Excitotoxic injury to developing oligodendrocytes

描述：兴奋性毒素在脑室周围白质软化（PVL）中的作用 将在本项目中使用体内和体外技术进行处理。的 研究者开始的假设是脑缺血导致 谷氨酸释放是PVL的初始触发物。由此产生的兴奋性毒性 对发育中的少突胶质细胞的损伤是项目IV的重点。谷氨酸盐 对白色物质的影响被假设为通过AMPA/红藻氨酸受体，因为没有 NMDA受体存在于少突胶质细胞（OL）上。AMPA/红藻氨酸受体连接 通道可以通过钠流介导细胞损伤和/或死亡，在一些实施方案中， 情况下的钙流量，并通过细胞去极化与所得的钙 通过依赖于电压的通道进入。该项目的重点是 AMPA/红藻氨酸盐对OL的毒性。 研究者将首先使用分离的OL培养物的体外系统， 通过不同发育阶段的标记物来测量AMPA/红藻氨酸受体 蛋白质表达的发育阶段。受体亚单位亚型和 关于钙的问题， 磁导率然后将这些实验在体内系统中重复进行比较 P3、P7和P4幼仔中AMPA/红藻氨酸受体蛋白表达。这些 实验包括具体目标一。 钙毒性将在发育阶段专门讨论 在AMP/红藻氨酸剂量反应曲线的设置中的特定培养物。标记 钙吸收将是终点。机制的药理学剖析 将使用AMPA的药理学抑制剂测定钙进入， 红藻氨酸盐、电压依赖性钙通道或Na/Ca转运蛋白。单独 实验将解决自由基毒性。 最后，形态学的注意力将被导向凋亡或 这些培养物中细胞死亡的坏死机制。电子显微镜和 Caspase-3的表达将是决定因素。这些实验构成了 具体目标2. 然后将在不同年龄的幼崽中研究AMPA/红藻氨酸盐的体内毒性， 注射白色物质的成年大鼠。损伤大小和单链DNA 损伤（原位末端标记-ISEL）将是形态学终点。频率 将通过双标记来评估OL发育阶段的DNA损伤， ISEL或胱天蛋白酶-3。额外的动物将被允许生存三年 周，用Luxol Fast Blue染色的大脑将用于评估 髓鞘形成不足的区域。将在P3和P7中重复AMPA/KA输注 大鼠在AMPA/红藻氨酸盐、AMPA或 红藻氨酸受体来仔细分析可能的受体。这些 实验构成具体目标3. 然后将在P3至成年期的动物中研究新生儿缺氧缺血 评估慢性髓鞘形成不足的脆弱性再次 在每个发育年龄，将通过双重评估， 用ISEL和标记蛋白染色用于OL期。其他大鼠将 允许存活至成年，并将评估OL密度的降低 并与注射AMPA/红藻氨酸盐的动物进行比较。最后 AMPA/红藻氨酸拮抗剂在缺氧缺血中的保护作用 将评估发展点以及自由基的潜力 食腐动物

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.