The long-term objective of the research project is to unravel basic mechanisms of reperfusion damage in the brain following global or focal brain ischemia and, thereby, to help developing a rational pharmacological therapy. The application is based on the assumption that reperfusion damage is, to a large extent, caused by production of reactive oxygen species (ROS), and on the possibility that mitochondrial dysfunction and damage are key events in the pathogenesis of disease due to stroke and related disease conditions. Two different models of ischemia are used to achieve the goals. In the first, transient global brain ischemia is induced in rats. In this model, neuronal necrosis is delayed by hours in the neocortex and caudoputamen, and by days in the CA1 sector of the hippocampus. The applicants propose that the initial ischemic period alters the pump-leak relationship for Ca2+ and causes a rise in the cytosolic calcium concentration (Ca2I) and, thereby, secondary mitochondrial calcium overload. In this model, the ischemic damage is ameliorated (or prevented) by cyclosporin A (CsA), an immunosuppressant drug which acts as a virtually specific inhibitor of the permeability transition (PT) pore of the inner mitochondrial membrane, a pore which is assembled under adverse conditions such as mitochondrial Ca2+ accumulation and oxidative stress. The objective of the study is to assess mitochondrial function, and calcium content, and the assembly of a CsA-sensitive PT pore in the interval between the initial ischemic transient and the delayed cell death. The other model involves transient middle cerebral artery occlusion (MCA) of 2 h duration. This period of ischemia usually leads to a large infarct, but amelioration is achieved by the spin-trap-2-phenyl-N-tert-butyl nitrone (PBN), by the immunosuppressant FK-506, and by antibodies to adhesion molecules for PMN leukocytes, all acting in the reperfusion period with a window of opportunity of up to 3 h. Since PBN prevents a secondary bioenergetic failure during reperfusion, and mitochondrial failure, the working assumption is that the delayed development of an infarct reflects mitochondrial calcium accumulation, and mitochondrial failure.
The aim of the investigation is to assess the nature of the mitochondrial dysfunction by measurements of cell and mitochondrial calcium content, mitochondrial respiratory function complexes, and the assembly of a mitochondrial PT pore.
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