Project I- Impact of Hypoxia-Ischemia and/or Inflammation on Microglia in Cerebellum

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PROJECT SUMMARY: Sensitive periods in neuronal development vary by brain region, sensitivity and vulnerability to disruptive agents. We have discovered that the second postnatal week in the rat is a sensitive period in cerebellar development to dysregulation by inflammation. This sensitive period is characterized by increased production of prostaglandins, in particular PGE2. This sensitive period coincides with a time of increased risk for perinatal H/I (e.g. birth in human infants). The source of the stunting of the Purkinje neuron dendritic tree following inflammation is unknown. Microglia are the brains innate immune cells and serve a variety of functions in both health and disease. We have observed tightly regulated and temporally controlled phagocytic activity of microglia in the developing cerebellum and we hypothesize this plays an important role in normal cerebellar development. We provide evidence that H/I at PN10 increases still further the peak in phagocytosis normally seen at PN17. One of the key signals regulating microglia phagocytic activity is the membrane lipid phosphatidylserine, which is externalized to the outer leaflet and promotes phagocytosis. Hypoxia has been known to stimulate phagocytosis by stressing cells sufficiently to externalize phosphotidylserine. The role of metabolism or lipid rafts in the externalization of this lipid has not been considered, nor have either of these variables been characterized in microglia in a surveying versus activated state. Hypoxic-Ischemic brain injury often occurs following an inflammatory insult, resulting in further injury than would otherwise occur. Whether this is true in the developing cerebellum has not been carefully explored nor have sex differences in hypoxic-ischemic brain or inflammatory injury been considered. We propose three Specific Aims focused on 1) morphometery of Purkinje neurons, granule cells and the deep cerebellar nuclei following H/I with and without inflammation, 2) the role of microglia in neuronal damage induced by H/I with and without inflammation and 3) the impact of tetracycline based antibiotic, Minocycline, as proof of principle and the glutamate transporter modifying antibiotic Ceftriaxone as a potential therapeutic. We will also explore the dietary supplement, choline and conduct behavioral and histological analyses to assess injury and recovery. Data generated by these experiments will provide novel insights into the impact of H/I on the cerebellum and the degree to which it contributes to the impaired cognitive and motor function seen in infants suffering hypoxic-ischemic encephalopathy.