Microdialysis Studies of Seizure-Induced Oxidative Stress

描述(申请人提供)：本项目的目的是调查癫痫发作在氧化应激中的作用。大量研究表明，癫痫发作会导致活性氧和氮物种(ROS和RNS)的形成，从而对DNA、脂质和蛋白质造成氧化损伤。兴奋性毒性事件在癫痫发作中的作用以及随后导致氧化应激的ROS和RNS的形成将被研究。在离散的大脑区域进行微透析采样，同时记录皮层脑电活动，将用于探测与癫痫发作相关的大脑化学和脑电活动的变化。这将提供将生物标记物水平与癫痫发作活动相关联的能力。将使用微渗析采样来 持续监测癫痫发作诱导前、中、后的几个生化途径。微透析实验将提供有关癫痫发作引起的氧化应激以及大脑对其反应的时间和空间信息。这种方法将提供对使用血浆和尿液采样以及标准组织采样技术仍不清楚的问题的洞察。PI实验室以前开发的方法将用于监测ROS和RNS的形成，DNA损伤和脂质过氧化的生物标记物，以及神经递质氨基酸和儿茶酚胺。将开发一种新的分析方法来检测花生四烯酸代谢产生的前列腺素类化合物。将在Wistar大鼠身上使用三种癫痫实验模型。前两种模型使用3-巯基丙酸(3-MPA)以受控的方式化学诱导癫痫发作。对于一种模型，3-MPA是全身给药，而在另一种模型中，它是通过微透析探头局部给药。然后将使用化学点燃模型，其中将使用低于阈值剂量的戊四氮(用于触发癫痫发作)来产生增加的癫痫发作 强度，最终导致全身性癫痫痉挛。此外，我们建议研究不同的调节剂在癫痫发作过程中对已知氧化应激物释放的作用。使用微透析采样和先进的分析方法，将调查从癫痫发作到氧化应激的神经事件。特别是，氧化应激(通过ROS和RNS的形成来衡量)和神经兴奋(通过GABA/谷氨酸和儿茶酚胺的比率来衡量)之间的时间关系将被确定，并与癫痫发作的持续时间和强度相关。

DESCRIPTION (provided by applicant): The purpose of this project is to investigate the role of epileptic seizures in oxidative stress. There is a wealth of research that demonstrates that epileptic seizures lead to the formation of reactive oxygen and nitrogen species (ROS and RNS), which cause oxidative damage to DNA, lipids, and proteins. The role of excitotoxic events during epileptic seizures and the subsequent formation of ROS and RNS leading to oxidative stress will be investigated. Microdialysis sampling in discrete brain regions along with simultaneous recording of electrocorticographic (ECoG) activity will be used to probe chemical and electrographic activity changes in the brain associated with seizures. This will provide the ability to correlate biomarker levels with seizure activity. Microdialysis sampling will be used to continuously monitor several biochemical pathways prior to, during, and after induction of seizures. Microdialysis experiments will provide both temporal and spatial information about oxidative stress caused by seizures and the brains response to them. This approach will provide insight into questions that have remained unclear using plasma and urine sampling and standard tissue sampling techniques. Methods previously developed in the PI's laboratory will be used to monitor formation of ROS and RNS, biomarkers for DNA damage and lipid peroxidation, and the neurotransmitter amino acids and catecholamines. A new analytical method will be developed to detect prostanoids resulting from arachidonic acid metabolism. Three experimental models of epilepsy will be used in Wistar rats. The first two models use 3-mercaptopropionic acid (3-MPA) to chemically induce seizures in a controlled manner. For one model the 3-MPA is systemically administered and in the other it is locally administered through the microdialysis probe. A chemical kindling model will then be used, where a sub-threshold dose (for triggering seizures) of pentylenetetrazol will be used to generate seizures of increasing intensity, culminating in a generalized seizure convulsion. In addition, we propose to investigate the role of various modulation agents on the release of known oxidative stress agents during epileptic seizures. Using microdialysis sampling coupled to the advanced analytical methodology, the neurological events leading from seizures to oxidative stress will be investigated. In particular, the temporal relationship between oxidative stress (as measured by ROS and RNS formation), and neuroexcitation (as measured by the GABA/glutamate ratio and the catecholamines) will be determined and correlated to the duration and intensity of seizure episodes.