Effect of Inducible Antioxidants on Hemoglobin Toxicity

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Hemorrhage accompanies most significant brain trauma, and is the primary event in about 15% of strokes. A growing body of evidence suggests that hemoglobin (Hb) release from lysing erythrocytes may contribute to oxidative injury to tissue surrounding a hematoma. Prior studies have demonstrated that Hb toxicity is mediated by transfer of its heme moieties to nearby cells, which is favored by its oxidation to methemoglobin. Hemin, the oxidized form of heme, is then catabolized by the heme oxygenase enzymes to iron, carbon monoxide, and biliverdin. The cytoprotective effects of the latter two products may be overwhelmed if iron sequestration is inadequate. However, the iron detoxification strategies of neurons and astrocytes remain largely undefined. Preliminary experiments suggest that neurons export extracellular iron, where it must be sequestered to prevent oxidative membrane injury;astrocytes induce ferritin, consistent with intracellular storage. The goal of this project is to define the role of ferritin and transferrin in mitigating heme-mediated injury to neurons and astrocytes, and to devise a strategy to optimize this process. Our experimental aims are as follows: 1) Specifically increase expression of H or L-ferritin subunits in cultured neurons and astrocytes by adenoviral transfer of their genes. Alternatively, culture astrocytes and neurons from iron regulatory protein (IRP) knockout mice, which have markedly increased cell ferritin. Determine the effect of ferritin gene transfer or IRP knockdown on culture reactive oxygen species (ROS) formation, protein oxidation, and cell viability after Hb or hemin exposure. 2) Treat cultured neurons and astrocytes with [55Fe]hemin. Quantify subsequent 55Fe release into the culture medium or deposited in cell ferritin. Assess the effect of apotransferrin and holotransferrin on cell vulnerability to heme-mediated injury. 3) Stereo- tactically inject blood into the striata of hypotransferrinemic mice or non-mutant littermates. Alternatively, increase ferritin expression in blood-injected mice by adenoviral transfer of ferritin genes or shRNA to IRP's. At 72 and 144 hours, determine the effect on cell viability, protein and lipid oxidation, and axonal injury in surrounding tissue. The information gained in this project may lead to new treatments for victims of hemorrhagic stroke and head trauma. The ultimate goal is to reduce brain injury in tissue surrounding a blood clot, and to thereby improve the likelihood of survival and return to an independent, productive life.

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