T2059 Persistent Epithelial Barrier Alterations in a Rat Model of Post-Inflammatory Gut Dysfunction
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can contribute to disrupted motility in the inflamed colon and that dampening the hyperexcit- ability of AH neurons can restore colonic motor function. These findings support the concept that increases in sensory neuron activity (reflected in changes to AH cell excitability) in a given region of the bowel can have a deleterious effect on motor function. T2058 Colitis Alters the Function of Chromaffin Cells of the Adrenal Medulla Mark K. Lukewich, Alan Lomax Inflammatory bowel diseases (IBD) are characterized by chronic inflammation of the gastroin- testinal (GI) tract. Catecholamines, which are released synaptically released by the axons of postganglionic sympathetic neurons and systemically by adrenal chromaffin cells (ACC), modulate GI motility, secretion, blood flow and inflammation, and may therefore play an important role in IBD. The present study tested the hypothesis that colitis impairs the release of catecholamines from ACC by examining ACC excitability and voltage-dependent Ca2+ influx with patch clamp and intracellular Ca2+ imaging techniques. Colitis was induced in male CD1 mice by 5% dextran sulfate sodium (DSS) in drinking water for 5 days followed by 2 days of normal drinking water. Controls were given normal drinking water for 7 days. In other experiments, colitis was induced by the intracolonic injection of 4.5 mg of trinitrobenzene sulfuonic acid (TNBS) in 40% ethanol, and saline injected mice were used as controls. ACC were enzymatically dissociated from the adrenal medullae and isolated cells were cultured overnight before recordings commenced. Population data were compared using unpaired t-tests, the Mann Whitney test or two-way ANOVA, as appropriate. Statistical significance was reached when P < 0.05. The n value refers to the number of cells. RESULTS and CONCLUSIONS: The resting membrane potential of ACC isolated from DSS-treated mice was significantly hyperpolarized compared to controls (DSS: -61.2 ± 1.8 mV, n = 32; Control: -50.7 ± 1.6 mV n = 24). Colitis significantly enhanced voltage-gated K+ current (DSS: n = 13 cells; Control: n = 30 cells). Importantly, voltage-gated Ca2+ current was significantly decreased in ACC from DSS-treated mice at membrane potentials between -25 and +25 mV (DSS: n = 11 cells; Control: n = 16 cells). Ca2+ influx in response to KCl- induced depolarization was significantly reduced to 62.2% of the control response during colitis (DSS: n = 114 cells; Control n = 61 cells). Similar to our findings in the DSS model, Ca2+ influx in ACC from TNBS-treated mice was significantly reduced to 68.3% of the control response (TNBS: n = 323 cells; Control: n = 330 cells). Our findings indicate that ACC become hyperpolarized and voltage-dependent Ca2+ influx is reduced during colitis. Given the important roles of cellular excitability and intracellular Ca2+ signaling in secretion, these alterations will likely result in decreased systemic catecholamine levels, which may play an important role in IBD. These data also provide further evidence that localized inflammation of the GI tract during colitis can alter the physiology of distant tissues. T2059 Persistent Epithelial Barrier Alterations in a Rat Model of Post-Inflammatory Gut Dysfunction Joan Antoni Fernandez-Blanco, Vicente Martinez, Maite Martin, Patri Vergara Introduction: Altered epithelial barrier function (EBF) and mucosal mast cells (MMCs) contribute to the initiation and perpetuation of defective intestinal immune responses as a pathogenic basis for gastrointestinal inflammatory and functional disorders. Aim: To charac- terize alterations in the intestinal EBF in a model of post-inflammatory gut dysfunction in rats. Methods: Male SD rats were infected with Trichinella spiralis (TS) (7500 larvae/rat, PO). At the post-inflammatory phase (day 30±2 post-infection) animals were euthanized and jejunal EBF assessed in Ussing chambers. Transepithelial voltage (Vte), short-circuit current (SCC), conductance (G) and FITC-dextran (4 & 40 kD) flux were determined as measures of EBF. Responses to 5-HT (10 -7 -10 -4 M), substance P (SP, 3.3x10 -8 -10 -6 M), the PAR-2 agonist SL-NH 2 (10 -6 -3x10 -5 M), and capsaicin (2x10 -6 M) were evaluated. Histological alterations and MMCs (immunohistochemistry for RMCPII) were also assessed. Results: During the post-inflammatory phase basal EBF was significantly altered (Table). 5-HT, SL- NH 2 and SP induced concentration-dependent increases in SCC. In TS-infected rats, secretory responses to 5-HT [SCC increase (μA/cm 2 ): TS-infected:12.7±2.8; Control:17.5±1.7; N=11- 13; P<0.05] and SL-NH 2 [SCC increase (μA/cm 2 ): TS-infected: 28.2±3.3; Control: 55.3±8.6; N=8-12; P<0.05] were reduced, while responses to SP were unaffected [SCC increase (μA/ cm 2 ): TS-infected: 35.7±1.8; Control: 36.5±2.5; N=10 each]. Only responses to SP in TS- infected animals were affected by pretreatment with tetrodotoxin (TTX) (μA/cm2; TS- infected+TTX: 22.18±3.6 P<0.05 vs control+TTX: 34.0±4.0; N=10 each). Capsaicin aug- mented SCC similarly in both groups. No histological signs of active inflammation were observed; however, the number of MMCs was increased in TS-infected animals (39.1±2.3 MMCs/villi P<0.0001 vs control: 13.9±0.9 MMCs/villi; N=7-9). Conclusions: TS infection in rats results in persistent, post-infective intestinal barrier dysfunctions and mucosal masto- cytosis without signs of active inflammation. Hyporesponsiveness to 5-HT and SL-NH 2 and altered permeability suggest alterations of epithelial function likely associated to increased release of MMC mediators. Decreased responses to SP after neuronal blockage suggests an enteric nervous system remodelling. These alterations might contribute to the epithelial barrier dysfunctions observed in IBD and IBS patients. Basal EBF during the post-inflammtory phase *: P<0.05 vs control S-623 AGA Abstracts T2060 The Clinical Course of Post-Infectious Irritable Bowel Syndrome: An Eight- Year Follow-up Study Hyun Chul Lim, Jie-Hyun Kim, Young Hoon Youn, Hyojin Park, Sang In Lee Background and Aims: Bacterial gastroenteritis has been known as a risk factor of irritable bowel syndrome(IBS). The incidence of post-infectious IBS (PI-IBS) was known in the range of 7-31%, but few studies have long term follow up results. The aims of this study were to evaluate the clinical course, risk factor, and prognosis of PI IBS 8 years after shigella infection. Methods: We recruited 133 patients with shigellosis during its outbreak and 105 healthy controls. We used a questionnaire to investigate their current bowel habits and other functional bowel disorders (FBDs). The shigella-exposed group was consisted of hospital employees who experienced abdominal pain, diarrhea, or fever during the shigellosis outbreak and control group was consisted of age and sex matched hospital employees who corre- sponded with patient and were not infected with shigella. Results: Complete data were obtained from 71 patients (53.4%) and 65 healthy controls (61.9%) and there was no significant demographic difference between both group. The prevalence of IBS in the shigella- exposed group and control group was 11(15.4%) and 6(9.2%) in 8 years after shigella infection. The incidence of newly developed IBS in the shigella-exposed group and control group was 4/71(5.6%) and 3/61(4.61%) in 8 years after shigella infection. There was no significant difference in incidence of IBS in 8 years after shigellosis. In shigella-exposed group, 2 out of 12 cases of PI-IBS showed persistent IBS after 8 years. The prevalence of PI-IBS after 8 years in the previous FBDs groups was 26.7%, whereas the prevalence of PI- IBS without history of FBDs group was 10.7%(p<0.05). Previous FBDs history, female sex, and presence of fever at the time of infection were risk factors of IBS after 8 years follow up study. Conclusions: Two in 12 patients (18.8%) of PI-IBS patients had persistent IBS over 8 year. . Overall prevalence of IBS was higher in shigella-exposed group compared with control group, but there was no difference in IBS incidence after 3 years. Shigella infection and previous functional bowel disorder history, female sex, and fever during admission were risk factors of IBS after 8 years follow up study. T2061 The Proinflammatory Cytokines Expression in Colon Can Be Induced by Chronic Variable Stress and Can Be Prevented by High Caloric Food in Rat Sang Kyun Noh, Yong Sung Kim, Moon Young Lee, Jung Taek Oh, Young Woo Sohn, Yong Leol Oh, Han Seung Ryu, Suck Chei Choi Backgrounds and Aims : Stress plays an important role in functional bowel disorder and inflammatory bowel disease. The purpose of this study was to evaluate the effect of stress on the hypothalamic-pituitary-adrenal (HPA) axis and colonic inflammatory cytokines according to stress mode and duration. We also want to evaluate the effect of high caloric (sweet) food on changes of behavior, HPA axis and cytokines induced by stress. Materials and Methods: Twenty-seven adult male rats, weighing about 290 g, were divided into 5 groups: control(CON), restraint stress with regular food (Res A), restraint stress with sweet food (Res B), chronic variable stress with regular food (CVS A), chronic variable stress with sweet food (CVS B). Before and after the experiment, all rats were evaluated and analyzed their exploring behavioral activity using activity monitor system. After 6 weeks of stress period, the weight of body adrenal gland,and the levels of the plasma ACTH and corticos- terone were measured. Plasma and colonic levels of pro- and anti-inflammatory cytokines (TGF-β, IL-2, IL-10 and interferon-γ) were measured by Western blotting assay. Results: Body weight gain was decreased in Res A, CVS A and CVS B group compared to control group. The amount of food consumption was lower in CVS A or CVS B group compared to other groups, however in terms of caloric intake, there was no difference between all groups. All stress groups showed significantly increase in the relative adrenal weight compared to the control group. Although there was no significant difference in the plasma corticosterone concentration between all groups, the levels of the plasma ACTH concentration in CVS A group was significantly decreased and therefore corticosterone/ACTH ratio was significantly higher than other groups. In open field test before the experiment, there were no activity differences between all groups. However, after the experiment, CVS B group showed signific- antly increase their exploring activity compared to CVS A and control group. In western blotting assays of plasma and colon, IL-2 and interferon-γ were significantly increased in CVS A group, but Res A, Res B and CVS B group did not show this change. There was no difference in TGF- β and IL-10 expression between all groups. Conclusions: Chronic non- adaptable stress, not simple adaptable stress, could induce the proinflammatory cytokines in plasma and colon as well as behavioral change. High caloric food ingestion could reduce the production of proinflammatory cytokines induced by the chronic non-adaptable stress. T2062 Clinical Evidence of Autonomic Dysfunction in Inflammatory Bowel Disease Jutta Keller, Viola Andresen, Peter H. Layer Background: The autonomic nervous system (ANS) is a critical regulator of gastrointestinal motility and much evidence now exists on cellular and molecular levels to suggest that the ANS also plays an important role in the development of inflammatory bowel disease (IBD). Clinical evidence of autonomic dysfunction and its role for regulation of motility in IBD is sparse. Aims: To investigate autonomic function in patients with Crohn's disease (CD) and ulcerative colitis (UC) compared with healthy volunteers and to associate it with gastric emptying (GE) parameters. Methods: 13 healthy subjects (CON), 12 CD and 10 UC patients underwent standardized tests of parasympathetic (Ewing test (ET): heart rate variation (ratio of 30th and 15th RR-interval) in response to tiliting) and sympathetic function (sustained handgrip test (SHGT): increase in diastolic blood pressure in response to sustained muscle contraction). Within 2 weeks all subjects also underwent a standardized 13C-octanoic acid gastric emptying breath test. Results: Patients with inflammatory bowel disease (IBD) were in remission or had mild to moderate disease activity (Crohn's disease activity index: 29- 252, Clinical Acitivity Index for UC: 5-13). Compared with healthy controls, mean diastolic blood pressure after 5 min of SHGT was significantly decreased in CD and UC patients AGA Abstracts
Transcript of T2059 Persistent Epithelial Barrier Alterations in a Rat Model of Post-Inflammatory Gut Dysfunction
can contribute to disruptedmotility in the inflamed colon and that dampening the hyperexcit-ability of AH neurons can restore colonic motor function. These findings support the conceptthat increases in sensory neuron activity (reflected in changes to AH cell excitability) in agiven region of the bowel can have a deleterious effect on motor function.
T2058
Colitis Alters the Function of Chromaffin Cells of the Adrenal MedullaMark K. Lukewich, Alan Lomax
Inflammatory bowel diseases (IBD) are characterized by chronic inflammation of the gastroin-testinal (GI) tract. Catecholamines, which are released synaptically released by the axons ofpostganglionic sympathetic neurons and systemically by adrenal chromaffin cells (ACC),modulate GI motility, secretion, blood flow and inflammation, and may therefore play animportant role in IBD. The present study tested the hypothesis that colitis impairs the releaseof catecholamines from ACC by examining ACC excitability and voltage-dependent Ca2+influx with patch clamp and intracellular Ca2+ imaging techniques. Colitis was induced inmale CD1 mice by 5% dextran sulfate sodium (DSS) in drinking water for 5 days followedby 2 days of normal drinking water. Controls were given normal drinking water for 7days. In other experiments, colitis was induced by the intracolonic injection of 4.5 mg oftrinitrobenzene sulfuonic acid (TNBS) in 40% ethanol, and saline injected mice were usedas controls. ACC were enzymatically dissociated from the adrenal medullae and isolatedcells were cultured overnight before recordings commenced. Population data were comparedusing unpaired t-tests, the Mann Whitney test or two-way ANOVA, as appropriate. Statisticalsignificance was reached when P < 0.05. The n value refers to the number of cells. RESULTSand CONCLUSIONS: The resting membrane potential of ACC isolated from DSS-treatedmice was significantly hyperpolarized compared to controls (DSS: -61.2 ± 1.8 mV, n = 32;Control: -50.7 ± 1.6 mV n = 24). Colitis significantly enhanced voltage-gated K+ current(DSS: n = 13 cells; Control: n = 30 cells). Importantly, voltage-gated Ca2+ current wassignificantly decreased in ACC from DSS-treated mice at membrane potentials between -25and +25 mV (DSS: n = 11 cells; Control: n = 16 cells). Ca2+ influx in response to KCl-induced depolarization was significantly reduced to 62.2% of the control response duringcolitis (DSS: n = 114 cells; Control n = 61 cells). Similar to our findings in the DSS model,Ca2+ influx in ACC from TNBS-treated mice was significantly reduced to 68.3% of thecontrol response (TNBS: n = 323 cells; Control: n = 330 cells). Our findings indicate thatACC become hyperpolarized and voltage-dependent Ca2+ influx is reduced during colitis.Given the important roles of cellular excitability and intracellular Ca2+ signaling in secretion,these alterations will likely result in decreased systemic catecholamine levels, which mayplay an important role in IBD. These data also provide further evidence that localizedinflammation of the GI tract during colitis can alter the physiology of distant tissues.
T2059
Persistent Epithelial Barrier Alterations in a Rat Model of Post-InflammatoryGut DysfunctionJoan Antoni Fernandez-Blanco, Vicente Martinez, Maite Martin, Patri Vergara
Introduction: Altered epithelial barrier function (EBF) and mucosal mast cells (MMCs)contribute to the initiation and perpetuation of defective intestinal immune responses as apathogenic basis for gastrointestinal inflammatory and functional disorders. Aim: To charac-terize alterations in the intestinal EBF in a model of post-inflammatory gut dysfunction inrats. Methods: Male SD rats were infected with Trichinella spiralis (TS) (7500 larvae/rat,PO). At the post-inflammatory phase (day 30±2 post-infection) animals were euthanizedand jejunal EBF assessed in Ussing chambers. Transepithelial voltage (Vte), short-circuitcurrent (SCC), conductance (G) and FITC-dextran (4 & 40 kD) flux were determined asmeasures of EBF. Responses to 5-HT (10-7-10-4M), substance P (SP, 3.3x10-8-10-6M), thePAR-2 agonist SL-NH2 (10-6-3x10-5M), and capsaicin (2x10-6M) were evaluated. Histologicalalterations and MMCs (immunohistochemistry for RMCPII) were also assessed. Results:During the post-inflammatory phase basal EBF was significantly altered (Table). 5-HT, SL-NH2 and SP induced concentration-dependent increases in SCC. In TS-infected rats, secretoryresponses to 5-HT [SCC increase (μA/cm2): TS-infected:12.7±2.8; Control:17.5±1.7; N=11-13; P<0.05] and SL-NH2 [SCC increase (μA/cm2): TS-infected: 28.2±3.3; Control: 55.3±8.6;N=8-12; P<0.05] were reduced, while responses to SP were unaffected [SCC increase (μA/cm2): TS-infected: 35.7±1.8; Control: 36.5±2.5; N=10 each]. Only responses to SP in TS-infected animals were affected by pretreatment with tetrodotoxin (TTX) (μA/cm2; TS-infected+TTX: 22.18±3.6 P<0.05 vs control+TTX: 34.0±4.0; N=10 each). Capsaicin aug-mented SCC similarly in both groups. No histological signs of active inflammation wereobserved; however, the number of MMCs was increased in TS-infected animals (39.1±2.3MMCs/villi P<0.0001 vs control: 13.9±0.9 MMCs/villi; N=7-9). Conclusions: TS infectionin rats results in persistent, post-infective intestinal barrier dysfunctions and mucosal masto-cytosis without signs of active inflammation. Hyporesponsiveness to 5-HT and SL-NH2 andaltered permeability suggest alterations of epithelial function likely associated to increasedrelease of MMC mediators. Decreased responses to SP after neuronal blockage suggests anenteric nervous system remodelling. These alterations might contribute to the epithelialbarrier dysfunctions observed in IBD and IBS patients.Basal EBF during the post-inflammtory phase
*: P<0.05 vs control
S-623 AGA Abstracts
T2060
The Clinical Course of Post-Infectious Irritable Bowel Syndrome: An Eight-Year Follow-up StudyHyun Chul Lim, Jie-Hyun Kim, Young Hoon Youn, Hyojin Park, Sang In Lee
Background and Aims: Bacterial gastroenteritis has been known as a risk factor of irritablebowel syndrome(IBS). The incidence of post-infectious IBS (PI-IBS) was known in the rangeof 7-31%, but few studies have long term follow up results. The aims of this study were toevaluate the clinical course, risk factor, and prognosis of PI IBS 8 years after shigella infection.Methods: We recruited 133 patients with shigellosis during its outbreak and 105 healthycontrols. We used a questionnaire to investigate their current bowel habits and otherfunctional bowel disorders (FBDs). The shigella-exposed group was consisted of hospitalemployees who experienced abdominal pain, diarrhea, or fever during the shigellosis outbreakand control group was consisted of age and sex matched hospital employees who corre-sponded with patient and were not infected with shigella. Results: Complete data wereobtained from 71 patients (53.4%) and 65 healthy controls (61.9%) and there was nosignificant demographic difference between both group. The prevalence of IBS in the shigella-exposed group and control group was 11(15.4%) and 6(9.2%) in 8 years after shigellainfection. The incidence of newly developed IBS in the shigella-exposed group and controlgroup was 4/71(5.6%) and 3/61(4.61%) in 8 years after shigella infection. There was nosignificant difference in incidence of IBS in 8 years after shigellosis. In shigella-exposedgroup, 2 out of 12 cases of PI-IBS showed persistent IBS after 8 years. The prevalence ofPI-IBS after 8 years in the previous FBDs groups was 26.7%, whereas the prevalence of PI-IBS without history of FBDs group was 10.7%(p<0.05). Previous FBDs history, female sex,and presence of fever at the time of infection were risk factors of IBS after 8 years followup study. Conclusions: Two in 12 patients (18.8%) of PI-IBS patients had persistent IBSover 8 year. . Overall prevalence of IBS was higher in shigella-exposed group comparedwith control group, but there was no difference in IBS incidence after 3 years. Shigellainfection and previous functional bowel disorder history, female sex, and fever duringadmission were risk factors of IBS after 8 years follow up study.
T2061
The Proinflammatory Cytokines Expression in Colon Can Be Induced byChronic Variable Stress and Can Be Prevented by High Caloric Food in RatSang Kyun Noh, Yong Sung Kim, Moon Young Lee, Jung Taek Oh, Young Woo Sohn,Yong Leol Oh, Han Seung Ryu, Suck Chei Choi
Backgrounds and Aims : Stress plays an important role in functional bowel disorder andinflammatory bowel disease. The purpose of this study was to evaluate the effect of stresson the hypothalamic-pituitary-adrenal (HPA) axis and colonic inflammatory cytokinesaccording to stress mode and duration. We also want to evaluate the effect of high caloric(sweet) food on changes of behavior, HPA axis and cytokines induced by stress. Materialsand Methods: Twenty-seven adult male rats, weighing about 290 g, were divided into 5groups: control(CON), restraint stress with regular food (Res A), restraint stress with sweetfood (Res B), chronic variable stress with regular food (CVS A), chronic variable stress withsweet food (CVS B). Before and after the experiment, all rats were evaluated and analyzedtheir exploring behavioral activity using activity monitor system. After 6 weeks of stressperiod, the weight of body adrenal gland,and the levels of the plasma ACTH and corticos-terone were measured. Plasma and colonic levels of pro- and anti-inflammatory cytokines(TGF-β, IL-2, IL-10 and interferon-γ) were measured by Western blotting assay. Results:Body weight gain was decreased in Res A, CVS A and CVS B group compared to controlgroup. The amount of food consumption was lower in CVS A or CVS B group comparedto other groups, however in terms of caloric intake, there was no difference between allgroups. All stress groups showed significantly increase in the relative adrenal weight comparedto the control group. Although there was no significant difference in the plasma corticosteroneconcentration between all groups, the levels of the plasma ACTH concentration in CVS Agroup was significantly decreased and therefore corticosterone/ACTH ratio was significantlyhigher than other groups. In open field test before the experiment, there were no activitydifferences between all groups. However, after the experiment, CVS B group showed signific-antly increase their exploring activity compared to CVS A and control group. In westernblotting assays of plasma and colon, IL-2 and interferon-γ were significantly increased inCVS A group, but Res A, Res B and CVS B group did not show this change. There was nodifference in TGF- β and IL-10 expression between all groups. Conclusions: Chronic non-adaptable stress, not simple adaptable stress, could induce the proinflammatory cytokinesin plasma and colon as well as behavioral change. High caloric food ingestion could reducethe production of proinflammatory cytokines induced by the chronic non-adaptable stress.
T2062
Clinical Evidence of Autonomic Dysfunction in Inflammatory Bowel DiseaseJutta Keller, Viola Andresen, Peter H. Layer
Background: The autonomic nervous system (ANS) is a critical regulator of gastrointestinalmotility and much evidence now exists on cellular and molecular levels to suggest that theANS also plays an important role in the development of inflammatory bowel disease (IBD).Clinical evidence of autonomic dysfunction and its role for regulation of motility in IBD issparse. Aims: To investigate autonomic function in patients with Crohn's disease (CD) andulcerative colitis (UC) compared with healthy volunteers and to associate it with gastricemptying (GE) parameters. Methods: 13 healthy subjects (CON), 12 CD and 10 UC patientsunderwent standardized tests of parasympathetic (Ewing test (ET): heart rate variation (ratioof 30th and 15th RR-interval) in response to tiliting) and sympathetic function (sustainedhandgrip test (SHGT): increase in diastolic blood pressure in response to sustained musclecontraction). Within 2 weeks all subjects also underwent a standardized 13C-octanoic acidgastric emptying breath test. Results: Patients with inflammatory bowel disease (IBD) werein remission or had mild to moderate disease activity (Crohn's disease activity index: 29-252, Clinical Acitivity Index for UC: 5-13). Compared with healthy controls, mean diastolicblood pressure after 5 min of SHGT was significantly decreased in CD and UC patients
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