A hypoxic-ischemic (H/I) event in the perinatal brain results in an infarcted region of the brain characterized by cell death. In the penumbra there is neuronal cell death and astrogliosis. The extent to which the size of the infarcted area can be minimized and cells in the penumbra rescued post-insult will significantly influence the extent of recovery and quality of life. There is controversy regarding whether the neuronal cell death subsequent to H/I is a result of oxygen/substrate deficiency or excitotoxicity. Astrocytes are ideally situated to mediate either of these paths of neurodegeneration. We proposal as a global hypothesis that H/I induced damage to astrocytes will create will create an environment which promotes neuronal cell death. We have developed a cell culture model in which to study the effect of H/I on astrocytes. We hypothesize that H/I induces intracellular changes in astrocytes that are similar to oxidative stressors (Aim 1). We will examine antioxidants and pro- oxidants to test the hypothesis that these agents damage or protect astrocytes (Aims 3 and 4). Furthermore, because acute lesions in while matter are also frequently associated with perinatal H/IO insults and it has been argued that oligodendrocytes are vulnerable to oxidative stress, we will use culture to examine the intracellular events associated with H/I versus oxidative stress in oligodendrocytes (Aim 2) and compare the response of oligodendrocytes with astrocytes to pro-oxidants and antioxidants (Aims 3 and 4). Astrocytes also provide protection in oligodendrocytes, so diminished function in astrocytes is also relevant to oligodendrocytes. We proposed that mitochondria are the organelles most sensitive to H/I in astrocytes and oligodendrocytes. The analyses performed in the cell culture studies will include ATP production, mitochondrial membrane potential, intracellular calcium concentrations, superoxide production and cell death. A key relationship is expected between mitochondrial membrane potential and intracellular calcium, with iron status of the cell expected to be a critical variable. To test the hypothesis that mitochondrial dysfunction occurs in vivo following H/I, we will use the immature rat pup (Aim 5). Animals will be examined at various intervals into adulthood subsequent to the insult. We will determine cytochrome c release at the varying intervals and predict that there will be complex specific impairment of mitochondrial respiratory chain activity. These data will provide insight into the intracellular events associated with H/I in different cell types in vivo.
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