Sub-lethal oxidative stress triggers the protective effects of ischemic preconditioning in the mouse liver
Introduction
Much effort has focused over the past decade on novel protective strategies against ischemic injury in the liver [1], [2]. Ischemic preconditioning, i.e. a short cycle of ischemia and reperfusion prior to a prolonged ischemic insult, has emerged as an important strategy with protection demonstrated in various cold [3] and warm [4], [5] ischemia models, and recently in patients undergoing major liver resection under inflow occlusion (Pringle maneuver) [6]. While a number of protective mediators have been identified (e.g. adenosine [7], nitric oxide [7] or various kinases [8], [9]), the triggering mechanism of protection has remained unknown.
The protective effects of ischemic preconditioning could be mediated through two possible mechanisms. First, the short period of ischemia might directly interfere with the pathways leading to parenchymal injury. For example, Peralta et al. [7] have shown that ischemic preconditioning activates the enzyme nitric oxide synthase resulting in the production of nitric oxide, which binds and inactivates caspases [10]. Caspase inhibition prevents apoptosis of liver cells [11], [12]. A second possibility is that ischemic preconditioning confers sub-lethal stress to the liver leading to an ’adaptation’ including the development or enhancement of natural defense mechanisms.
Short intervals of ischemia as applied during ischemic preconditioning induce various types of stress such as the release of a variety of cytokines [13], decrease in energy levels [14], or the generation of oxidative stress [3]. While these changes can cause irreversible cell damage under many situations, they may trigger ’protective–adaptive’ mechanisms when applied moderately. For example, while a high concentration of oxidative stress causes irreversible tissue injury through peroxidation of membrane lipids [15] and proteins and DNA damage [16], low stress-intensity may induce self defense mechanisms [17], [18].
In this study, we hypothesize that protection of ischemic preconditioning occurs through a novel pathway involving oxidative stress. We show that a mild burst of oxidative stress during ischemic preconditioning is critical for the protective effect against long ischemic intervals.
Section snippets
Animals
Male wild-type mice (C57BL6, Harlan, Netherlands) were used in all experiments. Animals were fed on a laboratory diet with water and food ad libitum until use and were kept under constant environmental conditions with 12-h light–dark cycles. All procedures were performed in accordance with the institutional animal care guidelines.
Partial hepatic ischemia
A non-lethal model of segmental (70%) hepatic ischemia was used as previously described [19]. All animals were pretreated with either a dose of NAC (100 mg/kg, i.p.) 3
Is oxidative stress generated during the various intervals of ischemia and reperfusion?
The concentration of hydroperoxides was measured in tissue homogenates during the time course of ischemic preconditioning and the subsequent prolonged period of ischemia. A molecular probe (10-acetyl-3,7-dihydroxyphenoxazine) was used, which becomes fluorescent in presence of peroxides. First, we measured peroxide levels in liver tissue in sham-operated animals. Very low concentrations of peroxides were detected under these baseline conditions (2.2±0.3 μmol/mg protein). Next, we evaluated
Discussion
In this study, we have provided evidence that the generation of a sub-lethal oxidative stress after preconditioning of the liver with a short period of ischemia triggers protective mechanisms resulting in better tolerance to a subsequent prolonged period of ischemia. This novel pathway was substantiated by three main findings. First, a mild oxidative stress, not causing detectable injury such as lipid peroxidation, was consistently detected upon reperfusion of liver rendered ischemic for 10
Acknowledgements
Supported by grants from the NIH (DK54048-01A1) and to the Swiss National Science Foundation (SNF3200-061411) to PAC. HAR is supported by grants from the Roche Research Foundation Switzerland and the Olga Mayenfisch Foundation, Switzerland.
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