Lipid Peroxidation In Vivo Induced by Reversible Global Ischemia in Rat Brain

Abstract

It has been hypothesized that ischemia, followed by reperfusion, facilitates peroxidative free-radical chain processes in brain. To resolve this question, rats were subjected to reversible global ischemia. From coronal sections of brains frozen in situ, small (ca. 2 mg) amounts of tissue were sampled from neocortex, hippocampus, and thalamus of both cerebral hemispheres of four groups of rats exposed to 30 min cerebral ischemia followed by 0, 30, 60, and 240 min of reperfusion, and from a control group subjected to the same operative procedures, except for the induction of ischemia. Heptane-solubilized total lipid extracts from these samples were analyzed spectroscopically in the 190–330 nm range for content of isolated (nonconjugated) double bonds and of conjugated diene structures; the latter are formed from isolated double bonds during peroxidation of unsaturated fatty acids. Spectra derived from tissue regions of rats subjected to ischemia, or ischemia followed by reperfusion, were compared to averaged, region-specific control spectra and were normalized to the original content of isolated double bonds in the peroxidized samples. The resultant difference spectra were analyzed in terms of ratios of conjugated diene concentration to the concentration of isolated double bonds originally at risk in the specific tissue zones considered. The peak representing conjugated diene formation was centered at 238 ± 1 nm and was usually well resolved when the molar ratio [conjugated diene]/[isolated double bonds], expressed as a percentage ([CD]/[IDB]), was greater than 0.25%. The incidence of resolvable conjugated diene peaks was much greater in the reperfused groups (18 of 124 samples total; 0.25% [CD]/[IDB] 1.34%), compared to the nonreperfused group (1 of 38 samples; [CD]/[IDB] = 0.62%). No specific regional susceptibility to conjugated diene formation was observed. It also was shown by computer averaging over all the tissue zones sampled that, if one had used large samples consisting of pooled brain tissue for analysis of lipid peroxides and/or their reaction products, the conjugated diene peak would have been attenuated below distinctly resolvable levels. These results constitute the first demonstration of lipid peroxidation induced by reversible global ischemia. The observation that the effect is highly focal and not generalized throughout the brain suggests that special conditions of reperfusibility and reoxygenation must be satisfied for lipid peroxidation to be detectable in an ischemic milieu.