Microdialysis Studies of Seizure-Induced Oxidative Stress

我们建议研究癫痫发作在氧化应激中的作用。有大量的研究表明，癫痫发作导致活性氧（ROS）的形成，导致DNA，脂质和蛋白质的氧化损伤。我们打算研究癫痫发作过程中兴奋毒性儿茶酚胺释放的作用及其在ROS形成中的作用，从而导致氧化应激。我们将使用微透析采样在离散的大脑区域沿着，同时记录皮层电图（ECoG）活动，以探测化学和脑电图活动的变化，在大脑中与癫痫发作。这将使我们能够将生物标志物水平与癫痫发作活动相关联。在诱导癫痫发作之前、期间和之后，将使用微透析采样来连续监测几种生化途径。微透析实验将提供有关癫痫发作引起的氧化应激和大脑对其反应的时间和空间信息。这种双重方法将使我们能够解决使用血浆和尿液采样以及标准组织采样技术仍然不清楚的问题。 PI实验室以前开发的方法将用于监测ROS的形成，DNA损伤的生物标志物，以及神经递质氨基酸和儿茶酚胺。将开发新的分析方法来监测通过脂质过氧化产生的醛类、参与保护免受ROS影响的内源性硫醇和二硫化物以及花生四烯酸代谢产生的前列腺素类。最初，我们将使用3-巯基丙酸（3-MPA）的化学诱导癫痫发作模型。结合先进的分析方法和微透析采样，我们将研究从癫痫发作到氧化应激的神经系统事件。特别地，将确定氧化应激（如通过ROS形成测量的）和神经兴奋（如通过GABA/谷氨酸比率和儿茶酚胺测量的）之间的时间关系，并将其与癫痫发作的持续时间和强度相关联。

We propose to investigate the role of epileptic seizures in oxidative stress. There is a wealth of research that demonstrates epileptic seizures lead to the formation of reactive oxygen species (ROS), which cause oxidative damage to DNA, lipids, and proteins. We intend to investigate the role of excitotoxic catecholamine release during epileptic seizures and their role in the formation of ROS leading to oxidative stress. We will use microdialysis sampling in discrete brain regions along with simultaneous recording of electrocorticographic (ECoG) activity to probe chemical and electrographic activity changes in the brain associated with seizures. This will allow us 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 dual approach will allow us to resolve 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, biomarkers for DNA damage, and the neurotransmitter amino acids and catecholamines. New analytical methods will be developed to monitor aldehydes produced through lipid peroxidation, endogenous thiols and disulfides involved in protection from ROS, and prostanoids resulting from arachidonic acid metabolism. Initially, we will use a chemically-induced seizure model using 3-mercaptopropionic acid (3-MPA). Coupled to the advanced analytical methodology and microdialysis sampling, we will then investigate the neurological events leading from seizures to oxidative stress. In particular, the temporal relationship between oxidative stress (as measured by ROS 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.