Ischemic preconditioning protects against cold ischemic injury through an oxidative stress dependent mechanism

Abstract

Background/Aims: Ischemic injury in cold preserved livers is characterized by sinusoidal endothelial cell (SEC) detachment and matrix metalloproteinase activity. Upon reperfusion reversible ischemic injury becomes permanent with SEC rapidly undergoing apoptosis. Ischemic preconditioning prevents reperfusion injury after normothermic ischemia. We hypothesized that ischemic preconditioning, through an oxygen free radical burst, protects against injury during cold preservation and reperfusion.

Methods: Ischemic preconditioning was achieved in rats by clamping blood supply to the left and median lobes for 10 min followed by 15 min of reperfusion prior to preservation in cold University of Wisconsin solution for 30 h. In a second set of experiments, rats were pretreated with N-acetyl-cysteine (NAC). SEC apoptosis upon reperfusion was assessed in an isolated perfused rat liver (IPRL) model.

Results: SEC detachment and activities of matrix metalloproteinase were significantly reduced in preconditioned livers. A decrease of SEC apoptosis after 1 h of reperfusion in the IPRL was noted in preconditioned livers compared to controls. Pretreatment with NAC reversed the beneficial effects of ischemic preconditioning on SEC detachment and apoptosis.

Conclusions: Ischemic preconditioning is an effective strategy to prevent injury during cold preservation and after reperfusion. The protective effect is possibly mediated by oxygen free radicals.

Introduction

Ischemic preconditioning, a short period of ischemia and reperfusion prior to a prolonged ischemic insult, is a novel protective approach [1], [2], [3]. Preconditioning effectively prevents reperfusion injury in the normothermic ischemic liver [2], and reverses organ injuries incompatible with animal survival to non-lethal injury [3]. The mechanisms of protection include prevention of apoptosis of the sinusoidal endothelial cells (SECs) and hepatocytes with down-regulation of the caspase pathway [3]. We have recently shown similar protection in patients undergoing hepatic resections performed under inflow occlusion (Pringle maneuver) [2].

A particular form of injury inherent to transplantation is cold ischemia. This type of injury typically causes rounding and detachment of the SEC [4], [5], perhaps due to actin disassembly and activation of matrix metalloproteinases (MMPs) [6], [7], [8], [9]. While SEC remain alive during prolonged periods of cold ischemia, they undergo rapid apoptosis after oxygenated reperfusion [10]. The degree of apoptosis correlates with graft injury, and inhibition of apoptosis is highly protective [10], [11], [12], [13], [14]. Whether preconditioning confers protection in the cold preserved liver is unknown [15], [16].

Data on triggering mechanisms of hepatoprotection by ischemic preconditioning remain scarce. A short burst of oxygen radicals was found to protect endothelial cells in culture against the effects of pro-inflammatory cytokines suggesting that short sublethal oxidative stress may trigger adaptive responses in endothelial cells to subsequent injury [17]. Therefore, we hypothesized in this study that ischemic preconditioning protects SEC in the cold preserved liver, and that protection is mediated by a sublethal burst of oxidative stress.