Detection of Topoisomerase II Mediated DNA Damage in Stroke

描述（申请人提供）：检测和分析脑早期凋亡细胞死亡的方法对于开发和测试有效的治疗干预措施非常重要。本研究的长期目标是开发一种新的方法来可视化组织切片中的早期细胞凋亡，并利用它来评估脑缺血时细胞凋亡启动的动力学。本项目的具体目标是：1)开发一种原位检测细胞凋亡初始阶段的新技术。2)应用新方法分析实验性脑卒中后大鼠脑早期凋亡动态。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。该方法将利用牛痘拓扑异构酶的独特酶性质来标记拓扑异构酶II在细胞凋亡初始阶段产生的特征DNA断裂。在细胞凋亡开始时，由细胞拓扑异构酶II产生的具有4个碱基长的突出5'OH末端的双链DNA断裂类型将选择性地可视化。该技术将在不同的细胞凋亡和非细胞凋亡DNA损伤模型上进行测试，然后应用于大鼠局灶性脑缺血的研究。这种新的检测方法将具有单细胞灵敏度限制，并将成为细胞凋亡检测库的重要补充，填补了在组织切片格式中标记早期细胞凋亡的技术空白。这将有助于更好地评估缺血性细胞损伤，并有助于开发缺血性脑损伤的新疗法。它也将广泛适用于早期细胞凋亡检测的所有生物医学领域。

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.