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Background: The small intestine is one of the most sensitive organs to ischemia. L-arginine has been shown to reduce damage from ischemia and reperfusion injury. We hypothesize that explanted intestinal segments from rats will demonstrate reduced susceptibility to ischemic injury when perfused with l-arginine. Methods: 45 small intestinal segments were harvested from male Sprague-Dawley rats and connected to an ex vivo intestinal perfusion device. Ischemic damage was induced by perfusing the extraluminal side with Ringer-HEPES buffer saturated with 100% N2. All segments were then perfused intraluminally with and without l-arginine. We conducted a set of experiments with intraluminal perfusion with both l-arginine and N-nitroarginine methyl ester (L-NAME), an inhibitor of the nitric oxide – arginine pathway. Control segments were perfused extraluminally under non-ischemic conditions and intraluminally with and without l-arginine. The intraluminal perfusate contained FITC-inulin, and the fluorescence signal of FITC-inulin was measured to calculate average fluid secretion, which directly corresponds to the extent of ischemic injury. Results: Intestinal segments perfused with l-arginine had significantly decreased secretion over time in comparison to intestinal segments perfused without l-arginine (p<0.0001). Perfusion with L-NAME abrogated the protective effect of l-arginine. Conclusion: Intraluminal perfusion with l-arginine reduced ischemic damage to harvested intestine.