Optimizing Heme Oxygenase Activity after CNS Hemorrhage

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Intracerebral hemorrhage accompanies most CNS traumatic injuries and about 15% of strokes. Increasing 9vidence suggests that hemoglobin (Hb) release from lysing erythrocytes may contribute to oxidative stress n surrounding tissue. Because of its prolonged time course, Hb toxicity may be an ideal target for therapeutic intervention. Prior studies have demonstrated that Hb affects the survival of neurons and astrocytes by pathways that are largely dependent on the heme oxygenase enzymes (HO). HO-1 is inducible, and is found primarily in glia, where it is protective; HO-2 is expressed mainly in neurons, where it worsens injury. Unfortunately, HO inhibitors block both isoforms, and increase injury to astrocytes while protecting neurons. The goal of this project is to develop a strategy that will optimize HO activity in both cell types after hemorrhage. We hypothesize that this may be accomplished by two approaches: 1) exploiting differences in the regulation of HO-1 and HO-2 activation; 2) transferring sense and antisense HO genes with promoters that target transcription to specific cell types. Our experimental aims are as follows: 1) Decrease neuronal HO-2 activity in cortical cultures by inhibiting its activation by CK2, using specific benzotriazole inhibitors. Determine the effect of this treatment on culture HO activity, reactive oxygen species (ROS) formation, and cell viability after Hb or hemin exposure. 2) Construct an adenovirus encoding the HO-1 gene driven by the astrocyte-specific GFAP promoter. Quantify its expression in astrocytes and neurons, and determine its effect on heme-mediated ROS formation and cell death. 3) Construct an adenovirus containing the HO-2 gene in antisense orientation, driven by the neuron-specific synapsin-1 promoter. Quantify its effect on HO-2 expression in cultured astrocytes and neurons, and on heme-mediated oxidative injury. 4) Stereotactically inject blood into the striata of mice, alone or with sense HO-1, antisense HO-2, or both vectors. Alternatively, treat blood-injected mice with intraperitoneal CK2 inhibitors. At 72 and 144 hours, determine the effect on lesion volume using TTC staining. Quantify cellular protein and lipid oxidation 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.