Determination of Lipid Peroxidation Associated with a Focal Seizure Model through In Vivo Microdialysis Sampling

Abstract

Two methods were developed to evaluate the extent of lipid peroxidation in an animal model of focal seizures. Microdialysis was used to sample from the extracellular fluid in specific regions of the hippocampus. The first method was a CE-fluorescence method for the thiobarbituric acid derivatized malondialdehyde (MDA) fluorophore. The second was an HPLC-MS method for seven eicosanoids; five products of the cyclooxygenase (COX) pathway (6-ketoprostaglandin F1α, thromboxane B2, prostaglandin F2, prostaglandin E2 and prostaglandin D2), one product of the lipooxygenase pathway (leukotriene B4), and one free radical byproduct of prostaglandin formation (8-isoprostaglandin F2). In a focal seizure model, a microdialysis probe was implanted in either the CA1 or CA3 region of the hippocampus. The chemical convulsant 3-mercaptopropionic acid (3-MPA) was dosed directly into the hippocampus by perfusion through the microdialysis probe for 50 minutes to produce a focal seizure. There were significant increases (p 0.001 compared to control) in MDA from 20 minutes after the start through 30 minutes after the end of dosing. It is hypothesized that these increases in MDA were related to the COX pathway. Therefore, an LC-MS method was developed and applied to the same animal model. Interestingly, there were decreases in the detectable prostaglandins (E2, D2, F2, and TXB2) during the 3-MPA dosing followed by immediate increases after dosing. It is thought that these prostaglandins are rapidly depleted during dosing from excessive oxidative damage.