INDUCIBLE DNA REPAIR IN CEREBRAL ISCHEMIA

描述(改编自申请者摘要)：最近的研究 DNA氧化损伤在脑内介导神经元死亡中的作用 脑缺血和神经退行性疾病。人们对此知之甚少 神经元DNA修复机制对缺血损伤和氧化DNA的响应 损坏。该项目的广泛、长期目标是确定 诱导性DNA切除修复在细胞死亡调控中的作用 急性缺血性脑损伤。具体目标是：(1)确定 在体局灶性大鼠模型中可诱导的DNA切除修复活性 缺血和再灌流。(2)确定可诱导的DNA切除修复 机械学定义的神经元缺血体外模型的活性 和神经细胞死亡。(3)测定选定的DNA修复活性 大鼠局灶性脑缺血的酶活性及DNA修复基因的表达 以及缺血和神经细胞死亡的体外细胞培养模型。(4) 确定选择性DNA修复酶在诱导性DNA切除中的作用 局灶性缺血的修复以及缺血和神经元死亡的体外模型， 通过改变这些酶的活性。体外模型允许 需要进行精确的机理研究，但复杂的相互依存 活体缺血后发生的病理生理变化不能 完全是模特儿。因此，两种原代皮质神经元培养 并采用大鼠暂时性大脑中动脉闭塞模型。 单、双和总DNA断裂的检测方法将用于 研究这些模型中DNA损伤的演变。的能力 全细胞提取液通过碱基切除修复质粒DNA损伤 或核苷酸切除修复将用于检测DNA修复活性 体内和体外。DNA修复酶的表达和活性将 以及对这些酶的药理抑制作用 研究DNA损伤和神经元存活情况。

DESCRIPTION (Adapted from Applicant's Abstract): Recent studies have implicated oxidative DNA damage in mediating neuronal death in cerebral ischemia and in neurodegenerative diseases. Little is known about how neuronal DNA repair mechanisms respond to ischemic injury and oxidative DNA damage. The broad, long-term objective of this project is to identify the role of inducible DNA excision repair in the regulation of cell death from acute ischemic brain injury. The specific objectives are: (1) Determine inducible DNA excision repair activity in an in vivo rat model of focal ischemia and reperfusion. (2) Determine inducible DNA excision repair activity in mechanist-ically defined in vitro models of neuronal ischemia and neuronal cell death. (3) Determine activity of selected DNA repair enzymes and expression of DNA repair genes in vivo focal cerebral ischemia and in vitro cell culture models of ischemia and neuronal cell death. (4) Determine the role of selective DNA repair enzymes in inducible DNA excision repair in focal ischemia and in vitro models of ischemia and neuronal death, by altering the activity of these enzymes. in vitro models allow for precise mechanistic studies to be performed, but the complex inter-dependent pathophysiologic changes that occur after ischemia in vivo cannot be completely modeled. Accordingly, both primary cortical neuronal cultures and rat temporary middle cerebral artery occlusion models will be used. Methods for detection of single, double and total DNA breaks will be used to study the evolution of DNA damage in these models. The ability of whole-cell extracts to repair damage to plasmid DNA by base excision repair or nucleotide excision repair will be used to assay DNA repair activity in vivo and in vitro. The expression and activity of DNA repair enzymes will be studied and the effect of pharmacologic inhibition of these enzymes upon DNA damage and neuronal survival investigated.