (electrical field stimulation, isobutylmethylxanthine or carbachol) in Ussing chamber studies (Asfaha et al., 1999). Stress responses may affect

66

P.R. Saunders et al. / Autonomic Neuroscience: Basic and Clinical 124 (2006) 56 – 68

intestinal epithelial function via a number of neuroendocrine factors (Santos et al., 1999; Saunders et al., 2002) and if the epithelial response to these signals is altered as a result of a previous inflammation, then the epithelium may be more susceptible to stress. In our study, hexamethonium which is a classical peripheral nicotinic cholinoceptor antagonist that blocks nicotinic neurotransmission in sympathetic ganglia, parasympathetic ganglia as well as enteric ganglia (Taylor, 2001) was found to be effective in preventing stress-induced reactivation of the colitis. The beneficial effects of hexamethonium have also been demonstrated in rats where hexamethonium prevented smoke induced exacerbation of acute dinitrobenzene sulfonic acid colitis Galeazzi et al. (1999). Taken together these data suggest that stressinduced reactivation of colitis that was clinically similar to the acute colitis, was mediated through nicotinic cholinergic neural pathways. What cannot be excluded is whether an effect of hexamethonium on intestinal blood flow contributed to the beneficial response. Since sympathetic and parasympathetic activity directed to the colon could not be measured in conscious animals, cardiac sympathovagal tone was assessed as an approximation of the overall autonomic activity. In response to stress, all animals demonstrated a typical and dramatic increase in sympathetic activity (sympathetic dominance, i.e. the flight, fight response) as evidenced by the significant increase in heart rate and LF : HF ratio. Whereas, the post stress recovery response was associated with parasympathetic arousal that was characterized by a significant decrease in LF : HF ratio which returned back to baseline. The magnitudes of the overall increases in LF : HF ratios during stress and the subsequent decreases post stress appeared to be greater in the rats that had recovered from the initial colitis than in rats not exposed to dinitrobenzene sulfonic acid but this failed to reach significance. Functional studies in patients with inflammatory bowel disease have demonstrated autonomic nervous system dysfunction (Lindgren et al., 1991, 1993; Straub et al., 1997). Typically parasympathetic activation is associated with a decrease in heart rate, however during the recovery period the heart rate remained significantly elevated in both groups of animals suggesting ongoing increased sympathetic activity. A definitive explanation for the ongoing increased sympathetic activity during the recovery period remains to be determined but likely reflects a parallel sympathetic and parasympathetic activation (described in other situations, Berntson et al., 1991) rather than a pure dominance of parasympathetic control. It is probably that had a later time point been chosen post stress, a return of the heart rate back to baseline might have been observed suggesting a shift in autonomic balance back toward neutrality. While neither atropine nor bretylium when administered alone prevented stress-induced reactivation of colitis, both compounds when administered prior to stress, significantly inhibited the increase in myeloperoxidase activity and

attenuated the macroscopic damage. In contrast, hexamethonium effectively prevented the stress-induced reactivation of the colitis. A possible explanation for the inability of atropine and bretylium to completely prevent the reactivation of colitis might be secondary to the differences in pharmacologic blockade and effect on neural transmission at the various sites. Previous studies have demonstrated that both sympathetic and cholinergic neural pathways are synergistically involved in mediating stress-induced increases in colonic permeability, since both atropine and bretylium have been shown to independently prevent the increase in colonic permeability due to stress or the administration of corticotropin releasing hormone (which mimics the effects of stress), (Santos et al., 1999; Saunders et al., 2002). Moreover the presence of muscarinic cholinergic receptors on lymphocytes and adrenergic receptors on granulocytes suggests a receptor mediated interaction between sympathetic and cholinergic nerves and immune cells with regulation of immune cell function (Bronzetti et al., 1996; Tayebati et al., 1999; Abo and Kawamura, 2002). Alternatively the combination of the agents could have resulted in a local anaesthetic effect on non-adrenergic, non-cholinergic nerves within the colon. Taken together these observations suggest that both noradrenergic and cholinergic neural pathways are involved, and that attenuating the stress mediated response of both pathways is required to prevent stress-induced reactivation of colitis. Further studies are required to determine the contribution of the autonomic nervous system and enteric nervous system in mediating the stress response. While the intestinal mucosal mechanisms associated with reactivation of colitis were not examined, available data suggest that alteration in epithelial barrier function with increased permeability to luminal dinitrobenzene sulfonic acid was a likely factor. These models may provide insight into mechanisms responsible for stress-induced relapses in patients with inflammatory bowel disease (Duffy et al., 1991; Levenstein et al., 2000). Moreover, this study raises the intriguing possibility of developing novel therapeutic options aimed at modulating stress-induced neural mediation of intestinal immune mechanisms and/or local intestinal physiological responses that result in reactivation of the intestinal inflammatory process. These therapeutic approaches might prove particularly useful when used prophylactically at the appropriate times and in the appropriate clinical context within that subgroup of inflammatory bowel disease patients with known relapse provoking factors, such as examination stress. In summary, this study demonstrates a rat model of restraint stress that is associated with reactivation of a clinically relevant colitis. Furthermore, this study demonstrates the critical involvement of both noradrenergic and cholinergic neural pathways in mediating the stress responses responsible for precipitating the relapse of colitis.

P.R. Saunders et al. / Autonomic Neuroscience: Basic and Clinical 124 (2006) 56 – 68

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 fellowship from the Norman Cousins Center of Psychoneuroimmunology at UCLA. Dr. M.V. Kamath received support from the Natural Sciences and Engineering Research Council and the DeGroote Foundation. This research was supported by a CAG/MRC Young Investigator Award and by a grant from CCFC (Canada) award to Dr. K. Jacobson. Dr Jacobson is a Clinician Scientist supported by the Children with Intestinal and Liver Disorders (C.H.I.L.D.) and the British Columbia Children’s Hospital Foundation.

References Abo, T., Kawamura, T., 2002. Immunomodulation by the autonomic nervous system: therapeutic approach for cancer, collagen diseases, and inflammatory bowel diseases. Ther. Apher. 6 (5), 348 – 357. Appleyard, C.B., Wallace, J.L., 1995. Reactivation of hapten-induced colitis and its prevention by anti-inflammatory drugs. Am. J. Phsyiol. 269, G119 – G125. Asfaha, S., Bell, C.J., Wallace, J.L., MacNaughton, W.K., 1999. Prolonged colonic epithelial hyporesponsiveness after colitis: role of inducible nitric oxide synthase. Am. J. Physiol. 276, G703 – G710. Berntson, G.G., Caccioppo, J.T., Quigley, K.S., 1991. Autonomic determinism: the modes of autonomic control, the doctrine of autonomic space and laws of autonomic constraint. Psychol. Rev. 98, 459 – 487. Bjorck, S., Dahlstrom, A., Ahlman, H., 1989. Topical treatment of ulcerative colitis with lidocaine. Scand. J. Gastroenterol. 24 (9), 1061 – 1072. Boughton-Smith, N.K., Wallace, J.L., Whittle, B.J., 1998. Relationship between arachidonic acid metabolism, myeloperoxidase activity and leukocyte infiltration in a rat model of inflammatory bowel disease. Agents Actions 25, 115 – 123. Bozkurt, A., Cakir, B., Ercan, F., Yegen, B.C., 2003. Anti-inflammatory effects of leptin and cholecystokinin on acetic acid-induced colitis in rats: role of capsaicin-sensitive vagal afferent fibres. Regul. Pept. 15 (116(1 – 3)), 109 – 118. Bronzetti, E., Adani, O., Amenta, F., Felici, L., Mannino, F., Ricci, A., 1996. Muscarinic cholinergic receptor subtypes in human peripheral blood lymphocytes. Neurosci. Lett. 208 (3), 211 – 215. Brown, J.H., Taylor, P., 2001. Chapter 7. Muscarinic receptor agonists and antagonists. In: Hardman, J.G., Gilman, A.G., Limbird, L.E. (Eds.), Goodman and Gilman’s The Pharmacological Basis of Therapeutics, Ninth edition. McGraw-Hill, New York. Cabarrocas, J., Savidge, T.C., Liblau, R.S., 2003. Role of enteric glial cells in inflammatory bowel disease. Glia 41 (1), 81 – 93. Castagliuolo, I., Lamont, J.T., Qiu, B., Fleming, S.M., Bhaskar, K.R., Nikulasson, S.T., Kornetsky, C., Pothoulakis, C., 1996. Acute stress causes mucin release from rat colon: role of corticotropin releasing factor and mast cells. Am. J. Physiol. 271 (5 Pt 1), G884 – G892. Cerutti, C., Gustin, M.P., Paultre, C.Z., Lo, M., Julien, C., Vincent, M., Sassard, J., 1991. Autonomic nervous system and cardiovascular

67

variability in rats: a spectral analysis approach. Am. J. Physiol. 261, H1292 – H1299. Collins, S.M., McHugh, K., Jacobson, K., Khan, I., Riddell, R., Murase, K., Weingarten, H.P., 1996. Previous inflammation alters the response of the rat colon to stress. Gastroenterology 111, 1509 – 1515. Colon, A.L., Madrigal, J.L., Menchen, L.A., Moro, M.A., Lizasoain, I., Lorenzo, P., Leza, J.C., 2004. Stress increases susceptibility to oxidative/nitrosative mucosal damage in an experimental model of colitis in rats. Dig. Dis. Sci. 49 (10), 1713 – 1721. Dennis, C., Eddy, F.D., Frykman, M.H., McCarthy, A.M., Westover, D., 1946. The response to vagotomy in idiopathic ulcerative colitis and regional enteritis. Ann. Surg. 128, 479 – 496. Dominguez-Gerpe, L., Lefkovits, I., 1996. Lymphocyte protein synthesis: evidence that murine T cells are more affected by stress than B cells. Immunol. Lett. 52, 109 – 123. Duchmann, R., Kaiser, I., Hermann, E., Mayet, W., Ewe, K., Meyer zum Buschenfelde, K.-H., 1995. Tolerance exists towards resident intestinal flora but is broken in active inflammatory bowel disease. Clin. Exp. Immunol. 102, 448 – 455. Duffy, L.C., Zielezny, M.A., Marshall, J.R., Byers, T.E., Weiser, M.M., Phillips, J.F., Calkins, B.M., Ogra, P.L., Graham, S., 1991. Relevance of major stress events as an indicator of disease activity prevalence in inflammatory bowel disease. Behav. Med. 17, 101 – 110. Endoh, K., Kao, J., Baker, M., Leung, F., 1992. Intragastric nicotine protection against 40% ethanol injury in the stomach. Role of ganglionic stimulation or blockade. Dig. Dis. Sci. 37, 1840 – 1846. Fujino, K., Takami, Y., de la Fuente, S.G., Ludwig, K.A., Mantyh, C.R., 2004. Inhibition of the vanilloid receptor subtype-1 attenuates TNBScolitis. J. Gastrointest. Surg. 8 (7), 842 – 847. Galeazzi, F., Blennerhassett, P.A., Qiu, B., O’Byrne, P.M., Collins, S.M., 1999. Cigarette smoke aggravates experimental colitis in rats. Gastroenterology 117, 877 – 883. Gue, M., Bonbonne, C., Fioramonti, J., More, J., Del Rio-Lacheze, C., Comera, C., Bueno, L., 1997. Stress-induced enhancement of colitis in rats: CRF and arginine vasopressin are not involved. Am. J. Physiol. 272 (1 Pt 1), G84 – G89. Gulpinar, M.A., Ozbeyli, D., Arbak, S., Yegen, B.C., 2004. Antiinflammatory effect of acute stress on experimental colitis is mediated by cholecystokinin-B receptors. Life Sci. 75 (1), 77 – 91. Haglund, U., Hellman, A., Haggendal, E., Haggendal, J., Lundberg, D., 1980. Effects of adrenergic neuron and ganglion blockers on hemodynamics and plasma catecholamine levels after corticosteroids during hemorrhagic shock in the dog. Acta Anaesthesiol. 24, 444 – 450. Hassani, H., Lucas, G., Rozell, B., Enfors, P., 2005. Attenuation of acute experimental colitis by preventing NPY Y1 receptor signaling. Am. J. Physiol. Gastrointest. Liver. Physiol. 288 (3), G550 – G556. Jacobson, K., McHugh, K., Collins, S.M., 1997. The mechanism of altered neural function in a rat model of acute colitis. Gastroenterology 112, 156 – 162. Kamath, M.V., Fallen, E.L., 1993. Power spectral analysis of heart rate variability: a noninvasive signature of cardiac autonomic function. Crit. Rev. Biomed. Eng. 21, 245 – 311. Kihara, N., de la Fuente, S.G., Fujino, K., Takahashi, T., Pappas, T.N., Mantyh, C.R., 2003. Vanilloid receptor-1 containing primary sensory neurones mediate dextran sulphate sodium induced colitis in rats. Gut 52 (5), 713 – 719. Kyosola, K., Penttila, O., Salaspuro,, 1977. Rectal mucosal adrenergic innervation and enterochromaffin cells in ulcerative colitis and irritable colon. Scand. J. Gastroenterol. 12 (3), 363 – 367. Kuwahara, M., Yayou, K., Ishii, K., Hashimoto, S., Tsubone, H., Sugano, S., 1994. Power spectral analysis of heart rate variability as a new method for assessing autonomic activity in the rat. J. Electrocardiol. 27, 333 – 337. Lashner, B.A., Hanauer, S.B., Silverstein, M.D., 1990. Testing nicotine gum for ulcerative colitis patients Experience with single patient trial. Dig. Dis. Sci. 35, 827 – 832.

68

P.R. Saunders et al. / Autonomic Neuroscience: Basic and Clinical 124 (2006) 56 – 68

Lechin, F., van der Dijs, B., Insausti, C.L., Gomez, F., Villa, S., Lechin, A.E., Arocha, L., Oramas, O., 1985. Treatment of ulcerative colitis with clonidine. J. Clin. Pharmacol. 25 (3), 219 – 226. Levenstein, S., Prantera, C., Varvo, V., Scribano, M.L., Andreoli, A., Luzi, C., Arca, M., Berto, E., Milite, G., Marcheggiano, A., 2000. Stress and exacerbation in ulcerative colitis: a prospective study of patients enrolled in remission. Am. J. Gastroenterol. 95, 1213 – 1220. Lindgren, S., Lilia, B., Rosen, I., Sundkvist, G., 1991. Disturbed autonomic nerve function in patients with Crohn’s Disease. Scand. J. Gastroenterol. 26, 361 – 366. Lindgren, S., Stewenius, J., Sjolund, K., Lilja, B., Sundvist, G., 1993. Autonomic vagal nerve dysfunction in patients with ulcerative colitis. Scand. J. Gastroenterol. 28, 638 – 642. Martinez, V., Wang, L., Million, M., Rivier, J., Tache, Y., 2004. Peptides 25, 1733 – 1744. Mazelin, L., Theodorou, V., More, J., Fioramonti, J., Bueno, L., 1998. Protective role of vagal afferents in experimentally-induced colitis in rats. J. Auton. Nerv. Syst. 73, 38 – 45. McCafferty, D.M., Wallace, J.L., Sharkey, K.A., 1997. Effects of chemical sympathectomy and sensory nerve ablation on experimental colitis in the rat. Am. J. Physiol. 272 (2 Pt 1), G272 – G280. Miceli, P.C., Jacobson, K., 2003. Cholinergic pathways modulate experimental dinitrobenzene sulfonic acid colitis in rats. Auton. Neurosci. 30 (105(1)), 16 – 24. Milde, A.M., Murison, R., 2002. A study of the effects of restraint stress on colitis induced by dextran sulphate sodium in singly housed rats. Integr. Physiol. Behav. Sci. 37 (2), 140 – 150. Million, M., Tache, Y., Anton, P., 1999. Susceptibility of Lewis and Fischer rats to stress-induced worsening of TNB-colitis: protective role of brain CRF. Am. J. Physiol. 276 (4 Pt 1), G1027 – G1036. Nguyen, C., Coelho, A.M., Grady, E., Compton, S.J., Wallace, J.L., Hollenberg, M.D., Cenac, N., Garcia-Villar, R., Bueno, L., Steinhoff, M., Bunnett, N.W., Vergnolle, N., 2003. Colitis induced by proteinaseactivated receptor-2 agonists is mediated by a neurogenic mechanism. Can. J. Physiol. Pharmacol. 81 (9), 920 – 927. Palmen, M.J.H.J., Dieleman, L.A., van der Ende, M.B., Uyterlinde, A., Pen˜a, A.S., Meuwissen, S.G.M., van Rees, E.P., 1995. Non-lymphoid and lymphoid cells in acute, chronic and relapsing experimental colitis. Clin. Exp. Immunol. 99, 226 – 232. Palmen, M.J.H.J., Wijburg, O.L.C., Kunst, I.H., Kroes, H., van Rees, E.P., 1998. CD4+ T cells from 2,4,6-trinitrobenzene sulfonic acid (TNBS)induced colitis rodents migrate to the recipient’s colon upon transfer; down regulation by CD8+ T cells. Clin. Exp. Immunol. 112, 216 – 225. Pfeiffer, C.J., Qiu, B., Lam, S.K., 2001. Reduction of colonic mucus by repeated short-term stress enhances experimental colitis in rats. J. Physiol. Paris 95 (1 – 6), 81 – 87. Pullan, R.D., Rhodes, J., Ganesh, S., Mani, V., Morris, J.S., Williams, G.T., Newcombe, R.G., Russell, M., Feyerabend, C., Thomas, G., Sawe, U.,

1994. Transdermal nicotine for active ulcerative colitis. N. Engl. J. Med. 24 (330(12)), 811 – 815. Qiu, B.S., Vallance, B.A., Blennerhassett, P.A., Collins, SM., 1999. The role of CD4+ lymphocytes in the susceptibility of mice to stress-induced reactivation of experimental colitis. Nat. Med. 5, 1178 – 1182. Santos, J., Saunders, P.R., Hanssen, N.P., Yang, P.C., Yates, D., Groot, J.A., Perdue, M.H., 1999. Corticotropin-releasing hormone mimics stressinduced colonic epithelial pathophysiology in the rat. Am. J. Physiol. 277, G391 – G399. Saunders, P.R., Hanssen, N.P., Perdue, M.H., 1997. Cholinergic nerves mediate stress-induced intestinal transport abnormalities in Wistar – Kyoto rats. Am. J. Physiol. 273 (2 Pt 1), G486 – G490. Saunders, P.R., Santos, J., Hanssen, N.P.M., Yates, D., Groot, J.A., Perdue, M.H., 2002. Physical and psychological stress in rats enhances colonic epithelial permeability via peripheral CRH. Dig. Dis. Sci. 47, 208 – 215. Shafiroff, G.P., Hinton, J., 1950. Denervation of the pelvic colon for ulcerative colitis. Surg. Forum 1, 134 – 139. Smith, J.W., Castro, G.A., 1978. Relation of peroxidase activity in gut mucosa to inflammation. Am. J. Physiol. 235, R72 – R79. Soderholm, J.D., Yang, P.C., Ceponis, P., Vohra, A., Riddell, R., DSherman, P.M., Perdue, M.H., 2002. Chronic stress induces mast cell-dependent bacterial adherence and initiates mucosal inflammation in rat intestine. Gastroenterology 123 (4), 1099 – 1108. Straub, R.H., Antoniou, E., Zeuner, M., Gross, V., Scho¨lmerich, J., Andus, T., 1997. Association of autonomic nervous hyperreflexia and systemic inflammation in patients with Crohn’s disease and ulcerative colitis. J. Neuroimmunol. 80 (1 – 2), 149 – 157. Tayebati, S.K., Codini, M., Gallai, V., Mannino, F., Parnetti, L., Ricci, A., Sarchielli, P., Amenta, F., 1999. Radioligand binding assay of M1 – M5 muscarinic cholinergic receptor subtypes in human peripheral blood lymphocytes. J. Neuroimmunol. 99 (2), 224 – 229. Taylor, P., 2001. Chapter 9. Agents acting at the neuromuscular junction and autonomic ganglia. In: Hardman, J.G., Gilman, A.G., Limbird, L.E. (Eds.), Goodman and Gilman’s The Pharmacological Basis of Therapeutics, (Ninth edition)McGraw-Hill, New York. Thorek, P., 1951. Vagotomy for idiopathic ulcerative colitis and regional enteritis. JAMA 145, 140 – 146. Vallance, B.A., Hewlett, B.R., Snider, D.P., Collins, S.M., 1998. T cellmediated exocrine pancreatic damage in major histocompatibility complex class II-deficient mice. Gastroenterology 115, 978 – 987. Wang, J., Snider, D.P., Hewlett, B.R., Lukacs, N.W., Gauldie, J., Liang, H., Xing, Z., 2000. Transgenic expression of granulocyte – macrophage colony-stimulating factor induces the differentiation and activation of a novel dendritic cell population in the lung. Blood 95, 2337 – 2345. Wilson, L.M., Baldwin, A.L., 1999 (Sep.). Environmental stress causes mast cell degranulation, endothelial and epithelial changes, and edema in the rat intestinal mucosa. Microcirculation 6 (3), 189 – 198.