GLUTAMATE NEUROTOXICITY IN NEURONAL NOS KNOCKOUTS

描述：(改编自申请者摘要)原代神经元 培养、N-甲基-D-天冬氨酸(NMDA)神经毒性及体外模型 脑缺血的发生，部分是由一氧化氮(NO)介导的。 尽管大量研究表明NO参与谷氨酸介导的 神经毒性，有证据表明NO可能在NMDA中没有作用 具有神经毒性，实际上可能具有神经保护作用。不，可能是 通过亚硝化和失活NMDA受体来保护神经。 没有S的双刃剑可能与S的氧化还原状态有关，与没有 自由基是有毒的，而亚硝基离子具有神经保护作用。在……里面 为了更好地了解NO在多种生理过程中的作用 NO参与调节的过程包括谷氨酸 神经毒性，申请者产生的小鼠携带选择性 胚胎干细胞中神经元型一氧化氮合酶基因的靶向缺失突变 细胞。利用一氧化氮合酶基因敲除小鼠及其野生型对照， 提出了一系列实验来阐明潜在的机制。 和NO在神经毒性中的参与，并寻找替代 毒性的途径。脑缺血引起的神经元损伤可能 通过过量的NO生产而发生。联合氧糖剥夺 在神经元培养和兴奋性氨基酸给药中 将其作为脑缺血的体外模型。易感性 对神经元损伤的影响将在神经元一氧化氮合酶基因敲除和 与野生型对照相比。将进行实验，以 确定靶向删除神经元型一氧化氮合酶是否会导致额外的 可能解释对谷氨酸介导的抗性的变化 毒性。谷氨酸受体的分布和密度将是 已评估。45Ca~(2+)的累积将被检测以响应 神经元型一氧化氮合酶基因敲除中的兴奋性氨基酸及其与野生型的比较 键入控件。NADPH黄递酶或NOS神经元对NMDA具有抵抗力 类型的神经毒性，对红藻氨酸和奎斯奎因高度敏感 神经毒性。一氧化氮合酶和NADPH黄递酶神经元与 生长抑素和神经肽Y。一氧化氮合酶基因敲除具有正常的神经肽Y和 生长抑素神经元。因此，这些神经元对 将在NOS淘汰赛中检测NMDA、红藻氨酸和奎斯奎因，而不是 野生型对照以确定是否这些易感性 在一氧化氮合酶基因敲除中，神经元发生了变化。此外，分布还包括 将检测一氧化氮合酶神经元上的谷氨酸受体，以评估 谷氨酸受体的差异表达解释了 对毒性的不同敏感性。其他可能的途径 由于一氧化氮合酶抑制剂只有部分保护作用，因此存在神经毒性。这个 神经元型一氧化氮合酶基因敲除为研究其他 谷氨酸神经毒性的潜在途径而不受 不是的。因此，谷氨酸的神经毒性和缺氧-葡萄糖剥夺 神经元培养将在神经元一氧化氮合酶基因敲除和 与野生型对照相比，在给药后 神经毒性的其他潜在途径的抑制剂。这些特工 将包括超氧阴离子清除剂和自由基。在……里面 此外，磷脂酶A2抑制剂环氧合酶的作用 抑制剂和脂氧合酶抑制剂将被检查。

DESCRIPTION: (adapted from Applicant's Abstract) In primary neuronal cultures, N-methyl-D-aspartate (NMDA) neurotoxicity, and in vitro model of cerebral ischemia, is mediated, in part, by nitric oxide (NO). Despite numerous studies implicating NO in glutamate mediated neurotoxicity, there is evidence that NO may play no role in NMDA neurotoxicity and may in fact be neuroprotective. NO may be neuroprotective by nitrosylating and inactivating the NMDA receptor. NO s double edge sword may be related to it s redox state, with the NO radical being toxic and the nitrosonium ion being neuroprotective. In order to better understand the role of NO in a variety of physiologic processes in which NO has been implicated to regulate including glutamate neurotoxicity, the applicants have generated mice carrying a selective mutation in the neuronal NOS gene by targeted deletion in embryonic stem cells. Utilizing the NOS knockout mice and their wild type controls, a series of experiments are proposed to clarify the potential mechanisms and involvement of NO in neurotoxicity and to identify alternative pathways of toxicity. Neuronal damage due to cerebral ischemia may occur through excess NO production. Combined oxygen-glucose deprivation in neuronal cultures as well as excitatory amino acid administration will be used as in vitro model of cerebral ischemia. The susceptibility to neuronal injury will be evaluated in neuronal NOS knockouts and compared to wild type controls. Experiments will be performed to determine whether targeted deletion of neuronal NOS leads to additional changes that might account for the resistance to glutamate mediated toxicity. The distribution and density of glutamate receptors will be evaluated. 45Ca2+ accumulation will be examined in response to excitatory amino acids in neuronal NOS knockouts and compared to wild type controls. NADPH diaphorase or NOS neurons are resistant to NMDA type neurotoxicity and are highly susceptible to kainate and quisqualate neurotoxicity. NOS and NADPH diaphorase neuronal colocalize with somatostatin and NPY. The NOS knockouts possess normal NPY and somatostatin neurons. As such the susceptibility of these neurons to NMDA, kainate and quisqualate will be examined in NOS knockouts versus wild type controls to determine whether the susceptibility of these neurons is altered in NOS knockouts. Additionally, the distribution of glutamate receptors will be examined on NOS neurons to evaluate whether the differential expression of glutamate receptors accounts for differential susceptibility to toxicity. Other potential pathways of neurotoxicity exist as NOS inhibitors are only partially protective. The neuronal NOS knockouts provide a unique opportunity to examine other potential pathways of glutamate neurotoxicity without the influence of NO. As such, glutamate neurotoxicity and oxygen-glucose deprivation in neuronal cultures will be examined in the neuronal NOS knockouts and compared to wild type controls after the administration of a variety of inhibitors of other potential pathways of neurotoxicity. These agents will include scavengers of the superoxide anion, and free radicals. In addition, the effects of phospholipase A2 inhibitors, cyclooxygenase inhibitors, and lipoxygenase inhibitors will be examined.