In Vivo Investigations Using Microdialysis Sampling

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Inflammatory bowel disease (IBD) is a chronic inflammation of the GI tract that includes Crohn's disease and ulcerative colitis. The major enzymes involved in mediating the inflammatory response in IBD are cyclooxygenase (COX), lipoxygenase (LOX) and nitric oxide synthase (NOS). The exact mechanisms by which these enzymes are induced and inflammation is caused are still not fully understood. The objective of this proposal is to develop in vivo microdialysis sampling in the colonic mucosa to be used in conjunction with microanalytical techniques to monitor the activity of these three enzyme systems in an animal model of IBD. Three analytical methods will be developed as part of this project. First, the COX products prostaglandin E2 and thromboxane B2 and the LOX products the hydroxyeicosatetraenoic acids, 5-HETE and 15-HETE, and leukotriene B4 will be determined by CE-LIF after derivitization with either 9anthryldiazomethane (ADAM) or 2-(2, 3-naphthalimino) ethyltrifluoromethanesulphonate (NE-OTf). Second, the NOS and arginase products citrulline and ornithine will be determined using CE-LIF after derivatization by naphthalene-2,3-dicarboxaldehyde/cyanide (NDA/CN). Third, nitrate and nitrite will be used to monitor NOS activity and determined directly by CE-UV or by CE-LIF following derivatization with 2,3�diaminoaphtalene (DAN). The animal model of IBD to be used will be by chemical induction using 2,4,6-trinitrobenzene sulfonic acid in the rat. Implementation of this model will involve development of microdialysis probe implantation protocols, evaluation of any tissue response due to probe implantation, and verification that the probe is located in the inflamed region. Microdialysis sampling and the developed analytical methods will then be used to continuously monitor the enzyme activity in the model as the animal develops IBD and in control animals.