Currently there are more than 2 million citizens in this country who are disabled by stroke. Stroke caused by cerebral ischemia is the third leading cause of death and the leading cause of neurological disability. The annual direct and indirect loss from stroke is estimated to be $25 billion. Partial recovery of function is frequently noted in stroke patients. The molecular mechanism of functional recovery, however, is poorly understood. In a well developed stroke model of the rodents, cerebral ischemia causes an increase in superoxide and hydrogen peroxide, which are converted to hydroxyl radicals. Hydroxyl radical, a known mutagen, induces DNA repair synthesis and the expression of repair enzyme DNA polymerase-beta in cell culture. Therefore, hydroxyl radical could cause activation of a genetic program leading to delayed neuronal death (apoptosis). To reduce apoptosis in the brain is central to functional recovery after stroke. The hypothesis is that apoptosis is mediated via DNA damage by the elevated oxygen free radicals during and after cerebral ischemia. Preliminary studies from our laboratory using th stroke model of focal cerebral ischemia of 30 min demonstrated a disappearance of S1 nuclease sensitive sites and an increase in the expression of DNA polymerase-beta mRNA within 2 hr after ischemia, a 6-fold elevation in gene mutation frequency within 8 hr in the lacl gene of Big Blue transgenic mice, and an appearance of DNA fragmentation, a characteristics of apoptosis, 3 days after ischemia. It is known from studies by others and ours that gene mutations are results from errors introduced during DNA repair synthesis. Mutations could lead to apoptosis in the nervous system as a results of ischemia. The goal of this proposal is to determine the contribution of hydroxy radicals to cerebral gene mutation and apoptosis after cerebral ischemia. In this proposal, we will: (1) analyze the sequence change in the DNA of 150 lacl mutants from ischemia and 30 lacl mutants from normal brain samples; (2) determine DNA repair in the transcribed strand of neurotrophin, DNA polymerase-beta, p53 and actin genes; (3) measure the increase in 8-hydroxyldeoxyguanosine (oh8dG) in DNA from cortical cells after ischemia; (4) determine if there is a positive correlation between the formation of hydroxyl radicals and the increase in the physiological parameter of mutation frequency, DNA repair, oh8dG and apoptosis using intravenous infusion of superoxide dismutase plus catalase before ischemia. The mutation spectrum that is obtained from this project will be a direct indicator of DNA damage by hydroxyl radicals after cerebral ischemia. This model will be suitable for exploring pathobiological effect of oxygen stress, and for testing drugs that reduce or abolish the damaging effect of hydroxyl radical after stroke.
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