In Vivo Microdialysis Studies of Oxidative Stress

描述（由申请方提供）：本提案的重点是使用微透析采样进行体内氧化应激研究。氧化应激产生活性氧物质，最终产生自由基，对DNA和其他生物分子造成损害。我们建议在心脏和大脑缺血和再灌注期间，在特定组织部位使用体内微透析取样来研究氧化应激的生理学。微透析将用于监测氧自由基（OFR），一种可作为保护剂（谷胱甘肽）的内源性抗氧化剂，以及氧化性DNA损伤（8-羟基脱氧鸟苷）和脂质过氧化（丙二醛）的生物标志物。为了进行拟议的研究，有必要首先开发一套分析方法来确定微透析样品中的每种目标分析物。微透析取样产生几微升或更少的样品。因此，基于毛细管电泳（CE）的方法， 胶束电动色谱（MEKC）和毛细管电动色谱（CEC）将得到发展。修饰的核苷和抗氧化剂是带电的和电活性的。将使用通过微透析探针引入的自由基捕获剂来稳定OFR，所述自由基捕获剂产生电活性产物。所有这些分析物都将使用电化学方法进行检测。将丙二醛衍生为荧光分析物，并通过激光诱导荧光法进行测定。OFR生产的作用，然后将在体内研究缺血和再灌注期间在心脏和大脑中使用微透析采样和新开发的分析方法。研究的目的是阐明缺血和再灌注过程中OFR形成的各种机制的作用。特别是，黄嘌呤氧化酶在脑中OFR的产生中所起的作用将被研究。将在心脏和大脑中研究作为儿茶酚胺释放的函数的OFR形成。微透析将用于局部施用黄嘌呤氧化酶抑制剂以及体内儿茶酚胺释放和吸收抑制剂，以确定这些途径的作用。

DESCRIPTION (provided by applicant): The focus of this proposal is the study of oxidative stress in vivo using microdialysis sampling. Oxidative stress produces reactive oxygen species and ultimately free radicals that cause damage to DNA and other biomolecules. We propose to study the physiology of oxidative stress using in vivo microdialysis sampling at specific tissue sites during ischemia and reperfusion in the heart and brain. Microdialysis will be used to monitor oxygen free radicals (OFRs), an endogenous antioxidant that may serve as a protective agent (glutathione), and biomarkers for oxidative DNA damage (8-hydroxydeoxyguanosine) and lipid peroxidation (malondialdehye). In order to carry out the proposed studies, it will be necessary to first develop a suite of analytical methods to determine each of the target analytes in microdialysis samples. Microdialysis sampling results in samples of a few microliters or less. Therefore, methods based on capillary electrophoresis (CE), micellar electrokinetic chromatography (MEKC), and capillary electrokinetic chromatography (CEC) will be developed. The modified nucleosides and antioxidants are charged and electroactive. OFRs will be stabilized using radical trapping agents introduced through the microdialysis probe that produce electroactive products. All of these analytes will be detected using electrochemical methods. Malondialdehyde will be derivatized to a fluorescent analyte and determined by laser induced fluorescence. The role of OFR production will then be studied in vivo during ischemia and reperfusion in the heart and brain using microdialysis sampling and the newly developed methods of analysis. Studies will be aimed at elucidating the role of various proposed mechanisms of OFR formation during ischemia and reperfusion. In particular, the role xanthine oxidase plays in the production of OFRs in the brain will be investigated. OFR formation as a function of catecholamine release will be studied in both the heart and brain. Microdialysis will be used to locally administer inhibitors of xanthine oxidase and of catecholamine release and uptake in vivo to determine the role of these pathways.