Starting from simple clinical statistics, the spectrum of methods used in epilepsy research in the Institute of Physiology of the Czechoslovak (now Czech) Academy of Sciences progressively increased. Professor Servít used electrophysiological methods for study of brain activity in lower vertebrates, neuropathology was focused on electronmicroscopic study of cortical epileptic focus and ion-sensitive microelectrodes were used for studies of cortical direct current potentials. Developmental studies used electrophysiological methods (activity and projection of cortical epileptic foci, EEG under the influence of convulsant drugs, hippocampal, thalamic and cortical electrical stimulation for induction of epileptic afterdischarges and postictal period). Extensive pharmacological studies used seizures elicited by convulsant drugs (at first pentylenetetrazol but also other GABA antagonists as well as agonists of glutamate receptors). Motor performance and behavior were also studied during brain maturation. The last but not least molecular biology was included into the spectrum of methods. Many original data were published making a background of position of our laboratory in the first line of laboratories interested in brain development.
- MeSH
- Academies and Institutes MeSH
- Biomedical Research trends MeSH
- History, 20th Century MeSH
- History, 21st Century MeSH
- Epilepsy * physiopathology MeSH
- Humans MeSH
- Brain drug effects physiology growth & development MeSH
- Animals MeSH
- Check Tag
- History, 20th Century MeSH
- History, 21st Century MeSH
- Humans MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Historical Article MeSH
- Review MeSH
- Geographicals
- Czech Republic MeSH
The present study aimed to elucidate the effect of sulforaphane (a natural isothiocyanate) on oxidative stress and mitochondrial dysfunction during and at selected periods following status epilepticus (SE) induced in immature 12-day-old rats by Li-pilocarpine. Dihydroethidium was employed for the detection of superoxide anions, immunoblot analyses for 3-nitrotyrosine (3-NT) and 4-hydroxynonenal (4-HNE) levels and respiratory chain complex I activity for evaluation of mitochondrial function. Sulforaphane was given i.p. in two doses (5 mg/kg each), at PD 10 and PD 11, respectively. The findings of the present study indicate that both the acute phase of SE and the early period of epileptogenesis (1 week and 3 weeks following SE induction) are associated with oxidative stress (documented by the enhanced superoxide anion production and the increased levels of 3-NT and 4-HNE) and the persisting deficiency of complex I activity. Pretreatment with sulforaphane either completely prevented or significantly reduced markers of both oxidative stress and mitochondrial dysfunction. Since sulforaphane had no direct anti-seizure effect, the findings suggest that the ability of sulforaphane to activate Nrf2 is most likely responsible for the observed protective effect. Nrf2-ARE signaling pathway can be considered a promising target for novel therapies of epilepsy, particularly when new compounds, possessing inhibitory activity against protein-protein interaction between Nrf2 and its repressor protein Keap1, with less "off-target" effects and, importantly, with an optimal permeability and bioavailability properties, become available commercially.
- Keywords
- Immature rats, Mitochondrial dysfunction, Oxidative stress, Protection, Status epilepticus, Sulforaphane,
- MeSH
- NF-E2-Related Factor 2 * metabolism MeSH
- Isothiocyanates pharmacology MeSH
- Kelch-Like ECH-Associated Protein 1 metabolism MeSH
- Rats MeSH
- Mitochondria metabolism MeSH
- Oxidative Stress MeSH
- Status Epilepticus * metabolism MeSH
- Sulfoxides metabolism pharmacology MeSH
- Superoxides metabolism MeSH
- Animals MeSH
- Check Tag
- Rats MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Names of Substances
- NF-E2-Related Factor 2 * MeSH
- Isothiocyanates MeSH
- Kelch-Like ECH-Associated Protein 1 MeSH
- sulforaphane MeSH Browser
- Sulfoxides MeSH
- Superoxides MeSH
Status epilepticus (SE) is a common paediatric emergency with the highest incidence in the neonatal period and is a well-known epileptogenic insult. As previously established in various experimental and human studies, SE induces long-term alterations to brain metabolism, alterations that directly contribute to the development of epilepsy. To influence these changes, organic isothiocyanate compound sulforaphane (SFN) has been used in the present study for its known effect of enhancing antioxidative, cytoprotective, and metabolic cellular properties via the Nrf2 pathway. We have explored the effect of SFN in a model of acquired epilepsy induced by Li-Cl pilocarpine in immature rats (12 days old). Energy metabolites PCr, ATP, glucose, glycogen, and lactate were determined by enzymatic fluorimetric methods during the acute phase of SE. Protein expression was evaluated by Western blot (WB) analysis. Neuronal death was scored on the FluoroJadeB stained brain sections harvested 24 h after SE. To assess the effect of SFN on glucose metabolism we have performed a series of 18F-DG μCT/PET recordings 1 h, 1 day, and 3 weeks after the induction of SE. Responses of cerebral blood flow (CBF) to electrical stimulation and their influence by SFN were evaluated by laser Doppler flowmetry (LDF). We have demonstrated that the Nrf2 pathway is upregulated in the CNS of immature rats after SFN treatment. In the animals that had undergone SE, SFN was responsible for lowering glucose uptake in most regions 1 h after the induction of SE. Moreover, SFN partially reversed hypometabolism observed after 24 h and achieved full reversal at approximately 3 weeks after SE. Since no difference in cell death was observed in SFN treated group, these changes cannot be attributed to differences in neurodegeneration. SFN per se did not affect the glucose uptake at any given time point suggesting that SFN improves endogenous CNS ability to adapt to the epileptogenic insult. Furthermore, we had discovered that SFN improves blood flow and accelerates CBF response to electrical stimulation. Our findings suggest that SFN improves metabolic changes induced by SE which have been identified during epileptogenesis in various animal models of acquired epilepsy.
- Keywords
- brain, cerebral blood flow (CBF), glucose metabolism, immature rat, pilocarpine, status epilepticus, sulforaphane, μCT/PET,
- Publication type
- Journal Article MeSH
The aim of the present study was to elucidate the effect of resveratrol (natural polyphenol) on seizure activity, production of ROS, brain damage and mitochondrial function in the early phase of status epilepticus (SE), induced in immature 12 day-old rats by substances of a different mechanism of action (Li-pilocarpine, DL-homocysteic acid, 4-amino pyridine, and kainate). Seizure activity, production of superoxide anion, brain damage and mitochondrial function were assessed by EEG recordings, hydroethidium method, FluoroJadeB staining and Complex I activity measurement. A marked decrease of complex I activity associated with the acute phase of SE in immature brain was significantly attenuated by resveratrol, given i.p. in two or three doses (25 mg/kg each), 30 min before, 30 or 30 and 60 min after the induction of SE. Increased O2 .- production was completely normalized, brain damage partially attenuated. Since resveratrol did not influence seizure activity itself (latency, intensity, frequency), the mechanism of protection is likely due to its antioxidative properties. The findings have a clinical relevance, suggesting that clinically available substances with antioxidant properties might provide a high benefit as an add-on therapy during the acute phase of SE, influencing also mechanisms involved in the development of epilepsy.
There are numerous studies supporting the contribution of oxidative stress to the pathogenesis of epilepsy. Prolonged oxidative stress is associated with the overexpression of ATP-binding cassette transporters, which results in antiepileptic drugs resistance. During our studies, three 1,2,4-triazole-3-thione derivatives were evaluated for the antioxidant activity and anticonvulsant effect in the 6 Hz model of pharmacoresistant epilepsy. The investigated compounds exhibited 2-3 times more potent anticonvulsant activity than valproic acid in 6 Hz test in mice, which is well-established preclinical model of pharmacoresistant epilepsy. The antioxidant/ROS scavenging activity was confirmed in both single-electron transfer-based methods (DPPH and CUPRAC) and during flow cytometric analysis of total ROS activity in U-87 MG cells. Based on the enzymatic studies on human carbonic anhydrases (CAs), acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), one can assume that the herein investigated drug candidates will not impair the cognitive processes mediated by CAs and will have minimal off-target cholinergic effects.
- Keywords
- 6 Hz psychomotor seizures, carbonic anhydrases, cholinesterase inhibitors, mitochondrial potential, total ROS activity,
- MeSH
- Acetylcholinesterase metabolism MeSH
- Anticonvulsants chemical synthesis chemistry pharmacology MeSH
- Antioxidants chemical synthesis chemistry pharmacology MeSH
- Biphenyl Compounds antagonists & inhibitors MeSH
- Butyrylcholinesterase metabolism MeSH
- Cholinesterase Inhibitors chemical synthesis chemistry pharmacology MeSH
- Epilepsy drug therapy metabolism MeSH
- Carbonic Anhydrase Inhibitors chemical synthesis chemistry pharmacology MeSH
- Carbonic Anhydrases metabolism MeSH
- Humans MeSH
- Models, Molecular MeSH
- Molecular Structure MeSH
- Mice MeSH
- Cell Line, Tumor MeSH
- Oxidative Stress drug effects MeSH
- Picrates antagonists & inhibitors MeSH
- Reactive Oxygen Species metabolism MeSH
- Triazoles chemical synthesis chemistry pharmacology MeSH
- Cell Survival drug effects MeSH
- Dose-Response Relationship, Drug MeSH
- Structure-Activity Relationship MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Mice MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Names of Substances
- 1,1-diphenyl-2-picrylhydrazyl MeSH Browser
- 1,2,4-triazole MeSH Browser
- Acetylcholinesterase MeSH
- Anticonvulsants MeSH
- Antioxidants MeSH
- Biphenyl Compounds MeSH
- Butyrylcholinesterase MeSH
- Cholinesterase Inhibitors MeSH
- Carbonic Anhydrase Inhibitors MeSH
- Carbonic Anhydrases MeSH
- Picrates MeSH
- Reactive Oxygen Species MeSH
- Triazoles MeSH
The potency of three nerve agents (sarin, soman, tabun) to induce oxidative damage of DNA in lymphocytes, liver and brain during lethal or sublethal poisoning was investigated. The single strand breaks or oxidative base DNA damage was evaluated with the help of Comet assay and a specific enzyme able to detect oxidative bases of DNA (endonuclease III). While sarin and soman administered at sublethal doses corresponding to 50% of their LD50 values were not able to induce oxidative damage of DNA, their lethal dose (LD50) induced the significant increase of the number of oxidative bases in DNA of hepatocytes. In addition, tabun administered at lethal dose (LD50) induced significant increase of the number of single strand breaks and oxidative bases of DNA in glial cells isolated from pontomedullar brain region. Thus, some nerve agents were able to induce oxidative damage in the peripheral as well as central compartment but only in the case of severe poisoning caused by lethal doses of nerve agents. This non-cholinergic effect of nerve agents has probably consequences with nerve agents-induced hypoxic status during acute cholinergic crisis and it can contribute to their long-term toxic effects.
- Keywords
- Comet assay, DNA, Oxidative damage, Rats, Sarin, Soman, Tabun,
- Publication type
- Journal Article MeSH
Epilepsy is characterized by the regular occurrence of seizures, which follow a stereotypical sequence of alterations in the electroencephalogram. Seizures are typically a self limiting phenomenon, concluding finally in the cessation of hypersynchronous activity and followed by a state of decreased neuronal excitability which might underlie the cognitive and psychological symptoms the patients experience in the wake of seizures. Many efforts have been devoted to understand how seizures spontaneously stop in hope to exploit this knowledge in anticonvulsant or neuroprotective therapies. Besides the alterations in ion-channels, transmitters and neuromodulators, the successive build up of disturbances in energy metabolism have been suggested as a mechanism for seizure termination. Energy metabolism and substrate supply of the brain are tightly regulated by different mechanisms called neurometabolic and neurovascular coupling. Here we summarize the current knowledge whether these mechanisms are sufficient to cover the energy demand of hypersynchronous activity and whether a mismatch between energy need and supply could contribute to seizure control.
- Keywords
- adenosine, lactate, neurometabolic coupling, neurovascular coupling, pericyte, seizure,
- Publication type
- Journal Article MeSH
- Review MeSH
The presence of oxidative stress in immature brain has been demonstrated during the acute phase of status epilepticus (SE). The knowledge regarding the long periods of survival after SE is not unequivocal, lacking direct evidence. To examine the presence and time profile of oxidative stress, its functional effect on mitochondria and the influence of an antioxidant treatment in immature rats during epileptogenesis, status epilepticus (SE) was induced in immature 12-day-old rats by Li-pilocarpine and at selected periods of the epileptogenesis; rat pups were subjected to examinations. Hydroethidine method was employed for detection of superoxide anion (O2.-), 3-nitrotyrosine (3-NT), and 4-hydroxynonenal (4-HNE) for oxidative damage of mitochondrial proteins and complex I activity for mitochondrial function. Natural polyphenolic antioxidant resveratrol was given in two schemes: "acute treatment," i.p. administration 30 min before, 30 and 60 min after induction of SE and "full treatment" when applications continued once daily for seven consecutive days (25 mg/kg each dose). The obtained results clearly document that the period of epileptogenesis studied (up to 4 weeks) in immature brain is associated with the significant enhanced production of O2.-, the increased levels of 3-NT and 4-HNE and the persisting deficiency of complex I activity. Application of resveratrol either completely prevented or significantly reduced markers both of oxidative stress and mitochondrial dysfunction. The findings suggest that targeting oxidative stress in combination with current antiepileptic therapies may provide a benefit in the treatment of epilepsy.
- Keywords
- Epileptogenesis, Immature rats, Mitochondrial dysfunction, Oxidative stress, Protection, Resveratrol, Status epilepticus,
- MeSH
- Survival Analysis MeSH
- Biomarkers metabolism MeSH
- Behavior, Animal drug effects MeSH
- Mitochondria drug effects metabolism pathology MeSH
- Brain pathology MeSH
- Oxidative Stress drug effects MeSH
- Rats, Wistar MeSH
- Electron Transport Complex I metabolism MeSH
- Resveratrol pharmacology MeSH
- Status Epilepticus pathology MeSH
- Superoxides metabolism MeSH
- Animals MeSH
- Check Tag
- Male MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Names of Substances
- Biomarkers MeSH
- Electron Transport Complex I MeSH
- Resveratrol MeSH
- Superoxides MeSH
Epilepsy is a neurologic disorder, particularly frequent in infants and children where it can lead to serious consequences later in life. Oxidative stress and mitochondrial dysfunction are implicated in the pathogenesis of many neurological disorders including epilepsy in adults. However, their role in immature epileptic brain is unclear since there have been two contrary opinions: oxidative stress is age-dependent and does not occur in immature brain during status epilepticus (SE) and, on the other hand, evidence of oxidative stress in immature brain during a specific model of SE. To solve this dilemma, we have decided to investigate oxidative stress following SE induced in immature 12-day-old rats by three substances with a different mechanism of action, namely 4-aminopyridine, LiCl-pilocarpine or kainic acid. Fluoro-Jade-B staining revealed mild brain damage especially in hippocampus and thalamus in each of the tested models. Decrease of glucose and glycogen with parallel rises of lactate clearly indicate high rate of glycolysis, which was apparently not sufficient in 4-AP and Li-Pilo status, as evident from the decreases of PCr levels. Hydroethidium method revealed significantly higher levels of superoxide anion (by ∼60%) in the hippocampus, cerebral cortex and thalamus of immature rats during status. SE lead to mitochondrial dysfunction with a specific pronounced decrease of complex I activity that persisted for a long period of survival. Complexes II and IV activities remained in the control range. Antioxidant treatment with SOD mimetic MnTMPYP or peroxynitrite scavenger FeTPPS significantly attenuated oxidative stress and inhibition of complex I activity. These findings bring evidence that oxidative stress and mitochondrial dysfunction are age and model independent, and may thus be considered a general phenomenon. They can have a clinical relevance for a novel approach to the treatment of epilepsy, allowing to target the mechanisms which play a crucial or additive role in the pathogenesis of epilepsies in infants and children.
- Keywords
- brain damage, immature rats, mitochondrial dysfunction, oxidative stress, protection, status epilepticus,
- Publication type
- Journal Article MeSH
