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.
Epilepsy affects over 1% of the world's population. The goal of this project is to elucidate the brain's response to oxidative stress resulting from seizure activity. Revised Budget The proposed specific aims for this two year funding period cover aims that were initially proposed to require 2.5 years to 3 years to accomplish. Four changes to the original budget request are necessary to accomplish this work. First, the work will be done by four graduate research assistants (GRAs) rather than one postdoctoral student and three graduate research assistants. The main reason for this change is that the four GRAs are already working in my laboratory and sufficiently trained to perform the research while a postdoctoral student would need to be hired which could take several months. The second change is to increase the PIs effort from 1 summer month (8.33%) to 2 summer months (16.66%). Because of the tighter timeline and not hiring a postdoctoral student, the PI will need to expend significantly more time directly supervising this project. These changes do not significantly change the net salary and fringe request of the budget but reflect the shorter timeline of the project. The third change is to move the purchase of the ECoG instrument from the second year to the first year in order to begin the initial animal studies earlier. This does not result in the net amount of funds requested but merely reallocates between the first and second year. The fourth change is to request additional equipment money in the first year to purchase an HPLC system. This will allow us to begin the animal studies using an existing method specific to glutathione and glutathione disulfide while the more general thiol/disulfide method is under development. The additional HPLC instrument will allow us to perform more analyses in parallel in order to increase throughput and begin routine analyses at an earlier time.
