Detection of Topoisomerase II Mediated DNA Damage in Stroke

描述（由申请人提供）：检测和分析脑中早期凋亡性细胞死亡的方法在开发和测试有效的治疗干预中是重要的。该提案的长期目标是开发一种新的方法，用于可视化组织切片中的早期细胞凋亡，并使用它来评估脑缺血期间细胞凋亡启动的动力学。本课题的主要目的是：1）建立一种原位检测细胞凋亡的新技术。2)应用新方法分析实验性脑卒中后早期细胞凋亡的动态变化。该方法将检测拓扑异构酶II的特定指纹，其参与早期细胞凋亡最近被证明。该方法将使用牛痘拓扑异构酶的独特酶性质来标记在细胞凋亡的初始阶段由拓扑异构酶II产生的特征性DNA断裂。细胞拓扑异构酶II在细胞凋亡开始时产生的具有4个碱基长的突出5'OH末端的先前不可检测类型的双链DNA断裂将被选择性地可视化。该技术将在凋亡和非凋亡DNA损伤的不同模型上进行测试，然后将应用于研究大鼠局灶性脑缺血。这种新的检测方法将有一个单细胞的灵敏度限制，并将是一个重要的除了凋亡检测武库，填补了差距的技术标记早期细胞凋亡的组织切片格式。它将允许更好地评估缺血性细胞损伤，并将有助于开发新的治疗缺血性脑损伤。它还将在早期细胞凋亡检测非常重要的所有生物医学领域中具有广泛的适用性。

DESCRIPTION (provided by applicant): Methods to detect and analyze early apoptotic cell death in brain are important in developing and testing efficient therapeutic interventions. The long-term objective of this proposal is to develop a new approach for visualization of early apoptosis in tissue sections and to use it to assess the dynamics of apoptosis initiation during brain ischemia. Specific Aims of this project are: 1) To develop a new technique for detection of initial stages of apoptosis in situ. 2) Use the newly developed approach to analyze the dynamics of early apoptosis in rat brain after experimental stroke. The approach will detect a specific fingerprint of topoisomerase II, whose involvement in early apoptosis was recently demonstrated. The method will use the unique enzymatic properties of vaccinia topoisomerase to label characteristic DNA breaks produced by topoisomerase II during the initial stages of apoptosis. The previously undetectable type of double-strand DNA breaks with 4 base-long protruding 5'OH ends, produced by cellular topoisomerase II at the start of apoptosis, will be selectively visualized. The technology will be tested on different models of apoptotic and nonapoptotic DNA damage, and will then be applied to study focal brain ischemia in rats. This new assay will have a single cell sensitivity limit and will be an important addition to the apoptosis detection arsenal, filing the gap in techniques for labeling early apoptosis in the tissue section format. It will permit better evaluation of ischemic cellular damage and will be helpful in development of new therapies for ischemic brain damage. It will also have a broad applicability in all fields of biomedicine where detection of early apoptosis is important.