Title page for ETD etd-06222008-193126


Type of Document Master's Thesis
Author Nagy, Amy Dae
URN etd-06222008-193126
Title Apoptosis in the equine small intestine following experimental ischemia-reperfusion injury
Degree Master of Science
Department Large Animal Clinical Sciences
Advisory Committee
Advisor Name Title
White, Nathaniel A. II Committee Chair
Blikslager, Anthony Committee Member
Crisman, Mark Virgil Committee Member
McCutcheon, L. Jill Committee Member
Tschetter, Jolynne Committee Member
Keywords
  • reperfusion
  • ischemia
  • apoptosis
  • equine
Date of Defense 2008-05-23
Availability restricted
Abstract
This study was aimed at characterizing the apoptotic response equine small intestine subjected to experimental ischemia-reperfusion injury and determining if use of an angiotensin converting enzyme inhibitor (enalaprilat) would ameliorate the apoptotic response. It was designed to determine if mucosal epithelial cells undergo apoptosis during the ischemia phase and also examined if apoptosis is significantly exacerbated by reperfusion. It also investigated whether administration of enalaprilat decreased reperfusion injury secondary to reduced enterocyte apoptosis. Injury was induced using a low flow model of I-R. During celiotomy a single loop of jejunum was isolated and arterial flow decreased to 20% of baseline for one hour and complete occlusion for a second hour. Reperfusion was monitored for 3 hours. A control group (n=6) were not treated while the treatment group (n=6) received 0.5 mg/kg enalaprilat in 0.9% NaCl immediately following ischemia. Jejunal samples were taken prior to the induction of ischemia, immediately post-ischemia and at 1, 2 and 3 hours of reperfusion. Samples were evaluated for gross tissue pathology with standard hematoxylin and eosin staining, the presence of apoptotic cells via TUNEL staining, and gene expression of three apoptosis related genes (bax, bcl-2, p53) using qPCR. Serum enalaprilat and ACE concentrations were determined from blood samples drawn concurrent with jejunal sampling using HPLC/MS and standard HPLC. Plasma enalaprilat concentrations were comparable to previous reports in awake horses. Enalaprilat appeared to have no effect on serum ACE concentrations, however a significant spike in ACE concentration occurred in the treatment group at 1 hour of reperfusion (P=0.0001). Grade of mucosal damage was not significantly different between control and treatment groups at any time point. Subjectively apoptotic index appeared to be higher in the treatment group at end ischemia and during reperfusion. There were no changes in expression of p53 or bcl-2 in either group. Bax expression was significantly decreased (P= 0.02) in the control group at 2 hours of reperfusion. Based on our data administration of an ACE inhibitor during anesthesia in horses with an ischemic segment of intestine confers no protective benefit and may be associated with increased intestinal injury and apoptosis. Lack of expression of p53, bax and bcl-2 suggests another apoptotic mechanism in equine ischemic intestine.
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