Effect of Inducible Antioxidants on Hemoglobin Toxicity

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DESCRIPTION (provided by applicant): Hemorrhage is the primary event in approximately 15% of strokes, and accompanies most significant brain trauma. A growing body of experimental evidence suggests that hemoglobin release from lysing erythrocytes may contribute to oxidative injury to tissue surrounding a hematoma. Prior studies have demonstrated that hemoglobin toxicity is mediated by transfer of its heme moieties to nearby cells, which is facilitated by its spontaneous 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. Astrocytes respond to hemoglobin exposure by rapidly inducing ferritin, a 24-mer heteropolymer with a capacity for over 4000 iron atoms in its mineral core. However, cortical neurons in cell culture and in vivo express very little ferritin after hemoglobin treatment. Cell ferritin levels are primarily regulated by iron regulatory proteins (IRP1 and IRP2), which bind to an iron responsive element (IRE) in the 5'-untranslated region of its mRNA and repress translation. In vitro studies using cell lines have demonstrated that inhibitors of IRP binding increase ferritin expression and protect against oxidative injury. In order to evaluate the therapeutic potential of this approach in attenuating hemoglobin neurotoxicity, we have established colonies of IRP1 and IRP2 knockout mice. In completed studies, we have observed that: 1) Ferritin is overexpressed by IRP2 knockout neurons at baseline and after oxidant exposure, while IRP1 knockout has a much weaker and variable effect; 2) IRP2 gene knockout markedly reduces neuronal vulnerability to hemoglobin and hydrogen peroxide; 3) Oxidative injury surrounding a striatal hematoma is attenuated in IRP2 knockout mice. The goal of this competitive revision application is to compare functional recovery in wild-type and IRP2 knockout mice after experimental intracerebral hemorrhage, using both direct blood injection and collagenase injection models. In addition to standard behavioral tests, we propose to quantify functional deficits via an approach that is novel in hemorrhagic stroke research: automated analysis of home cage locomotor behavior, testing proprietary software marketed by two U.S.-based companies. Specifically, mouse activity will be video recorded for 24 hour intervals. Activity levels, turning/circling, and stereotypic behaviors will be quantified with ANY-maze and HomeCageScan software. PUBLIC HEALTH RELEVANCE: 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 by detoxifying iron, and to thereby improve the likelihood of survival and return to an independent, productive life.