Excitotoxic injury to developing oligodendrocytes

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DESCRIPTION: The role of excitotoxins in periventricular leukomalacia (PVL) will be addressed in this project using an in vivo and in vitro techniques. The investigators begin with an assumption that brain ischemia with resultant glutamate release is the initial trigger of PVL. The resultant excitotoxic injury to developing oligodendrocytes is the focus of project IV. Glutamates effect on white matter is hypothesized to be via AMPA/kainate receptors as no NMDA receptors occur on oligodendrocytes (OLs). AMPA/kainate receptor gaited channels can mediate cell injury in and/or death via sodium flux, in some circumstances calcium flux, and via cell depolarization with resultant calcium entry via voltage dependent channels. The project focuses on the effects of AMPA/kainate toxicity on OLs. The investigators will first use an in vitro system of OLs cultures segregated by markers for various developmental stages to measure AMPA/kainate receptor protein expression by developmental stage. Receptor sub-unit isoforms and sub-unit editing will be addressed in regards to the issues of calcium permeability. The experiments will then be repeated in vivo systems to compare AMPA/kainate receptor protein expression in P3, P7, and P4 pups. These experiments comprise specific aim one. Calcium toxicity will then be specifically addressed in developmental stage specific cultures in the setting of AMP/kainate dose response curve. Labeled calcium uptake will be the end point. Pharmacologic dissection of the mechanism of calcium entry will be determined using pharmacologic inhibitors of AMPA, kainate, voltage dependent calcium channels or Na/Ca transporters. Separate experiments will address free radical toxicity. Finally, morphologic attention will be directed toward the apoptotic or necrotic mechanism of cell death in these cultures. Electronmicroscopy and Caspase-3 expression will be the determinant. These experiments constitute specific aim 2. AMPA/kainate toxicity in vivo will then be studied in pups at various ages and adult rats with white matter injections. Lesion sizes and single stranded DNA damage (in situ end labeling-ISEL) will be morphologic endpoints. The frequency of DNA damage by OL developmental stage will be assessed by double labeling for ISEL or Caspase-3. Additional animals will be permitted to survive for three weeks and their brains stained with Luxol Fast Blue will be used to assess areas of hypomyelination. The AMPA/KA infusions will be repeated in P3 and P7 rats in the presence of pharmacologic antagonists of AMPA/kainate, AMPA, or kainate receptors to pharmacologically dissect the probable receptor. These experiments constitute specific aim 3. Neonatal hypoxia ischemia will then be studied in animals from P3 to adulthood to assess the window of vulnerability for chronic hypomyelination. Again, the OL populations at risk will be assessed, at each developmental age, by double staining with ISEL and marker proteins for OL stage. Additional rats will be permitted to survive to adulthood and a decrease in OL density will be assessed and compared with those from the AMPA/kainate injected animals. Finally, the protective effects of AMPA/kainate antagonists in hypoxia ischemia at various developmental points will be assessed as well as the potential of free radical scavengers.