In vitro injury paradigms

将在原代培养的来自皮层、海马和纹状体的脑细胞的脑损伤的缺血样损伤中研究三组基因的保护潜力。首先，抗氧化剂策略将通过使用疱疹病毒和腺病毒载体过度表达铜锌超氧化物歧化酶（SOD 1）来测试，以实现在神经元和星形胶质细胞中的快速表达，以及逆转录病毒载体在星形胶质细胞中的长期稳定表达。急性表达是否可以提供保护将被测试。如果这不是保护性的，则将测试延长的稳定表达和使用双顺反子载体以快速表达SOD和下游抗氧化剂载体两者、快速表达SOD和下游抗氧化剂酶两者的效果。我们将测试保护作用，以及其他抗氧化酶的诱导。将确定保护是否与其他抗氧化酶的诱导相关。在保护条件下看到的程度的氧自由基的生产，脂质过氧化和谷胱甘肽水平的变化将被确定。该基因是否能防止坏死或凋亡形式的细胞死亡还有待确定。将确定该基因是否保护免于坏死或凋亡形式的细胞死亡，以及表达仍然可以保护的时间窗，因为重要的是开发在损伤后有效的治疗策略。其次，我们将研究Bcl-2表达在相同损伤模式中的保护能力。氧化状态和Bcl-2可以保护的时间窗口将被确定。第三，我们将研究诱导型热休克蛋白70（HSP 70）保护免受这些损伤的能力，再次分析该基因可以保护的时间窗以及它是否阻断凋亡或坏死细胞死亡。原代培养物对于分析缺血性脑损伤的机制和在细胞水平上的保护机制特别有用。神经元和神经胶质细胞的原代培养物和纯星形胶质细胞培养物将来自海马和纹状体。结果将与对新皮层原代培养物进行的平行研究进行比较。此外，星形胶质细胞培养物将从用不同基因剂量的SOD 1或MnSOD（SOD 2）制备的转基因小鼠中产生，包括缺乏这些基因的敲除，以确定这些酶对于缺血样损伤的星形胶质细胞存活的重要性。这些研究将提供新的见解，可能的机制，保护三个候选基因的抗缺血基因治疗测试在三个大脑区域。

The protective potential of three groups of genes will be studied in ischemia-like injury of brain injury of brain cells from cortex, hippocampus, and striatum, in primary culture. First an antioxidant strategy will be tested by over-expressing CuZn superoxide dismutase (SOD1) using herpes virus and adenoviral vectors to achieve rapid expression in neurons and astrocytes, and retroviral vectors for prolonged, stable expression in astrocytes. Whether acute expression can provide protection will be tested. If this is not protective, the effect of prolonged stable expression and the use of bicistronic vectors, to rapidly express both SOD and a downstream antioxidant vectors, to rapidly express both SOD and a downstream antioxidant enzyme, will be tested. We will test for protective effects, as well as for induction of other antioxidant enzymes. Whether protection correlates with induction of other antioxidant enzymes will be determined. Under conditions where protections seen the extent of oxygen radical production, lipid peroxidation and changes in level of glutathione will be determined. Whether this gene protects against necrotic or apoptotic forms of cell death will be determined. Whether this gene protects against necrotic or apoptotic forms of cell death will be determined, as well as the time window in which expression can still protect, since it is important to develop therapeutic strategies that are effective after insults. Second, we will study the ability of Bcl-2 expression to protect in the same injury paradigms. Oxidative status and the time window in which Bcl-2 can protect will be determined. Third we will study the ability of the inducible heat shock protein 70 (HSP70) to protect from these injury again analyzing the time window during which this gene can protect and whether it blocks apoptotic or necrotic cell death. Primary cultures are particularly useful for analyzing mechanisms of ischemic brain injury and mechanisms of protection at the cellular level. Primary cultures of neurons and glial cells and pure astrocyte cultures will be mad3e from hippocampus and striatum. Results will be compared with parallel studies carried out on primary cultures from neocortex. In addition, astrocyte cultures will be produced from transgenic mice made with different gene dosages of SOD1 or MnSOD (SOD2), including knockouts lacking these genes, to determine the importance of these enzymes for astrocyte survival of ischemia-like insults. These studies will provide fresh insight into possible mechanisms of protection by three candidate genes for anti- ischemic gene therapy tested in three brain regions.