Hirohisa Harada

Sexual dimorphism in reduced-size liver ischemia and reperfusion injury in mice: Role of endothelial cell nitric oxide synthase

We have recently reported that female mice are protected to a much greater extent from the injurious effects of reduced-size liver ischemia and reperfusion (RSL+I/R) than are males by an estrogen-dependent mechanism. The objective of this study was to examine the possibility that the protective effect observed in female mice depends on the up-regulation and/or activation of endothelial cell NO synthase (eNOS). Anesthetized female and male wild-type or eNOS-deficient C57BL/6 mice were subjected to 70% liver ischemia for 45 min followed by resection of the remaining 30% nonischemic lobes and reperfusion of ischemic tissue. Survival was monitored daily, whereas liver injury was quantified by using serum alanine aminotransferase determinations and histopathology. Hepatic eNOS mRNA, protein, and enzymatic activity were determined in male and female mice subjected to RSL+I/R. We found that liver injury was reduced and survival increased in female mice compared with males. This protective effect correlated with significant increases in hepatic eNOS message levels and enzyme activity but not protein expression compared with males subjected to the surgery. Furthermore, Nω-nitro-l-arginine methyl ester-treated or eNOS-deficient female mice responded to RSL+I/R with dramatic increases in liver injury and 100% mortality within 2 days of surgery. Finally, we found that pravastatin pretreatment significantly attenuated hepatocellular injury and increased survival of male mice, which was associated with enhanced expression of eNOS message. We conclude that the protective effect afforded female mice is due to the activation of hepatic eNOS activity and enhanced NO production.

Estrogen Receptor-α, Sexual Dimorphism and Reduced-Size Liver Ischemia and Reperfusion Injury in Mice

Estrogen (E2) exerts its effect on target organs principally by interacting with specific estrogen receptors (ER) such as ER-α or ER-β. The role that these E2 receptors play in mediating the protective effects observed in RSL+I/R induced injury remains to be defined. To study the role of ER-α, we anesthetized female and male wild type (wt; C57Bl/6) and ER-α-deficient (αERKO) mice and subjected them to 70% liver ischemia for 45 min followed by resection of the remaining 30% nonischemic lobes and reperfusion of the ischemic tissue. For some experiments, wt and αERKO male mice were injected with E2. Survival was monitored on a daily basis while liver injury was assessed by quantifying serum alanine aminotransferase (ALT) levels and histopathology. Hepatic eNOS mRNA levels were evaluated using semi-quantitative RT-PCR. Our data showed that untreated females or males treated with E2 survived RSL+I/R surgery indefinitely whereas all male mice given vehicle died within 3-5 days following surgery. This protective effect was diminished in αERKO female mice such that only 40% of αERKO females survived 7 d following RSL+I/R. Furthermore, liver injury was significantly higher in αERKO females compared with their wt counterparts and similar to those seen in wild type males and αERKO males. The protective effect observed in wild type females or E2 treated males correlated well with increases in hepatic eNOS message whereas both male and female αERKO mice exhibited significantly lower levels of eNOS mRNA. We conclude that this protection may in part be due to the E2/ER-α-mediated activation of eNOS.

Superoxide and Post-Ischemic Liver Injury: Potential Therapeutic Target for Liver Transplantation

Cessation of blood flow to the liver is required during liver transplantation and resectional surgery. A growing body of experimental evidence suggests that restoration of blood flow to the ischemic liver initiates hepatocellular injury which may lead, in some cases, to severe liver injury and graft failure. A large number of studies have implicated reactive oxygen species as potential mediators of post-ischemic tissue injury. Recent developments in genetic engineering as well as chemical modeling, have allowed for the production of novel free radical scavengers including mutated forms of superoxide dismutase (SOD) and low molecular weight SOD mimics with extended circulating half-lives and/or significant membrane permeabilitys. Application of these newly developed free radical scavengers show promising results in animal models of liver I/R and may become powerful tools in the treatment of post-ischemic liver injury that occurs in liver transplantation.