Evidence for resveratrol-induced preservation of brain mitochondria functions after hypoxia-reoxygenation

We have previously shown, as have other authors, that trans-resveratrol (E-resveratrol, 3,4,5-trihydroxy-E-stilbene) reduces reactive oxygen species (ROS) generation of mitochondria freshly isolated from healthy rat brains and that it also counteracts the effect of uncouplers (CCCP) on mitochondrial respiration and oxidative phosphorylation. Two main mechanisms have been shown: firstly, a scavenger effect toward O2- and secondly inhibition of complex III ROS generation. We now report on the effects of resveratrol in a pathological model that mimics the ischemia followed by the reperfusion process which may occur in the human brain. Isolated brain mitochondria were submitted first to hypoxia then to reoxygenation. The aim of this study was to determine the extent of mitochondrial damage induced by this experimental model, to demonstrate which mitochondrial functions were altered and to quantify the extent to which they were prevented by resveratrol. Resveratrol was either added to mitochondria freshly isolated from healthy rat brains or was injected by subcutaneous chronically implanted pumps (0.5, 2 and 10 mg/kg/day for 7 days). The rats were then sacrificed and mitochondria were extracted from brains. To evaluate the respective effects of hypoxia and reoxygenation on mitochondrial functions and the relevant effects of resveratrol, this drug was added (first protocol) either before the complete process (i.e., hypoxia and reoxygenation), or after anoxia before reoxygenation. We found that resveratrol prevented alterations of mitochondrial functions. This substance partly counteracted the decrease in respiratory control and the increase in ROS generation. It fully inhibited the alteration of membrane fluidity and the mitochondrial step of the apoptotic process (evidenced by cytochrome c release and membrane potential collapse). The effects of resveratrol were concentration-dependent (in vitro) or dose-dependent (ex vivo, second protocol). They were not significantly different when the drug was added before or after hypoxia, which suggests that in this model, reoxygenation was the most deleterious process and the stage at which resveratrol was most effective.