Allopurinol and ellagic acid decrease epileptiform activity and the severity of convulsive behavior in a model of status epilepticus.

BACKGROUND: During status epilepticus, severe seizures can occur, generating recurrent cycles of excitotoxicity and oxidative stress that cause neuronal damage and cell death. The administration of agents with antioxidant properties represents a therapeutic alternative aimed at reducing the severity of status epilepticus and mitigating the neurobiological consequences that precede them. OBJECTIVE: The objective of this work was to evaluate the antiseizure effect of the antioxidants allopurinol (ALL) and ellagic acid during status epilepticus induced by pilocarpine (PILO). METHODS: Male Wistar rats (200-250 g) were injected with ALL (50 mg/kg) or ellagic acid (50 mg/kg), 30 min before PILO administration (pretreatment) or 60 min after the beginning of status epilepticus, to evaluate the antiseizure effect of these drugs on epileptiform activity and convulsive behavior. RESULTS: ALL or ellagic acid administration before or after PILO significantly decreased the epileptiform activity and the severity of convulsive behavior. Better efficacy was observed when the drugs were administered as a pretreatment, increasing the latency time of the appearance of status epilepticus from 27.2 ± 2.6 to 45.8 ± 3.31 min, and significantly reducing the amplitude of epileptiform discharges by 53.5% with ALL and 68.9% with ellagic acid. CONCLUSION: The antioxidants ALL and ellagic acid showed an antiseizure effect, representing an alternative to reduce epileptiform activity and severity of convulsive behavior during status epilepticus, an effect that may be used as adjuvants to mitigate or reduce oxidative damage processes.

Ellagic acid and allopurinol decrease H2O2 concentrations, epileptiform activity and astrogliosis after status epilepticus in the hippocampus of adult rats.

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. The purpose of this study was to evaluate the effects of ellagic acid and allopurinol administered after SE on H2O2 concentrations, electrical activity and GFAP immunoreactivity in the hippocampus of rats evaluated on Day 18 after SE. H2O2 levels were measured using an online technique with high temporal resolution and simultaneous electrical activity recording. For this purpose, the lateral ventricles of male Wistar rats (200-250 g) were injected with pilocarpine (2.4 mg/2 µl) to induce SE. After SE, rats were injected with ellagic acid (50 mg/kg i.p., and two additional doses at 24 and 48 h) or allopurinol (50 mg/kg i.p., single dose). Administration of ellagic acid or allopurinol after SE significantly reduced the H2O2 concentrations and decreased the presence of epileptiform activity and GFAP immunoreactivity in the hippocampus 18 days after SE. In conclusion, the administration of antioxidants potentially reduces oxidative stress, which indicates the possible attenuation of the neurobiological consequences after SE.

Hydrogen peroxide extracellular concentration in the ventrolateral medulla and its increase in response to hypoxia in vitro: Possible role of microglia.

Hydrogen peroxide (H2O2) is a messenger involved in both damaging neuroinflammatory responses and physiological cell communication. The ventrolateral medulla, which regulates several vital functions including breathing and blood pressure, is highly influenced by hydrogen peroxide, whose extracellular levels could be determined by hypoxia and microglial activity, both of which modulate ventrolateral medulla function. Therefore, in this study we aimed to test whether different patterns of hypoxia and/or putative microglial modulators change extracellular hydrogen peroxide in the ventrolateral medulla by using an enzymatic reactor online sensing procedure specifically designed for this purpose. With this new technique, we detected extracellular levels of hydrogen peroxide in the ventrolateral medulla in vitro, which spontaneously fluctuated. These fluctuations are reduced by minocycline, a putative microglial inhibitor, and by the microglial toxin liposomal clodronate. Suitably, lipopolysaccharide increases extracellular hydrogen peroxide, while minocycline and liposomal clodronate reduce this increase. Application of blue light to slices with microglia expressing channelrhodopsin-2 also increases extracellular hydrogen peroxide. Moreover, long-lasting and intermittent hypoxia (as well as subsequent reoxygenation) increase extracellular hydrogen peroxide to similar levels, which is partially prevented by minocycline. The effect of long-lasting hypoxia was reproduced in vivo. Overall, our data show that changes in oxygen concentration, and possibly microglial function, modulate extracellular H2O2 levels in the ventrolateral medulla, which could influence the function of this neural circuit under normal and pathological conditions related to inflammation and/or hypoxia.

γ-Aminobutyric acid quantification in small volume biological samples through enzymatically induced electrochemiluminescence.

γ-Aminobutyric acid (GABA) is a well-known neurotransmitter that regulates inhibitory neurotransmission in the mammalian central nervous system and participates in several processes outside the brain. A reliable quantification method is needed to determine its role in different physiological and pathological conditions. However, GABA measurements have several challenges because GABA is neither fluorescent nor electroactive, and it is difficult to detect using enzymatic reactions because no oxidases or dehydrogenases have been identified. Several methods have been developed to quantify GABA concentrations based on the instrumentation available, the sensitivity required, and the volume of samples analyzed. Most of these methods use high-performance liquid chromatography (HPLC). Here, we describe a method for quantifying GABA concentrations in small volume samples using enzymatically-induced electrochemiluminescence with the well-known GABAse complex, which produces glutamate for use in a luminescent reaction with glutamate oxidase and luminol in an electrochemiluminescence cell. The luminescence obtained was proportional to the GABA concentrations in the micromolar range (1-1000), with linear r2 values > 0.95. GABA standards were treated with the enzymatic reactors to generate glutamate (Glu), which was measured simultaneously with an HPLC technique, to validate this new procedure. The assay was further used to determine GABA concentrations in hippocampal extracts. This alternative may be used to quantify GABA levels in fluid samples, such as microdialysates, other perfusates and tissue extracts. Thus, the method presented here is a good alternative for monitoring GABA levels with good sensitivity compared with the traditional methods that are still in use.

A novel online fluorescence method for in-vivo measurement of hydrogen peroxide during oxidative stress produced in a temporal lobe epilepsy model.

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.

A microdialysis and enzymatic reactor sensing procedure for the simultaneous registration of online glutamate measurements at high temporal resolution during epileptiform activity.

Glutamate measurement in microdialysis samples has primarily been determined using HPLC methods, and several attempts have been made to establish a relationship between this neurotransmitter and EEG activity during altered brain function, such as epilepsy. However, classic microdialysis methods lack high temporal resolution. In this study, a new alternative is proposed to improve the time resolution and thus obtain a better understanding of the dynamics of Glu and its relationship with epileptiform activity. A new setup was designed to measure Glu online in microdialysates using enzymatic reactors and fluorescence detection. In this study, we performed EEG recordings and Glu measurements simultaneously in the hippocampus to establish their relationship with the epileptiform events that are induced by pentylenetetrazole in intact and epileptic rats. Basal Glu levels in intact and animals with spontaneous seizures were not significantly different. However, a significant increase in Glu levels was detected during the first pentylenetetrazole-induced seizure in both groups. EEG analysis showed that the amplitude of epileptiform activity was higher in rats with spontaneous seizures and that the frequency of this activity did not change. The results showed that this method can be used to determine Glu changes at high temporal resolution and that these changes can be related to seizure activity. In addition, this method can also be used to measure other neurotransmitters that generate fluorescent derivatives. Moreover, this new technique has the following advantages compared with classical neurochemical methods: easy setup, low training requirements, no need for separation, rapidity, and the experimental data can be obtained and analysis performed in a single session.

Increase in the extracellular glutamate level during seizures and electrical stimulation determined using a high temporal resolution technique.

BACKGROUND: Glutamate 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. METHODS: A 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. RESULTS: The 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. CONCLUSIONS: These 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.

Evaluation of behavioral parameters and mortality in a model of temporal lobe epilepsy induced by intracerebroventricular pilocarpine administration.

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.

An algorithm for on-line detection of high frequency oscillations related to epilepsy.

Recent studies suggest that the appearance of signals with high frequency oscillations components in specific regions of the brain is related to the incidence of epilepsy. These oscillations are in general small in amplitude and short in duration, making them difficult to identify. The analysis of these oscillations are particularly important in epilepsy and their study could lead to the development of better medical treatments. Therefore, the development of algorithms for detection of these high frequency oscillations is of great importance. In this work, a new algorithm for automatic detection of high frequency oscillations is presented. This algorithm uses approximate entropy and artificial neural networks to extract features in order to detect and classify high frequency components in electrophysiological signals. In contrast to the existing algorithms, the one proposed here is fast and accurate, and can be implemented on-line, thus reducing the time employed to analyze the experimental electrophysiological signals.

Effect of early glutamate exposure on EAAT-3 and GAT-1 protein expression in cells of the dentate gyrus and CA1 region of the adult rat hippocampus.

BACKGROUND AND AIMS: Glutamate and GABA transporters are cell surface proteins localized on neurons and glial cells that mediate the reuptake of glutamate and GABA from the extracellular space. In different models of the acquisition of epilepsy, important changes in the expression of these transporters have been demonstrated, although to date no such studies have been performed using the monosodium glutamate (MSG)-induced seizure model in neonatal rats. METHODS: Following repeated MSG administration, we used immunofluorescence techniques to quantify the number of cells expressing the EAAT-3 and GAT-1 transporters at postnatal days (PD) 14 and 60 in the dentate gyrus (DG) and CA1 region of the hippocampus. RESULTS: EAAT-3 and GAT-1 were expressed around the soma of granular cells and in the soma and dendrites of pyramidal cells in both experimental (MSG) and control (NaCl) rats. In the DG, MSG administration significantly increased the number of granular cells double-labelled for EAAT-3/Neun at PD 60 but not PD 14. No significant changes were observed at either PD 14 or 60 in terms of the number of cells expressing GAT-1 in the DG or CA1. CONCLUSIONS: The findings suggest that the selective long-term increase in EAAT-3 expression in granular cells following neonatal MSG treatment reflects an important compensatory or protective response to the excitotoxic and seizure-promoting effects of early glutamate exposure in adult animals.