Neurochemical Investigation of Locally Induced Epilepsy and Subsequent Oxidative Damage Using Microdialysis Sampling

Abstract

The goal of this research was to develop and understand an anesthetized, multiple-seizure rat model for local epilepsy. Local seizures are not as well understood as global seizures due to their specificity and unpredictability. Furthermore, patients are diagnosed with epilepsy after experiencing two or more unprovoked seizures. In this model, two separate seizure episodes were induced by locally administering the epileptic agent 3-mercaptopropionic acid through a microdialysis probe to the CA1 region of the hippocampus. Upon development of the model, attenuation in glutamate release was observed in the second seizure stimulation. To investigate neurochemical and biochemical pathways which may be responsible for the glutamate diminution, the perfusion fluid was spiked with either glucose, lactate, or dihydrokainic acid. Additionally, as it is well known that high levels of extracellular glutamate can result in excitotoxicity, neuronal staining was performed to determine the neuronal viability after the induction of the first seizure. It was determined that the attenuation in glutamate release in the second seizure episode was primarily due to a combination of mitochondrial starvation and cell damage. The local seizure model was then used to correlate local seizure induction to oxidative damage. Glutathione (GSH) and malondialdehyde (MDA) were selected as biomarkers of oxidative stress. Intracellular levels GSH were up regulated and down regulated in hopes of modifying the amount of seizure induced oxidative damage. There was no statistically significant change in MDA formation with changing GSH levels; however, GSH did appear to serve as a release modifier of the redox cycle. Extracellular GSH increased significantly during the seizure induction and returned to basal after the seizure ended. This increase in extracellular GSH concentration can be accounted for by astrocytes and glial cells releasing GSH to counteract reactive oxygen species produced during the seizure. Additional experiments need to be done in order to make further conclusions; however, it is evident that there is a correlation between seizures and oxidative stress. Finally, Appendix I describes a small in vitro pharmacokinetic project using microdialysis sampling to measure plasma protein binding values of commercially available drugs with the ultimate goal of applying the technique for in vivo studies.