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BackgroundGlutamate has been measured using different methods to determine its role under normal and pathological conditions. Although microdialysis coupled with HPLC is the preferred method to study glutamate, this technique exhibits poor temporal resolution and is time consuming. The concentration of glutamate in dialysis samples can be measured via glutamate oxidase using the Amplex Red method.MethodsA new device has been designed and constructed to rapidly deposit dialysis samples onto a polycarbonate plate at Cartesian coordinates (every five seconds). The samples were added to an enzymatic reaction that generates hydrogen peroxide from glutamate, which was quantified using fluorescence detection. Fluorescence emission was induced by laser excitation, stimulating each spot automatically, in addition to controlling the humidity, temperature and incubation time of the enzymatic reaction.ResultsThe measurement of standard glutamate concentrations was linear and could be performed in dialysis samples. This approach was used to determine the effect of the convulsant drugs bicuculline and 4-aminopyridine on the extracellular glutamate concentration. Seizure activity was associated with a considerable increase in glutamate that correlated with altered EEG patterns for both drugs.ConclusionsThese results indicate that this method is able to read samples with high temporal resolution, and it is easy to use compared with classical methods such as high-performance liquid chromatography, with the advantage that a large number of samples can be measured in a single experimental series. This method provides an alternative approach to determine the concentrations of neurotransmitters or other compounds that generate hydrogen peroxide as a reaction product.

The pilocarpine model of temporal lobe epilepsy (TLE) is a useful tool that is used to investigate the mechanisms underlying the generation and maintenance of seizures. Although this model has been modified significantly to reduce mortality and to promote the appearance of spontaneous recurrent seizures, to date, no detailed evaluation has been performed of the behavioral parameters and mortality in TLE induced by intracerebroventricular pilocarpine administration; therefore, this was the goal of the present study. A single dose of pilocarpine hydrochloride (2.4 mg in a total volume of 2 µl) was injected into the right lateral brain ventricle of rats; the convulsive behavior was rated using the Racine scale and the mortality was analyzed in these animals. We found that 30-90 min after animals received intracerebroventricular pilocarpine injections, 73% developed status epilepticus (SE) with an activity score of 4/5 on the Racine scale. Moreover, these seizures were associated with the propagation of epileptiform activity to different hippocampal regions. Of the animals that developed SE, spontaneous recurrent seizures were observed in 32.5% at different times after SE induction. A 35% mortality rate was observed, which included animals that died during pilocarpine injection and after SE induction. On the basis of these findings, and given the observed latency between the insult (SE induction by pilocarpine injection) and the manifestation of spontaneous recurrent seizures, we propose that this model is a useful tool for basic biomedical research of SE and TLE.

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Status epilepticus (SE) can result in an overproduction of hydrogen peroxide (H2O2), which contributes to oxidative stress and brain injury during different phases of epileptogenesis and seizures. In this study, we measured the extracellular H2O2 concentration in the rat hippocampus in a temporal lobe epilepsy model. A new fluorescent technique for measuring H2O2 in vivo simultaneously with electroencephalography recording was tested. The method consists of mixing microdialysate with an enzymatic reactor to produce a fluorescent compound. The fluorescence intensity was measured every second and was proportional to the H2O2 concentration. The results showed that H2O2 was released during SE; we detected a significant increase of up to five times over the baseline value that correlated with changes in electrical activity. We also observed that H2O2 was produced for days after SE and was associated with continuous neuronal death and seizure generation. Therefore, we monitored H2O2 48 h and 15 days after SE, observing increases of up to 96 and 124%, respectively, accompanied by changes in electrical activity with spontaneous discharges of large amplitude. These changes may reflect the oxidative stress generated during epileptogenesis that remains during the chronic period (458% increased) with the presence of large spikes, indicating that the H2O2 could also participate in the generation and maintenance of spontaneous recurrent seizures. There are no previous reports on the detection of H2O2 at this temporal resolution; thus, this study contributes a novel technique for studying and understanding epileptogenesis to develop new antioxidant strategies for the treatment of temporal lobe epilepsy.