Role of NADPH oxidase-derived superoxide in reduced size liver ischemia and reperfusion injury

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Abstract

Hepatic resection with concomitant periods of ischemia and reperfusion (I/R) is required to perform reduced size liver transplantation such as split liver or liver donor transplantation. Although great progress has been made using these types of surgeries, there remains substantial risk to both donors and recipients, with a significant number of patients developing liver injury and failure. The objective of this study was to assess the roles of superoxide (O2) and tumor necrosis factor-α (TNF-α) in the pathophysiology of a mouse model of reduced size liver combined with ischemia and reperfusion (RSL + I/R). We found that all male mice subjected to RSL + I/R died within 3–5 days following surgery. Mortality was always preceded by dramatic increases in liver injury and TNF-α expression in the absence of neutrophil infiltration. Using a long-lived, polycationic form of human manganese superoxide dismutase (pcMnSOD), NADPH oxidase-deficient mice (gp91−/−) or a monoclonal antibody directed against mouse TNF-α, we demonstrated that hepatocellular injury (and mortality) were significantly attenuated. In addition, we found that pcMnSOD administration or NADPH deficiency reduced expression of TNF-α. Taken together, our data suggest that NADPH oxidase-derived O2 plays an important role in the pathophysiology of RSL + I/R-induced liver injury via its ability to enhance expression of TNF-α. We propose that therapies directed toward scavenging of O2, inhibiting NADPH oxidase, and/or immuno-neutralizing TNF-α may prove useful in limiting the liver injury induced by surgical procedures that require resection and I/R such as split liver or living donor liver transplantation.

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Animals

Wild type (wt) male C57Bl/6 mice (18–24 g) were purchased from Harlan while mice genetically deficient in the gp91phox sub-unit of NADPH oxidase (gp91−/−) on a C57B/6 background were obtained from Jackson Laboratories (Bar Harbor, ME) and generated as described previously [24]. All animals were maintained on a standard laboratory diet with free access to food and water until the time of the experiment.

Animal model of reduced size liver ischemia–reperfusion

Fasted (16–18 h) mice were anesthetized with intramuscular ketamine (150 mg/kg) and xylazine (7.5 

Animal survival, tissue injury, and TNF-α expression following RSL + I/R

In a first series of studies, we wished to define what role superoxide (O2) and TNF-α play in a model of RSL + I/R. Fig. 1 presents the 7 day mortality percentages of wt mice subjected to RLS + I/R or sham surgery. We found that all (100%) mice died within 3–5 days following the surgery as we have previously described [25], [26]. This increased mortality was preceded by substantial and significant increases in liver injury as measured by increased serum ALT levels at 3 h post surgery (Fig. 1).

Acknowledgements

Some of this work was supported by a grant from the NIH (DK43875).

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