Tolerance to focal cerebral ischemia can be induced by different stimuli, if applied prior to the ischemic event. Known examples are brief global or focal ischemia or a series of cortical spreading depression. Maximal protection is generally achieved if the preconditioning stimulus is applied 2 to 3 days in advance of the second detrimental ischemic event. 3-NPA is a irreversible inhibitor of the succinate dehydrogenase and thus a mild inhibitor of the oxidative phosphorylation. We showed, that 3-NPA application induces a profound tolerance towards subsequent focal cerebral ischemia. The infarct volume was reduced to ¼ of it’s size compared to controls. 3-NPA also preconditions brain slices against hypoxia (Riepe et al.). We are addressing the question which molecular mechanisms lead to ischemic tolerance. Quantification of the temporal and spatial profile of succinate dehydrogenase inhibition by 3- NPA will help to understand the initial steps in tolerance induction. Detection of cortical free radical generation with a chemiluminescence technique will clarify whether superoxide anions are a necessary prerequisite in the generation of tolerance. Immunfluorescence microscopy for proteins like bcl2 and bax will be used to determine expression of pro- or antiapoptotic genes. The Northern blot technique is used to determine expression levels of candidate genes, relevant for preconditioning, while the SAGE approach is used to screen for relevant genes.
Northern blot technique; SAGE; Immunfluorescence; free radical detection by a chemiluminescence technique; . Densitometric and photometric quantification of the temporal and spatial profile of succinate dehydrogenase inhibition, Brint model of focal cerebral ischemia
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