Hemoglobin-based compounds are being developed as blood substitutes to be used in surgery or emergency medicine, yet the consequences of placing hemoglobin-based compounds in direct contact with CNS tissue are unknown. These studies will continue to develop a cell culture model that can be used to assess the toxicity of hemoglobin or hemoglobin-based compounds to the CNS. Additionally, this model can be utilized to identify and test possible therapeutic approaches for the treatment of hemoglobin-mediated cellular injury. Previous results suggest that hemoglobin is neurotoxic. Oxyhemoglobin causes cerebral vasospasm, and intracortical injections of hemoglobin have been used to generate animal models of epilepsy. Preliminary experiments have demonstrated that both hemoglobin and a hemoglobin-based oxygen carrier are toxic to cultured murine neurons in a concentration-dependent fashion. This toxicity developed over time and progressed even after hemoglobin was removed. None of the hemoglobin solutions tested have appeared to be toxic to murine glial cells. The current proposal will address hemoglobin-mediated injury in cultures of rat cerebellar neurons, cortical neurons, pure astrocytes, and co- cultures of astrocytes and microglia. The concentration dependence and time course of hemoglobin-mediated injury will be delineated. Specific plasma proteins will be evaluated as protectants, emphasizing proteins that bind and facilitate the clearance of hemoglobin or heme--haptoglobin or hemopexin. Other compounds that may have therapeutic potential for the treatment of hemoglobin-mediated injury will be evaluated. These compounds will include antioxidants (such as Trolox, alpha tocopherol, and the 21- aminosteroids), chelators (such as deferoxamine and a novel deferoxamine derivative), as well as drugs that block the calcium entry into neurons (NMDA, kainate/AMPA, and metabotropic receptor antagonists and calcium channel blockers). Additional experiments will be conducted to test the hypotheses that ascorbate- and hemoglobin-dependent neurotoxicity are mediated through similar mechanisms and that ascorbate may exacerbate hemoglobin-mediated injury. Finally, the expression the of heat shock proteins HSP7O and heme oxygenase- l will be examined in neurons and astrocytes during the course of hemoglobin- and ascorbate-dependent injury. These proteins, which are thought to be protective, may be induced differently in different cells, partially explaining the differential sensitivity of neurons and astrocytes to hemoglobin.
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