Excitotoxic injury to developing oligodendrocytes

对发育中的少突胶质细胞的兴奋性毒性损伤

• 批准号: 6565276
• 负责人: Frances E Jensen
• 金额: $ 19.61万
• 依托单位: BOSTON CHILDREN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-12-01 至 2002-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6565276
• 关键词: AMPA receptors; age difference; calcium flux; cerebral ischemia /hypoxia; cytotoxicity; developmental neurobiology; free radicals; glutamate receptor; glutamates; laboratory rat; neuroprotectants; neurotoxicology; neurotransmitter antagonist; oligodendroglia; oxidative stress

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.

描述：兴奋毒素在脑室周围白质软化症 (PVL) 中的作用 将在该项目中使用体内和体外技术来解决。这 研究人员首先假设脑缺血会导致 谷氨酸释放是 PVL 的最初触发因素。由此产生的兴奋性毒性 发育中的少突胶质细胞损伤是项目 IV 的重点。谷氨酸盐 假设对白质的影响是通过 AMPA/红藻氨酸受体实现的，因为没有 NMDA 受体存在于少突胶质细胞 (OL) 上。 AMPA/红藻氨酸受体步态 在某些情况下，通道可以通过钠通量介导细胞损伤和/或死亡 环境钙通量，并通过细胞去极化产生钙 通过电压相关通道进入。该项目的重点是影响 AMPA/红藻氨酸盐对 OL 的毒性。 研究人员将首先使用分离的 OL 培养物的体外系统 通过不同发育阶段的标记来测量 AMPA/红藻氨酸受体 各发育阶段的蛋白质表达。受体亚基异构体和 子单元编辑将涉及钙的问题 渗透性。然后将在体内系统中重复实验以进行比较 P3、P7 和 P4 幼崽中的 AMPA/红藻氨酸受体蛋白表达。这些 实验包括具体目标一。 然后将在发育阶段专门解决钙毒性问题 AMP/红藻氨酸剂量反应曲线设置中的特定培养物。贴上标签 钙的吸收将是终点。机制的药理学剖析 钙进入量将使用 AMPA 药物抑制剂来确定， 红藻氨酸盐、电压依赖性钙通道或 Na/Ca 转运蛋白。分离 实验将解决自由基毒性问题。 最后，形态学注意力将集中于凋亡或 这些培养物中细胞死亡的坏死机制。电子显微镜和 Caspase-3 表达将是决定因素。这些实验构成 具体目标2. 然后将在不同年龄的幼犬中研究 AMPA/红藻氨酸的体内毒性 成年大鼠白质注射。病灶大小和单链 DNA 损伤（原位末端标记-ISEL）将是形态学终点。频率 OL 发育阶段的 DNA 损伤将通过双标记进行评估 ISEL 或 Caspase-3。额外的动物将被允许存活三年 几周后，他们的大脑将用 Luxol Fast Blue 染色来评估 髓鞘形成不足的区域。 AMPA/KA 输注将在 P3 和 P7 中重复进行 存在 AMPA/红藻氨酸、AMPA 或 AMPA 药物拮抗剂的大鼠 红藻氨酸受体以药理学剖析可能的受体。这些 实验构成具体目标3。 然后将在从 P3 到成年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窗口。再次， 将在每个发育年龄对处于危险中的 OL 人群进行双倍评估 用 ISEL 和 OL 阶段标记蛋白染色。额外的老鼠将 允许存活至成年并且将评估 OL 密度的降低 并与注射 AMPA/红藻氨酸的动物进行比较。最后， AMPA/红藻氨酸拮抗剂对不同条件下缺氧缺血的保护作用 将评估发育点以及自由基的潜力 拾荒者。