Detection of free radicals generated in NADPH-dependent lipid peroxidation in ischemic brain homogenate. Application of the spin trapping technique

T. Tominaga, S. Imaizumi, T. Yoshimoto, J. Suzuki, Y. Fujita

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The spin trapping technique has been applied to the detection of free radicals generated in the NADPH stimulated lipid peroxidation process in ischemic brain homogenate. Using male Wistar rats, complete cerebral ischemia for 30 min, 60 min or 120 min was produced by decapitation followed by preservation of the heads at 37°C. Global cerebral ischemia of 30 min or 60 min duration was induced by occlusions of three vessels (bilateral common carotid and basilar artery) in the ventilated rats. In some animals, bilateral carotid occlusions were released for 30 min following 30 min of ischemia to study the postischemic event. Two reaction mixtures containing brain homogenate, NADPH, Fe-EDTA and spin trapping reagent, phenyl-t-butylnitrone (PBN), were prepared from each brain sample - one to be incubated in air and the other to be incubated in nitrogen gas. After incubation for 20 min at 37°C, free radical adducts of PBN were measured by electron spin resonance (ESR). In preliminary experiments, no ESR signals were obtained from the reaction mixtures without the addition of NADPH and Fe-EDTA. The dependence of ESR signal intensity upon the NADPH concentrations was observed. The six-line signals (triplet of doublets), whose hyperfine splitting constants were A(N) = 16.2-16.5 G and Aβ(H) = 3.6-3.8 G, were obtained from both ischemic models. These signals were dependent upon the presence of oxygen in the reaction systems, as evidenced by the fact that the signal intensity obtained from aerobic incubation was consistently stronger than that obtained from anaerobic incubation in each brain sample. In decapitation models, the intensities of ESR spectra were sequentially increased with the ischemic period under aerobic incubation, and increased gradually for 60 min of the ischemic period and then decreased under anaerobic incubation. In three-vessel occlusion models, the intensities of ERS spectra reached their peaks at 30 min into the ischemic period under both aerobic and anaerobic incubations, and markedly increased during postischemic recirculation. The results imply that ischemic injury may make the brain very susceptible to lipid peroxidation and that, if there is sufficient supply of oxygen, the reaction will readily progress.

Original languageEnglish
Pages (from-to)169-175
Number of pages7
JournalBrain and Nerve
Issue number2
Publication statusPublished - 1986


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