In vitro neuroprotection against oxidative stress by pre-treatment with a combination of dihydrolipoic acid and phenyl-butyl nitrones

Abstract

One consequence of trauma to the CNS is the production and liberation, from damaged tissue, of large amounts of oxygen-centered free radicals or reactive oxygen species (ROS). An excessive production of ROS can overwhelm the endogenous antioxidant defense system resulting in lipid peroxidation, DNA strand breaks, protein denaturation and cross-linking. The brain is particularly vulnerable to oxidative injury, because it contains high concentrations of readily oxidizable poly-unsaturated fatty acids, has a high rate of oxygen consumption per unit mass, and has only a relatively modest antioxidant defense system. We have conducted studies in vitro to determine the feasibility of reducing ROS-mediated damage in neurons by bolstering endogenous neuronal antioxidant defenses. Primary cultures of neurons derived from embryonic rat forebrain were pre-treated with the free radical scavenger dihydrolipoic acid (DHLA), the reduced form of Alpha-lipoic acid (ALA), and then subjected to H(2)O(2)-mediated oxidative stress. Neuroprotection was determined using the colorimetric MTT reduction assay. As has been reported by others, pre-treatment of neurons with DHLA (4 h) provided dose-dependent neuroprotection against a subsequent exposure to H(2)O(2). The addition of spin trapping nitrones N-tert-butyl-Alpha-phenyl-nitrone (PBN) or its sulfonated analog N-tert-butyl-Alpha(2-sulfophenyl)-nitrone (SPBN) to the pre-treatment cocktail enhanced neuroprotection at every dihydrolipoate concentration. Greater therapeutic efficacy in antioxidant treatment might be realized by employing combinations of complementary antioxidants.