Tu1236 Participation of the Bile Acid in Low-Dose Aspirin-Induced Small Intestinal Mucosal Injury

barrier function in equine jejunum. In contrast, barrier integrity, mucosal repair, and the inflammatory response were not affected by lidocaine in the ischemic injured colon in horses.

AGA Abstracts

not different between WT and SERT KO rats at 24 and 72 hours; however, wound healing was impaired in the SERT KO rats at 120 hours as seen by a larger wound area (0.138 ± 0.053 vs 0.384 ± 0.086 mm2, P < 0.05). Higher 5-HT levels were found along the wound edge compared to normal mucosa (627 ± 228 vs 318 ± 143 nM, P < 0.0005). Conclusions: Decreased 5-HT uptake with increased extracellular 5-HT levels are associated with decreased mucosal growth and impaired wound healing. Focal damage to the mucosa results in elevated 5-HT along the wound edge. These results suggest an important role of 5-HT in regulating responses to damage and maintaining intestinal homeostasis. (Supported by the Division of Neonatology at Michigan State University and by an AAP Resident Research Grant to MP). Tu1236 Participation of the Bile Acid in Low-Dose Aspirin-Induced Small Intestinal Mucosal Injury Yukiko Uehara, Yuji Naito, Osamu Handa, Mayuko Morita, Katsura Mizushima, Tetsuya Okayama, Naohisa Yoshida, Kazuhiro Kamada, Kazuhiro Katada, Kazuhiko Uchiyama, Takeshi Ishikawa, Tomohisa Takagi, Hideyuki Konishi, Nobuaki Yagi, Yoshito Itoh, Toshikazu Yoshikawa [Background] Acethyl-salicylic acid (ASA) has been widely used for the secondary prevention of cerebro- and cardio-vascular events, based on its anti-platelet effect. Advances in enteroscopy techniques such as capsule endoscopy and balloon-assisted endoscopy now allow for the assessment of small intestinal mucosal injury. Using these technologies, it has been shown that ASA-induced small intestinal mucosal injury is quite common. Recently, it has been reported that indomethacin may form a complex with bile acids within the small intestinal epithelial cell lipid bilayer, and cause destruction of the cell membrane. However, the mechanism by which ASA-induced small intestinal mucosal injury and the role of bile acids in inducing this injury is not clear yet. We hypothesized that ASA along with bile acids induces apoptosis in small intestinal epithelial cells, and we used a rat small intestinal epithelial cell line (RIE1) to explore this hypothesis. [Materials and Methods] ASA (in doses ranging from 2.5 to 40mM) and/or the bile acid, tauro-deoxycholic acid (TDCA; in doses ranging from 0.25 to 2mM) were added to RIE1 and we assessed cell death using the MTT assay and LDH release into the cell culture medium. To further confirm the type of cell death, we qualified cell apoptosis by a double staining method with Hoechest 33342 and propidium iodide (PI) under fluorescence microscopy and we also quantified it by flow cytometry with Annexin V- FITC and PI. Oxidative stress was also evaluated by redoxsensitive fluorogenic probes under the fluorescent microscopy with time-lapse system. [Result] TDCA (0.25mM) alone or ASA (10mM) alone did not significantly increase cell death. When low concentrations of TDCA and ASA were added together to RIE1, cell viability was significantly decreased without increasing LDH release. RIE1 cells treated with low concentrations of both TDCA and ASA exhibited chromatin condensation and membrane blebbing, characteristics of early stage apoptosis as assessed by fluorescence microscopy, and flow cytometry study also showed increased apoptotic cells in the same setting. Antioxidant treatment reduced oxidative stress and apoptotic cell death induced by low concentrations of both ASA and TDCA. [Conclusion] In small intestinal epithelial cells, low concentrations of TDCA alone or ASA alone do not decrease cell viability. In contrast, the combination of low doses of TDCA and ASA significantly increase apoptosis, an event that can be prevented by antioxidant treatment. These findings suggest that TDCA might augment the cytotoxic effect of ASA on small intestinal epithelial cell injury and that agents with anti-oxidant properties might be useful to prevent ASA-induced small intestinal mucosal injury.

Tu1238 Fibrotic Human Precision-Cut Intestinal Slices: A New Ex Vivo Model for Established Intestinal Fibrosis Wouter T. van Haaften, Bao Tung Pham, Dorenda Oosterhuis, Hendrik S. Hofker, Gerard Dijkstra, Peter Olinga

Tu1237 Effects of a Continuous Rate Infusion of Lidocaine on Ischemic Injury and Epithelial Barrier Integrity, Hematological Changes, and Inflammation in Equine Small and Large Intestine Astrid Grosche, Alison J. Morton, A. Sarah Graham, Anje G. Bauck, Maristela D. SeudoLopes, David E. Freeman

Introduction - Intestinal fibrosis (IF) is a major complication that can occur in inflammatory bowel disease, after radiation therapy, or post transplantation. Until now, no antifibrotic therapies and no human model for intestinal fibrosis exist. Previously, we have shown that precision-cut (human) intestinal slices from healthy jejunum are viable up to 48 hours in culture, and can be used to study the early onset of fibrogenesis and to test the efficacy of antfibrotic drugs. However in Crohn's disease (CD), fibrosis is often already apparent before it is clinically discovered. Therefore, the effect of antifibrotic drugs on established fibrosis should be determined. The aim of this study was to evaluate if precision-cut intestinal slices from fibrotic tissue are viable during culture. Methods - Precision-cut intestinal slices (PCIS) were prepared from human fibrotic intestinal tissue obtained from resections due to stenosis of the bowel in CD patients. Slices were incubated up to 24, 48 and 72 hrs. To evaluate viability of PCIS, the ATP content was assessed. Total RNA was isolated and the gene expression of different fibrosis markers: α-smooth muscle actin (αSMA), pro-collagen1a1 (pro-col1a1), heat shock protein 47 (HSP47), synaptophysin (SYN) and fibronectin (FN2) was determined. Results - Up to 48 hours, the ATP content of the slices decreased 40% during incubation compared to directly after slicing. After 72 hours the viability was decreased by 70%. A significant decrease in αSMA gene expression (6 fold) in fibrotic PCIS (fPCIS) cultured up to 72h was found compared to freshly prepared fPCIS. The gene expression of SYN was not changed during incubation up to 72h. The extracellular matrix genes Procol1a1 and FN2 were significantly down-regulated (0.5 and 0.3 fold, respectively) after 24h of incubation compared to fPCIS directly after slicing, but after 48h and 72h of incubation these genes were not different from freshly prepared fPCIS. The gene expression of the collagen chaperone HSP47 tended to be increased during culture of fPCIS up to 72h of incubation. Conclusion - Fibrotic PCIS are successfully cultured up to 48 hours. Gene expression of most fibrosis markers are constant up to 48 hours of culture. Therefore, this ex vivo model for established human intestinal fibrosis may be an ideal model to test the efficacy of antifibrotic compounds in the intestine.

BACKGROUND: Ischemia is a major feature of intestinal strangulation in horses, and ischemic injury could be exacerbated by infiltration of neutrophils during reperfusion. Lidocaine is widely used in equine gastrointestinal surgery for its putative prokinetic properties. However, the apparent clinical benefit of intraoperative lidocaine in horses is currently attributed to novel antiinflammatory effects on neutrophils. AIM: To examine effects of a continuous rate infusion of lidocaine on transmural inflammation, mucosal barrier function, and hematological changes after ischemia and reperfusion in equine jejunum and colon. METHODS: In anesthetized horses, ischemia was induced in two contiguous segments of jejunum (30min; n=6) and colon (1h; n=11). By random assignment, one group received lidocaine and one group received saline throughout anesthesia. After ischemia, one segment was removed from each organ to examine the effects of ischemia on transepithelial resistance and mannitol flux in Ussing chambers. This was repeated in the remaining segments after 4h reperfusion. Full thickness biopsies were taken before and after ischemia, and after 1h and 4h reperfusion for assessment of neutrophil influx and morphometric changes. Jugular (JuB), and jejunal (JeB) and colonic venous blood (CB) from the injured segments were sampled simultaneously for hematological analysis. RESULTS: Colonic mucosa developed mild morphometric changes after ischemia associated with impaired barrier function in vitro in both treatment groups. Lidocaine did not affect these changes (Fig 1), and the ischemic injured mucosa recovered despite a massive influx of neutrophils into mucosa, muscle layers, and serosa during reperfusion irrespective of treatment. The mucosal damage was more profound in the jejunum after 30min ischemia accompanied by severe epithelial barrier dysfunction. Jejunal barrier integrity deteriorated after reperfusion in the saline group, but improved with lidocaine after ischemia and reperfusion (Fig 1). Lidocaine tended to decrease neutrophil influx into jejunal mucosa during reperfusion, but did not affect neutrophil accumulation in muscle and serosa (Fig 2). Ischemic metabolic changes (lactic acidosis, hyperkalemia, hyponatremia and hypoglycemia) were more profound in CB. Lidocaine caused hyperglycemia in JuB, JeB and CB before ischemia (130-137mg/dl), with less pronounced hypoglycemia after ischemia in JeB and CB. All laboratory changes returned to normal after reperfusion in both treatment groups. CONCLUSIONS: Lidocaine may have a beneficial effect on mucosal recovery and

S-791

AGA Abstracts