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P028. Establishment of a short time rat model of mucosal inflammation by in situ perfusion of an intestinal segment

A. Breitrueck, G. Sparmann, J. Emmrich

University of Rostock, Department of internal Medicine, Division of Gastroenterology, Rostock, Germany

Introduction: There is limited information about the first phase of mucosal reactions in inflammatory bowel diseases (IBD). We have established a short-time rat model using the in situ perfusion of a segment of the small intestine to show early alterations. Furthermore, this model was used to evaluate the therapeutical potency of probiotics during the initial phase of inflammation.

Material and Methods: Laparatomy of male Lewis rats (160–180 g) was performed under pentobarbital anaesthesia (50 mg/kg body weight). A jejunum segment (6 cm) with intact vascular system was isolated and cannulated proximally and distally. The proximal cannula was connected with a peristaltic pump. Experimental inflammation was induced by perfusion of the segment with 1% iodoacetamide (IA, 15'). Subsequently, the segment of the therapy group was perfused with the probiotic Mutaflor® (3x10E8 CFU) or Dexamethasone (1 mg/kg body weight). The treatment was followed by 2 hours DMEM perfusion (0.1 ml/min). Untreated segments obtained from the same animal were used as controls. At the end of the experiment, tissue samples were removed and prepared for histological, immunohistochemical (detection of infiltrating CD4, CD8, CD68, TCRα/β and TCRγ/δ positive cells) as well as RNA expression analyses. Transcript levels of cytokines (IL-6, IL-10, IL-1β, MCP-1) and the intestinal alkaline phosphatase (iAP) were investigated by reverse transcription PCR. In addition, enzymatic activity of AP was visualized histochemically using the phosphatase substrate BCIP/NBT (BM Purple).

Results: The histological examination revealed marked mucosal lesions caused by iodoacetamide. The morphology of control segments was not affected. The inflamed tissue was characterized by an increased infiltration of CD4, CD8, CD68 and TCRα/β positive cells, whereas TCRγ/δ positive cells were decreased. Furthermore, PCR results showed 2 to 6 fold higher mRNA-expression of the inflammatory cytokines IL-6, IL-1β, IL-10 and MCP-1 in the IA-treated segment compared to the control. Interestingly, iAP mRNA expression and consequently, the enzymatic phosphatase activity was dramatically reduced in the inflamed jejunum.

The perfusion with Mutaflor or Dexamethasone reduced the mucosal inflammatory reactions, shown by decreased leukocyte infiltrations and lower RNA-expression levels of cytokines in the treated segment. Furthermore, the decline of the iAP expression in the injured tissue was attenuated by both therapeutical substances.

Conclusions: We established a short-time rat model of early mucosal inflammation. The local in situ perfusion with iodoacetamide leads to epithelial cell lesions, the infiltration of leukocytes, an increased expression of proinflammatory cytokines and a decrease of the iAP transcript level. In addition, our model was shown to be suitable for studies regarding short-time effects of novel therapeutical concepts.