Effect of Inducible Antioxidants on Hemoglobin Toxicity

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EXCEED THE SPACE PROVIDED. Hemorrhage complicates many traumatic injuries to the CMSand about 20% of strokes. Over subsequent hours, erythrocytes lyse and release their contents into the extravascular space. The most abundant protein released is hemoglobin (Hb). A growing body of experimental evidence suggests that the oxidative toxicity of extracellular Hb contributes to the pathogenesisof hemorrhagic CNS injury. Moreover, because of its prolonged time course, Hb toxicity may be an ideal target for therapeutic intervention. Further insight into the cellular mechanisms and prevention of this toxicity therefore seems desirable. Cultured neurons are highly vulnerable to Hb, but astrocytes are resistant via a mechanism that requires protein synthesis. Preliminary experimentssuggest that this discrepancy may be explained in part by the effects of two inducible antioxidants: heme oxygenase(HO)-1 and ferritin. The former is rapidly induced by Hb and may facilitate synthesis of L-rich ferritin in astrocytes. In contrast, Hb decreases the expression of L-rich ferritin in neurons; iron released as a product of heme breakdown may then be toxic. ¿ This project will address the role of HO and ferritin in cell culture and in vivo models. Overexpression of HO-1 will be accompished in glial, neuronal, or mixed cultures via gene transfer; the relationship between activity, heme-mediated reactive oxygen species formation, and cell death will be established. Cellular vulnerability to Hb or hemin will then be compared in cultures prepared from wild-type, HO-1 knockout, and HO-2 knockout mice. Using antibodies that specifically recognize H- or L-ferritin, the subunit content of ferritin will be assessed at baseline and in response to Hb in these cultures. Expression of HasA, which binds to and may facilitate heme iron uptake, will also be determined. H and L-rich ferritin heteropolymers will be constructed from recombinant H or L-ferritin. Neuronal and glial uptake of these heteropolymers via receptor-mediated endocytosis will allow investigation of the effect of the H:L ratio on cellular vulnerability to herne-mediated injury. Finally, the putamen of wild type, HO-1 or HO-2 knockout, and transgenic mice that overexpress HO-1 will be injected with Hb, or with coflagenase to induce an endogenous hemorrhage. Surrounding neuronal loss, DMA cleavage, and caspase-3 activation will then be quantified at defined time points 12-96 hours after injection. PERFORMANCE S<nTE(S; (organization, city, state} v Thomas Jefferson University 1020 Walnut Street Philadelphia, PA 19107 KEY PERSONNEL ========================================Section End===========================================