Our goal is to define the role of complement in ischemic stroke. C9 is the major cytolytic component of complement, but the role of C9 in post-ischemic cerebral injury is not known. Recent experiments revealed that administration of human C9 to developmentally C9-deficlent neonatal rats increased ischemic cerebral infarct volume by 85% (P < 0.05). Therefore, we will test the Hypotheses stated in three Specific Aims: 1) in post-ischemic cerebra, plasma C9 crosses a compromised blood brain barrier, is activated, and is incorporated into complement membrane attack complexes (MAC) assembled on neurons and oligodendrocytes. Unilateral ischemic cerebral infarction will be induced in adult and neonatal rats. Assays will be performed using computer-based planimetry (cerebral infarct volume), post-ischemic cerebral measurement of Evans blue accumulation, cerebral quantification of C9 by ELISA and Western blot, and confocal double label fluorescence immunohistochemistry using antibodies specific for activated C9 incorporated in MAC. 2) Following cerebral ischemia, C9 deposition augments a) loss of the complement regulatory protein CD59 from neurons and b) synthesis of C9, by neurons, microglia, and astrocytes. ELISA, Western blot, ribonuclease protection assay, in situ hybridization and double label immunofluorescence will be employed to test this hypothesis in the post-ischemic cerebra of neonatal rats injected ip with human C9. 3) Incorporation of C9 into MAC on oligodendrocytes and neurons induces a) cytolysis or b) apoptosis. Oligodendrocytes and neurons will be cultured from neonatal and embryonic rat forebrain. The effect of C9 on cytolysis and apoptosis will be assessed by immunofluorescence microscopy (oligodendrocytes), lactic acid dehydrogenase release (neurons), measurement of three different markers of apoptosis (annexin V labeling, cytochrome c release, detection of single stranded DNA), and electron microscopy, in each specific aim, the neuroprotective effect of four different agents that inhibit C9 activity will be assessed. These studies will reveal an unrecognized, C9-medlated mechanism of ischemic cerebral injury and a novel strategy to reduce the injury by inhibiting C9-mediated neurotoxicity. ? ?
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