Nitric oxide metabolism in ischaemic preconditioning of the liver

Ischaemic preconditioning is a term used for the reduction of ischaemic damage to the liver by a transient period of ischaemia and reperfusion. This thesis evaluates the hypothesis that ischaemic preconditioning may protect against ischaemia reperfusion injury of the liver via nitric oxide formation. Two study models were used. In the first model, lobar ischaemia reperfusion injury in the rat was used to investigate the relationship of nitric oxide metabolism with hepatic oxygenation, microcirculation, and function with ischaemic preconditioning of the liver. As the fatty liver is less tolerant of ischaemic injury the second study model has evaluated ischaemic preconditioning in a steatotic liver model. In the first study model, Sprague Dawley rats were subjected to 45 mins lobar ischaemia followed by 2 hr reperfusion. Ischaemic preconditioning was performed with 5 min lobar ischaemia and 10 min reperfusion before the sustained ischaemia. L-arginine or Nω-nitro-L-arginine methyl ester (L-NAME) was administered to stimulate or block nitric oxide synthesis. Ischaemic preconditioning resulted in significantly increased hepatic intracellular oxygenation, microcirculation and hepatic tissue ATP, and decreased hepatocellular injury. Preconditioning significantly increased nitric oxide production measured by plasma nitrite/nitrate and cGMP. L-arginine treatment reproduced the protective effect of ischaemic preconditioning. Nitric oxide inhibition with L-NAME antagonized the protective effect of ischaemic preconditioning. Nitric oxide synthase detected by NADPH diaphorase was induced by ischaemic preconditioning. Immunohistochemistry and Western blotting showed this to be due to overexpression of the constitutive isoform eNOS rather than the inducible isoform iNOS. The second study involved model of hepatic steatosis in rats induced by a high fat diet. Liver histology in these animals showed moderate grade macrovesicular steatosis. The same protocol for ischaemic preconditioning resulted in increased intracellular oxygenation, microcirculation and ATP, and decreased hepatocellular injury. These results were similar to those observed with preconditioning in the normal livers. This thesis has confirmed a protective effect of hepatic ischaemic preconditioning. This is associated with increased eNOS derived nitric oxide production. Furthermore, the protective effect of ischaemic preconditioning can be applied to the steatotic liver. These data may have important implications in liver surgery and transplantation and may lead to the development of pharmacological strategies for protecting the liver from ischaemic injury