The proposed studies will investigate specific cellular and molecular mechanisms of hypoxic neuronal injury in the newborn by relating the degree of hypoxia to alterations in neuronal nuclear membrane and intranuclear Ca/++, as well as the expression of apoptotic and anti- apoptotic genes. We propose that the severity of hypoxia will have a specific impact on the function of the neuronal nuclear membrane calcium influx mechanisms and critical nuclear functions. The monitoring of the degree of brain tissue hypoxia in vivo will be achieved by the continuous measurement of brain high energy compounds with 31P nuclear magnetic resonance spectroscopy and confirmed biochemically by ATP and phosphocreatine. Histochemical methods will be performed to assess histopathologic changes. Experimental protocols will be carried out on newborn piglets investigating: (1) The relationship of quantitative tissue hypoxia to changes in neuronal calcium influx mechanisms: high affinity Ca/++-ATPase, inositol 1, 3, 4, 5 tetrakisphosphate (IP/4) receptor and inositol 1, 4, 5 triphosphate (IP/3) receptor; (2) The effect of hypoxia on increased intranuclear calcium concentration; (3) The relationship of increased neuronal intranuclear Ca/++ to transcription of bax (apoptotic) and bcl-2 (anti-apoptotic) genes, endonuclease activity, and the pattern of DNA fragmentation in response to graded tissue hypoxia; (4) Morphological changes indicative of neuronal death, particularly programmed cell death following hypoxia; and (5) The effect of inhibition of the NMDA receptor or pathways of free radical generation or hypoxia- induced changes in neuronal nuclei and programmed cell death. The proposed experiments will be performed by utilizing well established techniques. These studies will provide new insights into neuronal nuclear functions and will lead to a better understanding of the mechanisms of hypoxic brain injury. The elucidation of basic cellular mechanisms in response to hypoxic brain injury. The elucidation of basic cellular mechanisms in response to hypoxia will enable the development of novel strategies of preventing or attenuating the deleterious effects of hypoxia in the newborn.
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