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Introduction: One of the mechanisms of epileptgenesis is impairment of inhibitory neural circuits. Several studies have compared neural changes among subtypes of gamma-aminobutyric acid-related (GABAergic) neurons after acquired epileptic seizure. However, it is unclear that GABAergic neural modifications that occur during acquisition process of epileptic seizure., Methods: Male rats were injected with pentylenetetrazole (PTZ kindling: n = 30) or saline (control: n = 15) every other day to observe the development of epileptic seizure stages. Two time points were identified: the point at which seizures were most difficult to induce, and the point at which seizures were most easy to induce. The expression of GABAergic neuron-related proteins in the hippocampus was immunohistochemically compared among GABAergic subtypes at each of these time points., Results: Bimodal changes in seizure stages were observed in response to PTZ kindling. The increase of seizure stage was transiently suppressed after 8 or 10 injections, and then progressed again by the 16th injection. Based on these results, we defined 10 injections as a short-term injection period during which seizures are less likely to occur, and 20 injections as a long-term injection period during which continuous seizures are likely to occur. The immunohistochemical analysis showed that hippocampal glutamic acid decarboxylase 65 (GAD65) expression was increased after short-term kindling but unchanged after long-term kindling. Increased GAD65 expression was limited to somatostatin-positive (SOM + ) cells among several GABAergic subtypes. By contrast, GAD, GABA, GABA A R α1, GABA B R1, and VGAT cells showed no change following short- or long-term PTZ kindling., Conclusion: PTZ kindling induces bimodal changes in the epileptic seizure stage. Seizure stage is transiently suppressed after short-term PTZ injection with GAD65 upregulation in SOM + cells. The seizure stage is progressed again after long-term PTZ injection with GAD65 reduction to baseline level., Competing Interests: Declaration of competing interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: The study aimed to assess the influence of a single valproate (VPA) administration on inhibitory and excitatory neurotransmitter concentrations in the brain structures involved in epileptogenesis in pentylenetetrazol (PTZ)-kindled rats., Methods: Adult, male Wistar rats were kindled by repeated intraperitoneal (ip) injections of PTZ at a subconvulsive dose (30 mg/kg, three times a week). Due to the different times required to kindle the rats (18-22 injections of PTZ), a booster dose of PTZ was administrated 7 days after the last rats were kindled. Then rats were divided into two groups: acute administration of VPA (400 mg/kg) or saline given ip. The concentration of amino acids, kynurenic acid (KYNA), monoamines, and their metabolites in the prefrontal cortex, hippocampus, amygdala, and striatum was assessed by high-pressure liquid chromatography (HPLC)., Results: It was found that a single administration of VPA increased the gamma-aminobutyric acid (GABA), tryptophan (TRP), 5-hydroxyindoleacetic acid (5-HIAA), and KYNA concentrations and decreased aspartate (ASP) levels in PTZ-kindled rats in the prefrontal cortex, hippocampus, amygdala and striatum., Conclusions: Our results indicate that a single administration of VPA in the PTZ-kindled rats restored proper balance between excitatory (decreasing the level of ASP) and inhibitory neurotransmission (increased concentration GABA, KYNA) and affecting serotoninergic neurotransmission in the prefrontal cortex, hippocampus, amygdala, and striatum., (© 2024. The Author(s) under exclusive licence to Maj Institute of Pharmacology Polish Academy of Sciences.)

Epilepsy is a neurological disorder that remains difficult to treat due to the lack of a clear molecular mechanism and incomplete understanding of involved proteins. To identify potential therapeutic targets, it is important to gain insight into changes in protein expression patterns related to epileptogenesis. One promising approach is to analyze proteomic data, which can provide valuable information about these changes. In this study, to evaluate the changes in gene expression during epileptogenesis, LC-MC 2 analysis was carried out on hippocampus during stages of electrical kindling in rat models. Subsequently, progressive changes in the expression of proteins were detected as a result of epileptogenesis development. In line with behavioral kindled seizure stages and according to the proteomics data, we described epileptogenesis phases by comparing Stage3 versus Control (S3/C0), Stage5 versus Stage3 (S5/S3), and Stage5 versus Control group (S5/C0). Gene ontology analysis on differentially expressed proteins (DEPs) showed significant changes of proteins involved in immune responses like Csf1R, Aif1 and Stat1 during S3/C0, regulation of synaptic plasticity like Bdnf, Rac1, CaMK, Cdc42 and P38 during S5/S3, and nervous system development throughout S5/C0 like Bdnd, Kcc2 and Slc1a3.There were also proteins like Cox2, which were altered commonly among all three phases. The pathway enrichment analysis of DEPs was also done to discover molecular connections between phases and we have found that the targets like Csf1R, Bdnf and Cox2 were analyzed throughout all three phases were highly involved in the PPI network analysis as hub nodes. Additionally, these same targets underwent changes which were confirmed through Western blotting. Our results have identified proteomic patterns that could shed light on the molecular mechanisms underlying epileptogenesis which may allow for novel targeted therapeutic strategies., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Epilepsy is a chronic condition characterized by recurrent spontaneous seizures. The interaction between astrocytes and neurons has been suggested to play a role in the abnormal neuronal activity observed in epilepsy. However, the exact way astrocytes influence neuronal activity in the epileptogenic brain remains unclear. Here, using the PTZ-induced kindling mouse model, we evaluated the interaction between astrocyte and synaptic function by measuring astrocytic Ca 2+ activity, neuronal excitability, and the excitatory/inhibitory balance in the hippocampus. Compared to control mice, hippocampal slices from PTZ-kindled mice displayed an increase in glial fibrillary acidic protein (GFAP) levels and an abnormal pattern of intracellular Ca 2+ -oscillations, characterized by an increased frequency of prolonged spontaneous transients. PTZ-kindled hippocampal slices also showed an increase in the E/I ratio towards excitation, likely resulting from an augmented release probability of excitatory inputs without affecting inhibitory synapses. Notably, the alterations in the release probability seen in PTZ-kindled slices can be recovered by reducing astrocyte hyperactivity with the reversible toxin fluorocitrate. This suggests that astroglial hyper-reactivity enhances excitatory synaptic transmission, thereby impacting the E/I balance in the hippocampus. Altogether, our findings support the notion that abnormal astrocyte-neuron interactions are pivotal mechanisms in epileptogenesis.

Epileptogenesis is a potential process. Mossy fiber sprouting (MFS) contributes to epileptogenesis. Silencing of the dentate gyrus (DG) suppressed spontaneous seizures model of epilepsy and hyperactivity of granule cells resulted in MFS in vitro. However, the role of DG's excitability in epileptogenesis have not yet been well explored, and underlying mechanisms has not been elucidated. Using chemical genetics, we studied whether MFS and epileptogenesis could be modulated by silencing of DG in the PTZ kindling rat model of epilepsy. MFS and protein expression was measured by Timm staining, Western blotting, and Immunofluorescence. Previous studies demonstrated that MFS and epileptogenesis could be modulated by a regulator of axonal growth (e.g. RGMa, PTEN). NDR2 kinase regulate neuronal polarity and prevents the formation of supernumerary axons in the hippocampus. We experimentally confirmed chemogenetic inhibition in DG resulted in decreased MFS and NDR2 expression, and alleviated epileptogenesis. Furthermore, our results showed that injection of AVV vector expressing NDR2 into DG induced upregulation of NDR2 in the hippocampus, and over expression of NDR2 in the hippocampus promote MFS and block protective effect of chemogenetic silencing of DG on epileptogenesis. Overall, we concluded that silencing of DG inhibits MFS and prevents epileptogenesis through NDR2 in the hippocampus in the PTZ kindling rat model of TLE, thereby providing a possible strategy to prevent epileptogenesis., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Zhang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Epilepsy can severely affect the quality of life of patients, who are often at higher risk of mortality. However, the molecular mechanisms and pathogenesis underlying epileptogenesis are poorly understood. In this study, we performed a proteomic analysis of the hippocampus in pentylenetetrazole (PTZ)-kindled epileptic rats to explore the molecular mechanisms of epileptogenesis. We established an epileptic model in Sprague Dawley rats by injecting PTZ intraperitoneally and applied isobaric tags for relative and absolute quantification (iTRAQ) technology integrated with liquid chromatography-tandem mass spectrometry (LC-MS/MS) to identify differentially expressed proteins (DEPs) in the hippocampus. A total of 99 proteins, comprising 93 upregulated and 6 downregulated proteins, were identified based on a fold change >1.2 (or <0.83) and a p-value < .05. A further bioinformatics analysis suggested that the candidate proteins were mainly involved in the ubiquitin ligase complex or metabolite homeostasis or acted as intrinsic components of the membrane. A Kyoto Encyclopedia of Gene and Genomes (KEGG) pathway enrichment analysis identified a series of representative pathological pathways, including the calcium signaling pathway, neuroactive ligand-receptor interaction pathway, and the NF-kappa B pathway. The mass spectrometry results were further confirmed by assessing five representative proteins (Akt1, Syvn1, Amfr, Lamb1, and Cox17) using western blotting and immunohistochemistry. These results may help to reveal the molecular mechanisms underlying epileptogenesis and provide new directions or targets for epilepsy research., (© 2020 International Society for Developmental Neuroscience.)

Genetic Absence Epilepsy Rats from Strasbourg (GAERS) is a well-known animal model of absence epilepsy and they are resistant to electrical kindling stimulations. The present study aimed to examine possible differences in gamma-aminobutyric acid (GABA) levels and synapse counts in the substantia nigra pars reticulata anterior (SNRa) and posterior (SNRp) regions between GAERS and Wistar rats receiving kindling stimulations. Animals in the kindling group either received six stimulations in the amygdala and had grade 2 seizures or they were kindled, having grade five seizures. Rats were decapitated one hour after the last stimulation. SNR regions were obtained after vibratome sectioning of the brain tissue. GABA immunoreactivity was detected by immunogold method and synapses were counted. Sections were observed by transmission electron microscope and analyzed by Image J program. GABA density in the SNRa region of fully kindled GAERS and Wistar groups increased significantly compared to that of their corresponding grade 2 groups. The number of synapses increased significantly in kindled and grade 2 GAERS groups, compared to kindled and grade 2 Wistar groups, respectively, in the SNRa region. GABA density in the SNRp region of kindled GAERS group increased significantly compared to that of GAERS grade 2 group. In the SNRp region, both kindled and grade 2 GAERS groups were found to have increased number of synapses compared to that of GAERS control group. We concluded that both SNRa and SNRp regions may be important in modulating resistance of GAERS to kindling stimulations.

Although epilepsy is one of the most common chronic neurological disorders with a prevalence of approximately 1.0 %, the underlying pathophysiology remains to be elucidated. Understanding the molecular and cellular mechanisms involved in the development of epilepsy is important for the development of appropriate therapeutic strategy. In this study, we investigated the effects of status epilepticus on astrocytes, microglia, and extracellular matrix (ECM) molecules in the somatosensory cortex and piriform cortex of mice. Activation of astrocytes was observed in many cortices except the retrosplenial granular cortex after pentylenetetrazol (PTZ)-induced kindling in mice. Activated astrocytes in the cortex were found in layers 1-3 but not in layers 4-6. In the somatosensory and piriform cortices, no change was observed in the number of parvalbumin (PV)-positive neurons and PV-positive neurons covered with perineuronal nets. However, the amount of ECM in the extracellular space increased. The expression of VGLUT1- and GAD67-positive synapses also increased. Thus, in the PTZ-kindling epilepsy mice model, an increase in the number of ECM molecules and activation of astrocytes were observed in the somatosensory cortex and piriform cortex. These results indicate that PTZ-induced seizures affect not only the hippocampus but also other cortical areas. Our study findings may help to develop new therapeutic approaches to prevent seizures or their sequelae., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Objective: Development of novel therapies for temporal lobe epilepsy is hindered by a lack of models suitable for drug screening. While testing the hypothesis that "inhibiting inhibitory neurons" was sufficient to induce seizures, it was discovered that a mild electrical kindling protocol of VGAT-Cre mice led to spontaneous motor and electrographic seizures. This study characterizes these seizures and investigates the mechanism., Methods: Mice were implanted with electroencephalographic (EEG) headsets that included a stimulating electrode in the hippocampus before being electrically kindled. Seizures were evaluated by review of EEG recordings and behavior. γ-Aminobutyric acidergic (GABAergic) neurotransmission was evaluated by quantitative polymerase chain reaction, immunocytochemistry, Western blot, and electrophysiology., Results: Electrical kindling of VGAT-Cre mice induces spontaneous recurring seizures after a short latency (6 days). Seizures occur 1-2 times per day in both male and female mice, with only minimal neuronal death. These mice express Cre recombinase under the control of the vesicular GABA transporter (VGAT), a gene that is specifically expressed in GABAergic inhibitory neurons. The insertion of Cre disrupts the expression of VGAT mRNA and protein, and impairs GABAergic synaptic transmission in the hippocampus., Significance: Kindled VGAT-Cre mice can be used to study the mechanisms involved in epileptogenesis and may be useful for screening novel therapeutics., (© 2020 International League Against Epilepsy.)

Objective: Brain tissue oxygen (partial oxygen pressure [pO 2 ]) levels are tightly regulated to stay within the normoxic zone, with deviations on either side resulting in impaired brain function. Whereas pathological events such as ischemic attacks and brief seizures have previously been shown to result in pO 2 levels well below the normoxic zone, oxygen levels during prolonged status epilepticus (SE) and the subsequent endogenous kindling period are unknown., Methods: We utilized two models of acquired temporal lobe epilepsy in rats: intrahippocampal kainic acid infusion and prolonged perforant pathway stimulation. Local tissue oxygen was measured in the dorsal hippocampus using an optode during and for several weeks following SE., Results: We observed hyperoxia in the hippocampus during induced SE in both models. Following termination of SE, 88% of rats initiated focal self-generated spiking activity in the hippocampus within the first 7 days, which was associated with dynamic oxygen changes. Self-generated and recurring epileptiform activity subsequently organized into higher-frequency bursts that became progressively longer and were ultimately associated with behavioral seizures that became more severe with time and led to postictal hypoxia., Significance: Induced SE and self-generated recurrent epileptiform activity can have profound and opposing effects on brain tissue oxygenation that may serve as a biomarker for ongoing pathological activity in the brain., (© 2020 The Authors. Epilepsia published by Wiley Periodicals LLC on behalf of International League Against Epilepsy.)

Here, we investigated the participation of pro and anti-inflammatory cytokines in the spread of repeated audiogenic seizures from brainstem auditory structures to limbic areas, including the hippocampus. We used Wistar Audiogenic Rats (WARs) and Wistars submitted to the audiogenic kindling protocol with a loud broad-band noise. We measured pro and anti-inflammatory cytokines and nitrate levels in the hippocampus of stimulated animals. Our results show that all WARs developed audiogenic seizures that evolved to limbic seizures whereas seizure-resistant controls did not present any seizures. However, regardless of seizure severity, we did not observe differences in the pro inflammatory cytokines IL-1β, IL-6, TNF-α and IFN-α or in the anti-inflammatory IL-10 in the hippocampi of audiogenic and resistant animals. We also did not find any differences in nitrate content. Our data indicate that the spread of seizures during the audiogenic kindling is not dependent on hippocampal release of cytokines or oxidative stress, but the severity of brainstem seizures will be higher in animals with higher levels of cytokines and the oxidative stress marker, nitrate., Competing Interests: Declaration of Competing Interest Authors declare no conflicts of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Patients with Alzheimer's disease (AD) experience seizures at higher rates than the general population of that age, suggesting an underexplored role of hyperexcitability in AD. Genetic variants in presenilin (PSEN) 1 and 2 genes lead to autosomal dominant early-onset AD (ADAD); patients with PSEN gene variants also report seizures. Pharmacological control of seizures in AD may be disease-modifying. Preclinical efficacy of FDA-approved antiseizure drugs (ASDs) is well defined in young adult rodents; however, the efficacy of ASDs in aged rodents with chronic seizures is less clear. The mechanism by which ADAD genes lead to AD remains unclear, and even less studied is the pathogenesis of epilepsy in AD. PSEN variants generally all result in a biochemical loss of function (De Strooper, 2007). We herein determined whether well-established models of acute and chronic seizure could be used to explore the relationship between AD genes and seizures through investigating whether loss of normal PSEN2 function age-dependently influenced susceptibility to seizures and/or corneal kindling acquisition. PSEN2 knockout (KO) and age-matched wild-type (WT) mice were screened from 2- to 10-months-old to establish age-dependent focal seizure threshold. Additionally, PSEN2 KO and WT mice aged 2- and 8-months-old underwent corneal kindling such that mice were aged 3- and 9-months old at the beginning of ASD efficacy testing. We then defined the dose-dependent efficacy of mechanistically distinct ASDs on kindled seizures of young versus aged mice to better understand the applicability of corneal kindling to real-world use for geriatric patients. PSEN2 KO mice demonstrated early-life reductions in seizure threshold. However, kindling acquisition was delayed in 2-month-old PSEN2 KO versus WT mice. Young male WT mice took 24.3 ± 1.3 (S.E.M.) stimulations to achieve kindling criterion, whereas age-matched PSEN2 KO male mice took 41.2 ± 1.1 stimulations (p < .0001). The rate of kindling acquisition of 8-month-old mice was no longer different from WT. This study demonstrates that loss of normal PSEN2 function is associated with age-dependent changes in the in vivo susceptibility to acute seizures and kindling. Loss of normal PSEN2 function may be an underexplored molecular contributor to seizures. The use of validated models of chronic seizures in aged rodents may uncover age-related changes in susceptibility to epileptogenesis and/or ASD efficacy in mice with AD-associated genotypes, which may benefit the management of seizures in AD., (Copyright © 2019. Published by Elsevier Inc.)

Up-regulation of efflux transporters in brain capillaries may lead to the decreased therapeutic efficacy of antiepileptic drugs in patients with Drug Resistant Epilepsy. Adenosine receptor activation in brain capillaries can modulate blood-brain barrier permeability by decreasing the protein levels and function of efflux transporters. Therefore, we aimed to investigate whether the activation of adenosine receptors improves convulsions outcome in carbamazepine (CBZ) resistant animals and modulates the protein levels of efflux transporters (P-GP, MRP1, MRP2) in brain capillaries. We employed the window-pentylenetetrazol (PTZ) kindling model to develop CBZ resistant rats by CBZ administration during the post-kindling phase, and tested if these animals displayed subsequent resistance to other antiepileptic drugs. Crucially, we investigated if the administration of a broad-spectrum adenosine agonist (NECA) improves convulsions control in CBZ resistant rats. Of potential therapeutic relevance, in CBZ resistant rats NECA restored the anticonvulsant effect of CBZ. We also evaluated how the resistance to CBZ and the activation of adenosine receptors with NECA affect protein levels of efflux transporters in brain capillaries, as quantified by western blot. While CBZ resistance was associated with the up-regulation of both P-GP/MRP2 in brain capillaries, with the administration of NECA in CBZ resistant rats, we observed a decrease of P-GP and an increase of MRP2 levels, in brain capillaries. Since the activation of adenosine receptors improves the outcome of convulsions probably through the modulation of the efflux transporters protein levels in brain capillaries, adenosine agonists could be useful as an adjunct therapy for the control of Drug Resistant Epilepsy., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Seizure activity stimulates adult neurogenesis, the birth of new neurons, in the hippocampus. Many new neurons that develop in the presence of repeatedly induced seizures acquire abnormal morphological and functional characteristics that can promote network hyperexcitability and hippocampal dysfunction. However, the impact of seizure induced neurogenesis on behaviour remains poorly understood. In this study, we investigated whether adult-born neurons generated immediately before and during chronic seizures were capable of integration into behaviorally relevant hippocampal networks. Adult rats underwent pentylenetetrazole (PTZ) kindling for either 1 or 2 weeks. Proliferating cells were labelled with BrdU immediately before kindling commenced. Twenty-four hours after receiving their last kindling treatment, rats were placed in a novel environment and allowed to freely explore for 30 min. The rats were euthanized 90 min later to examine for behaviourally-induced immediate early gene expression (c-fos, Zif268). Using this approach, we found that PTZ kindled rats did not differ from control rats in regards to exploratory behaviour, but there was a marked attenuation in behaviour-induced expression of Fos and Zif268 for rats that received 2 weeks of PTZ kindling. Further examination revealed that PTZ kindled rats showed reduced colocalization of Fos and Zif268 in 2.5 week old BrdU + cells. The proportion of immature granule cells (doublecortin-positive) expressing behaviorally induced Zif268 was also significantly lower for PTZ kindled rats than control rats. These results suggest that chronic seizures can potentially disrupt the ability of adult-born cells to functionally integrate into hippocampal circuits important for the processing of spatial information., (Copyright © 2019 Elsevier Inc. All rights reserved.)

In our study, we assessed the potency of the brain-derived proteins ubiquitin carboxy-terminal hydrolase L1 (UCH-L1), matrix metalloproteinase 9 (MMP-9), glial fibrillary acidic protein (GFAP) and the immune activation indicators interleukin 1β (IL-1β) and interleukin 6 (IL-6) as peripheral biomarkers of different susceptibilities to kindling in a preclinical model. We observed increased plasma UCH-L1 levels in kindled vs. control animals. Furthermore, MMP-9 and IL-1β concentrations were the lowest in rats resistant to kindling. In summary, UCH-L1 is an indicator of neuronal loss and BBB disruption after seizure. MMP-9 and IL-1β may indicate resistance to kindling. UCH-L1, MMP-9 and IL-1β may have utility as peripheral biomarkers with translational potency in the clinic., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Glucose metabolism and serotonergic neurotransmission have been reported to play an important role in epileptogenesis. We therefore aimed to use neuroimaging to evaluate potential alterations in serotonin 5-HT 1A receptor and glucose metabolism during epileptogenesis in the rat electrical kindling model. To achieve this goal, we performed positron emission tomography (PET) imaging in a rat epileptogenesis model triggered by electrical stimulation of the hippocampus using 2-deoxy-2-[ 18 F]fluoro-D-glucose ( 18 F-FDG), a radiolabeled analog of glucose, and 2'-methoxyphenyl-(N-2'-pyridinyl)-p- 18 F-fluoro-benzamidoethylpiperazine ( 18 F-MPPF), a radiolabeled 5-HT 1A receptor ligand, to evaluate brain metabolism and 5-HT 1A receptor functionality. Since the 5-HT 1A receptor is also highly expressed in astrocytes, glial fibrillary acidic protein (GFAP) immunofluorescence was performed to detect astrogliosis arising from the kindling procedure once the study was finalized. Lastly, in vitro 18 F-MPPF autoradiography was performed to evaluate changes in 5HT 1A receptor expression. 18 F-FDG PET showed reduction of glucose uptake in cortical structures, whereas 18 F-MPPF PET revealed an enhancement of tracer binding potential (BP ND ) in key areas rich in 5-HT 1A receptor involved in epilepsy, including septum, hippocampus and entorhinal cortex of kindled animals compared to controls. However, in vitro 5-HT 1A receptor autoradiography showed no changes in densitometric signal in any brain region, suggesting that the augmentation in BP ND found by PET could be caused by reduction of synaptic serotonin. Importantly, astroglial activation was detected in the hippocampus of kindled rats. Overall, electrical kindling induced hypometabolism, astrogliosis and serotonergic alterations in epilepsy-related regions. Furthermore, the present findings point to 5-HT 1A receptor as a valuable epileptogenesis biomarker candidate and a potential therapeutic target., (Copyright © 2019 IBRO. Published by Elsevier Ltd. All rights reserved.)

Inflammatory responses involving Toll-like receptor signaling represent a key factor contributing to epileptogenesis. Thus, it is of particular interest to explore the relevance of toll-like receptor ligands and modulators, such as heat shock protein 70 (HSP70). Motivated by recent findings demonstrating an upregulation of HSP70 in a model of epileptogenesis, we analyzed the consequences of genetic and pharmacological targeting of HSP70 expression in a mouse kindling paradigm. Lack of inducible HSP70 resulted in increased prekindling seizure thresholds. However, at threshold stimulation the deficiency-promoted seizure spread, as indicated by an increased seizure severity. Subsequent kindling stimulations elicited more severe seizures in knockout mice, whereas endogenous termination of seizure activity remained unaffected with duration of behavioral and electrographic seizure activity comparable to that of wild-type mice. Interestingly, HSP70 deficiency resulted in enhanced microglia activation in the CA1 region. Next, we assessed a pharmacological targeting approach aiming to promote HSP70 expression. Celastrol treatment had no impact on kindling progression but reduced postkindling seizure thresholds and enhanced microglia activation in CA1 and CA3. In conclusion, the findings from HSP70-knockout mice support a protective role of HSP70 with an effect on microglia activation and spread of seizure activity. Unexpectedly, celastrol administration resulted in detrimental consequences. These findings should be considered as a warning about the general safety of celastrol as a drug candidate. The impact of pathophysiological mechanisms on the quality of celastrol effects requires comprehensive future studies exploring influencing factors. Moreover, alternate strategies to increase HSP70 expression should be further developed and validated.

Previously we demonstrated that p53 mediates dopaminergic neurotoxicity via inducing mitochondrial burdens and proapoptotsis. However, little is known about the role of p53 in the excitotoxicity induced by psychostimulant, such as cocaine. Cocaine-induced kindling (convulsive) behaviors significantly increased p53 expression in the brain. Cocaine-induced p53 expression was more pronounced in hippocampus than in striatum or prefrontal cortex. Genetic depletion of p53 significantly attenuated cocaine-induced convulsive behaviors, followed by c-Fos immunoreactivity, and oxidative burdens in the hippocampus of mice. The antioxidant potentials mediated by genetic depletion of p53 were more pronounced in the mitochondrial-than cytosolic-fraction. Depletion of p53 significantly attenuated the changes in mitochondrial transmembrane potential, intramitochondrial Ca 2+ level, and mitochondrial oxidative burdens induced by cocaine. Consistently, depletion of p53 significantly inhibited mitochondrial p53 translocation, and cleaved-PKCδ induced by cocaine. In addition, depletion of p53 protected from cytosolic cytochrome c release, and pro-apoptotic changes induced by cocaine. Importantly, the protective/anticonvulsant potentials by genetic depletion of p53 were comparable to those by pifithrin-μ (PFT), a p53 inhibitor. Our results suggest that depletion of p53 offers anticonvulsive and neuroprotective potentials mainly via attenuating mitochondrial oxidative burdens, mitochondrial dysfunction, and pro-apoptotic signalings against cocaine-induced convulsive neurotoxicity., (Copyright © 2018. Published by Elsevier Ltd.)

Compelling evidence indicates that oxidative stress contributes to cocaine neurotoxicity. The present study was performed to elucidate the role of the glutathione peroxidase-1 (GPx-1) in cocaine-induced kindling (convulsive) behaviors in mice. Cocaine-induced convulsive behaviors significantly increased GPx-1, p-IkB, and p-JAK2/STAT3 expression, and oxidative burdens in the hippocampus of mice. There was no significant difference in cocaine-induced p-IkB expression between non-transgenic (non-TG) and GPx-1 overexpressing transgenic (GPx-1 TG) mice, but significant differences were observed in cocaine-induced p-JAK2/STAT3 expression and oxidative stress between non-TG and GPx-1 TG mice. Cocaine-induced glial fibrillary acidic protein (GFAP)-labeled astrocytic level was significantly higher in the hippocampus of GPx-1 TG mice. Triple-labeling immunocytochemistry indicated that GPx-1-, p-STAT3-, and GFAP-immunoreactivities were co-localized in the same cells. AG490, a JAK2/STAT3 inhibitor, but not pyrrolidone dithiocarbamate, an NFκB inhibitor, significantly counteracted GPx-1-mediated protective potentials (i.e., anticonvulsant-, antioxidant-, antiapoptotic-effects). Genetic overexpression of GPx-1 significantly attenuated proliferation of Iba-1-labeled microglia induced by cocaine in mice. However, AG490 or astrocytic inhibition (by GFAP antisense oligonucleotide and α-aminoadipate) significantly increased Iba-1-labeled microglial activity and M1 phenotype microglial mRNA levels, reflecting that proinflammatory potentials were mediated by AG490 or astrocytic inhibition. This microglial activation was less pronounced in GPx-1 TG than in non-TG mice. Furthermore, either AG490 or astrocytic inhibition significantly counteracted GPx-1-mediated protective potentials. Therefore, our results suggest that astrocytic modulation between GPx-1 and JAK2/STAT3 might be one of the underlying mechanisms for protecting against convulsive neurotoxicity induced by cocaine., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Oxidative stress has been considered as one of pathogenesis of brain damage led by epilepsy. Reducing oxidative stress can ameliorate brain damage during seizures. However, expression levels of important antioxidative enzymes such as thioredoxin-1 (TRX1), thioredoxin-like 1 protein (TXNL1) and thioredoxin reductase 1 (TXNRD1) during seizures have not been investigated. In this study, we examined protein and mRNA expression levels of TRX1, TXNL1 and TXNRD1 in different brain regions in PTZ induced seizure model mice. We found that protein expression levels of TRX1, TXNL1 and TXNRD1 are simultaneously up-regulated by 2- or 3-fold in the cortex of both acute and chronic seizure model mice. But there is no unified expression pattern change of these enzymes in the hippocampus, cerebellum and diencephalon in the seizure model mice. Less extent up-regulation of mRNA expression of these enzymes were also observed in the cortex of seizure mice. These data suggest that antioxidative enzymes may provide a protective effect against oxidative stress in the cortex during seizures., Competing Interests: The authors have declared that no competing interests exist.

Glutathione (GSH) is an important antioxidant that can protect cells under oxidative stress. Thus, a non-invasive method to measure and map the distribution of GSH in live animals is needed. To image the distribution of GSH levels in specific brain regions, a new method using electron paramagnetic resonance (EPR) imaging with a nitroxide imaging probe was developed. Pixel-based mapping of brain GSH levels was successfully obtained by using the linear relationship between reduction rates for nitroxides in brains, measured by an in vivo EPR imager, and brain GSH levels, measured by an in vitro biochemical assay. The newly developed method was applied to a kindling mouse model induced with pentylenetetrazole (PTZ) to visualize changes in GSH levels in specific brain regions after seizure. The obtained map of brain GSH levels clearly indicated decreased GSH levels around the hippocampal region compared to control mice., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Kindling is a model for studying epileptogenesis and associated neuropsychiatric conditions. The antiepileptic drug levetiracetam (LEV) presents anti-kindling properties, but some severe neuropsychiatric events, especially depression, have been associated with its use in epileptic patients. The positive modulation of glucagon-like peptide-1 (GLP-1) receptors emerged as a potential target for the treatment of epilepsy and other neurological disorders. Here, we investigated behavioral and neurochemical effects of liraglutide (LIRA), a GLP-1 receptor agonist, alone or combined with LEV in mice subjected to PTZ-induced kindling. Male mice received PTZ on alternate days for 21 days. Before PTZ, the animals received LIRA, LEV (alone or in combination with LIRA) or saline. After seizures staging according to Racine's scale, behavioral evaluations were performed to verify anxiety-, depressive-like and cognitive performance. Brain oxidative alterations and BDNF levels were also measured. LEV showed anti-kindling properties, but aggravated depressive-like behavior in PTZ-kindling. In control conditions, LEV induced a pro-depressant effect and impaired avoidance memory retention. LIRA delayed but did not prevent the full kindling development. LIRA prevented the depressive-like behavior induced by PTZ kindling and PTZ + LEV. LEV + LIRA protected against PTZ-induced anxiety-like alterations and impairments in locomotion and cognition. Furthermore, LEV + LIRA reduced nitrite levels and lipid peroxidation in the hippocampus and prefrontal cortex, while it increased reduced glutathione levels in all evaluated brain areas. LIRA or LEV + LIRA increased hippocampal BDNF levels. In conclusion, our results showed that LIRA can be a promising adjunctive therapy for epilepsy-related neuropsychiatric comorbidities and to improve the management of antiepileptic drug associated behavioral adverse effects., (Copyright © 2018 Elsevier Masson SAS. All rights reserved.)

Clinical and experimental studies show that epilepsy affects cardiac function; however, the underlying molecular mechanism has not been fully elucidated. Inwardly-rectifying potassium (Kir) channels transport K+ ions into excitable cells such as neurons and cardiomyocytes; they control the cell excitability by acting towards the repolarization phase of cardiac action potential. Kir channel expression has been previously shown to vary in epileptic brains, at the same time seizures are known to affect the autonomic nervous system. Kir channel expression in cardiac tissue is a possible mechanism for the explanation of cardiac pathology in epilepsy. We investigate the expression of Kir channels in epileptic cardiac tissue by using pentylenetetrazole (PTZ)-kindling model in rats. Our molecular analyses showed significant decrease in cardiac Kir channel mRNA and protein expression of PTZ-kindled rats. Interestingly, both Kir2.x, which directs IK1 flux in ventricular tissue and Kir3.x, which is responsible for IKACh in the atria, were observed to decrease significantly. Kir channel expression also differs between females and males. This is the first study to our knowledge in epileptic cardiac tissue showing the expression of Kir channels. Our results show that Kir channels may play a role in cardiac pathology associated with epilepsy.

The objective of this study was to evaluate the effect of dexamethasone, on the severity of seizures and levels of pro-inflammatory interleukins in animals with kindling model induced by pentylenetetrazole (20 mg/kg) in alternated days for 15 days of treatment. The animals were divided into five groups: control group given saline, a group treated with diazepam (2 mg/kg) and groups treated with dexamethasone (1, 2 and 4 mg/kg). Open field test was conducted. The treatment with dexamethasone decreased the severity of seizures, also decreased TNF-alpha and Interleukin 1 beta levels in the hippocampus and TNF-alpha level in the serum., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Ribosome biogenesis, including the RNA polymerase 1 (Pol1)-mediated transcription of rRNA, is regulated by the pro-epileptogenic mTOR pathway. Therefore, hippocampal Pol1 activity was examined in mouse models of epilepsy including kainic acid- and pilocarpine-induced status epilepticus (SE) as well as a single seizure in response to pentylenetetrazole (PTZ). Elevated 47S pre-rRNA levels were present acutely after induction of SE suggesting activation of Pol1. Conversely, after a single seizure, 47S pre-rRNA was transiently downregulated with increased levels of unprocessed 18S rRNA precursors in the cornu Ammonis (CA) region. At least in the dentate gyrus (DG), the pilocarpine SE-mediated transient activation of Pol1 did not translate into long-term changes of pre-rRNA levels or total ribosome content. Unaltered hippocampal ribosome content was also found after a 20-day PTZ kindling paradigm with increasing pro-convulsive effects of low dose PTZ that was injected every other day. However, after selectively deleting the essential Pol1 co-activator, transcription initiation factor-1A (Tif1a/Rrn3) from excitatory neurons, PTZ kindling was impaired while DG total ribosome content was moderately reduced and no major neurodegeneration was observed throughout the hippocampus. Therefore, Pol1 activity of excitatory neurons is required for PTZ kindling. As seizures affect ribosome biogenesis without long-term effects on the total ribosome content, such a requirement may be associated with a need to produce specialized ribosomes that promote pro-epileptic plasticity.

Strong evidence exists that Toll-like receptor (TLR)-mediated effects on microglia functional states can promote ictogenesis and epileptogenesis. So far, research has focused on the role of high-mobility group box protein 1 as an activator of TLRs. However, the development of targeting strategies might need to consider a role of additional receptor ligands. Considering the fact that heat shock protein A1 (hsp70) has been confirmed as a TLR 2 and 4 ligand, we have explored the consequences of its overexpression in a mouse kindling paradigm. The genetic modulation enhanced seizure susceptibility with lowered seizure thresholds prior to kindling. In contrast to wildtype (WT) mice, HSPA1A transgenic (TG) mice exhibited generalized seizures very early during the kindling paradigm. Along with an increased seizure severity, seizure duration proved to be prolonged in TG mice during this phase. Toward the end of the stimulation phase seizure parameters of WT mice reached comparable levels. However, a difference between genotypes was still evident when comparing seizure parameters during the post-kindling threshold determination. Surprisingly, HSPA1A overexpression did not affect microglia activation in the hippocampus. In conclusion, the findings demonstrate that hsp70 can exert pro-convulsant effects promoting ictogenesis in naïve animals. The pronounced impact on the response to subsequent stimulations gives first evidence that genetic HSPA1A upregulation may also contribute to epileptogenesis. Thus, strategies inhibiting hsp70 or its expression might be of interest for prevention of seizures and epilepsy. However, conclusions about a putative pro-epileptogenic effect of hsp70 require further investigations in models with development of spontaneous recurrent seizures., (Copyright © 2018 IBRO. Published by Elsevier Ltd. All rights reserved.)

Objective: Aberrant myelination and developmental delay have been reported in epilepsy. However, it is unclear whether these are linked to intrinsic mechanisms that support a predisposition toward seizures and the development of epilepsy. Thus, we compared rates of myelination and neurodevelopment in male rats selectively bred for enhanced susceptibility to kindling epileptogenesis (FAST) with male rats bred for resistance (SLOW)., Methods: Myelin-specific gene expression was compared in the brainstem, cerebellum, and cerebral hemisphere of FAST and SLOW rats on postnatal days (PNDs) 5, 11, 17, 23, and 90 to determine strain-specific myelination rates. Myelin protein levels were also compared at PNDs 5 and 23 in the brainstem. Relative rates of neurodevelopment were evaluated between PNDs 5 and 21 using physical growth landmarks and neuromotor tests including righting reflex, cliff avoidance, negative geotaxis, and locomotor activity., Results: Myelin-specific mRNA expression was significantly down-regulated in FAST rats on PNDs 5 and 11 in all 3 brain structures, indicating relatively delayed myelination. Likewise, corresponding protein levels were significantly lower in FAST brainstem on PND 5. Developmental delay was evident in the FAST strain such that only 9% of FAST pups, compared to 81% of SLOW, had open eyes by PND 13, locomotor activity was significantly reduced between PNDs 12 and 16, and neuromotor task acquisition was delayed between PNDs 5 and 10., Significance: Relative delays in myelination and neurodevelopment co-occurred in the seizure-prone FAST strain in the absence of seizures. These findings suggest these symptoms are not seizure-induced and may be mechanistically linked to an underlying pathophysiology supporting a predisposition toward developing epilepsy., (Wiley Periodicals, Inc. © 2018 International League Against Epilepsy.)

Epilepsy is a chronic brain disease characterized by repeated unprovoked seizures. Currently, no drug therapy exists for curing epilepsy or disease modification in people at risk. Despite several emerging mechanisms, there have been few studies of epigenetic signaling in epileptogenesis, the process whereby a normal brain becomes progressively epileptic because of precipitating factors. Here, we report a novel role of histone deacetylation as a critical epigenetic mechanism in epileptogenesis. Experiments were conducted using the histone deacetylase (HDAC) inhibitor sodium butyrate in the hippocampus kindling model of temporal lobe epilepsy (TLE), a classic model heavily used to approve drugs for treatment of epilepsy. Daily treatment with butyrate significantly inhibited HDAC activity and retarded the development of limbic epileptogenesis without affecting after-discharge signal. HDAC inhibition markedly impaired the persistence of seizure expression many weeks after epilepsy development. Moreover, subchronic HDAC inhibition for 2 weeks resulted in a striking retardation of epileptogenesis. HDAC inhibition, unexpectedly, also showed erasure of the epileptogenic state in epileptic animals. Finally, butyrate-treated animals exhibited a powerful reduction in mossy fiber sprouting, a morphologic index of epileptogenesis. Together these results underscore that HDAC inhibition prevents the development of TLE, indicating HDAC's critical signaling role in epileptogenesis. These findings, therefore, envisage a unique novel therapy for preventing or curing epilepsy by targeting the epigenetic HDAC pathway., (Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.)

Objective: There is an urge to identify new molecules which can modulate process of epileptogenesis, since currently available drugs act symptomatically and one-third of the patients remain refractory to the disease. Hence, the present study was conducted to evaluate the effects of Resveratrol (RESV) on epileptogenesis in pentylenetetrazole (PTZ)-induced kindling in mice., Method: Swiss albino mice were administered RESV (10, 20 and 40 mg/kg,p.o) in acute study. On the seventh day animals were subjected to various neurological and neurobehavioral tests viz, Increasing Current Electroshock Test (ICES), PTZ-induced seizures, passive avoidance response, and elevated plus maze test. For the development of kindling PTZ was administered in a dose of 25 mg/kg, i.p. on every alternate day and RESV in all the three doses was administered daily. Seizure score was continuously monitored till the development of kindling and cognition tests were performed in the end of the study. The animals were sacrificed and levels of inflammatory biomarkers viz., IL-1β, interleukin-1 receptor antagonist (IL1-Ra), IL-6, and TNF-α were assessed in the hippocampus and cortex of the kindled animals., Results: RESV in all three doses increased the seizure threshold to hind limb extension in the ICES test. RESV in all the tested doses suppressed the development of kindling and reduced the levels of IL-1β, IL1-Ra, IL-6, and TNF-α in kindled mice., Conclusion: RESV suppressed the development of kindling in mice and decreased the levels of inflammatory biomarkers in their hippocampus. RESV modified brain inflammation during epileptogenesis and found to possess nootropic activity in the kindled mice.

The biologic processes underlying epileptogenesis following a brain insult are not fully understood, but several lines of evidence suggest that hyperphosphorylation of tau may be an important factor in these processes. To provide further insight into the causal relationship between tau and epileptogenesis, this study applied amygdala kindling to rTg4510 mice that, concurrent with other pathologies, overexpress phosphorylated tau, tau knockout mice, or their respective wild-type controls. Mice were electrically stimulated twice daily, 5 days per week for 3 weeks. Electroencephalography was recorded to measure the primary afterdischarge duration, and the behavioral progression of kindling-induced seizures was assessed. rTg4510 mice (n = 10) had increased primary afterdischarge durations (p < 0.001), and significantly more rapid progression of kindling (p < 0.001), compared with wild-type mice (n = 10). Tau knockout mice (n = 7), however, did not differ from their wild-type counterparts (n = 8) on any of the seizure outcomes. These results suggest that Tg4510 mice are more vulnerable to epileptogenesis, but that the presence of tau itself is not necessary for kindling epileptogenesis to occur., (Wiley Periodicals, Inc. © 2017 International League Against Epilepsy.)

Introduction: In kindling, repeated electrical stimulation of certain brain areas causes progressive and permanent intensification of epileptiform activity resulting in generalized seizures. We focused on the role(s) of glutamate and a negative regulator of glutamate release, STXBP5/tomosyn-1, in kindling., Methods: Stimulating electrodes were implanted in the amygdala and progression to two successive Racine stage 5 seizures was measured in wild-type and STXBP5/tomosyn-1 -/- (Tom -/- ) animals. Glutamate release measurements were performed in distinct brain regions using a glutamate-selective microelectrode array (MEA)., Results: Naïve Tom -/- mice had significant increases in KCl-evoked glutamate release compared to naïve wild type as measured by MEA of presynaptic release in the hippocampal dentate gyrus (DG). Kindling progression was considerably accelerated in Tom -/- mice, requiring fewer stimuli to reach a fully kindled state. Following full kindling, MEA measurements of both kindled Tom +/+ and Tom -/- mice showed significant increases in KCl-evoked and spontaneous glutamate release in the DG, indicating a correlation with the fully kindled state independent of genotype. Resting glutamate levels in all hippocampal subregions were significantly lower in the kindled Tom -/- mice, suggesting possible changes in basal control of glutamate circuitry in the kindled Tom -/- mice., Conclusions: Our studies demonstrate that increased glutamate release in the hippocampal DG correlates with acceleration of the kindling process. Although STXBP5/tomosyn-1 loss increased evoked glutamate release in naïve animals contributing to their prokindling phenotype, the kindling process can override any attenuating effect of STXBP5/tomosyn-1. Loss of this "braking" effect of STXBP5/tomosyn-1 on kindling progression may set in motion an alternative but ultimately equally ineffective compensatory response, detected here as reduced basal glutamate release.

In view of well-evidenced antiepileptic effects of melatonin and few reports of anticonvulsant action of agomelatine, the present study investigated whether agomelatine protects against pentylenetetrazole (PTZ)-induced kindling in mice and kindling-associated oxidative stress, depression, and impairment of spatial memory. In order to explore whether effects are mediated by melatonergic or serotonergic mechanisms, 1-(m-chlorophenyl) piperazine (mCPP), selective 5HT2c receptor agonist and luzindole, melatonergic receptor antagonist, were taken as pharmacological tools. In view of few hepatotoxic reports on agomelatine, the study evaluated effects on hepatic enzyme levels. Swiss strain albino mice were injected with PTZ (25mg/kg, i.p.) once every two days for 5weeks to induce kindling. The effects of agomelatine (10mg/kg, p.o.) alone and in combination with luzindole (2.5mg/kg, i.p.) or mCPP (7mg/kg, i.p.) on seizure severity during induction and % incidence of animals kindled at the end of 5weeks were recorded. Modified forced swim test was used for studying depression-like behavior while spontaneous alternation behavior was used for studying effects on spatial memory. Serum AST and ALT concentrations, cortical and hippocampal malondialdehyde, and reduced glutathione were measured. Agomelatine 10mg/kg, p.o. effectively delayed development of kindling, reduced seizure severity, and decreased % incidence. Luzindole reversed the protective effects of agomelatine while mCPP failed to show such a reversal, indicating melatonergic (and not serotonergic) mechanisms in the observed effects. Agomelatine also showed antioxidant effects that can partially contribute to its anticonvulsant action. In addition, it alleviated PTZ-kindling-associated behavioral despair and favorably modulated liver enzymes. Its effects on improvement of kindling-associated spatial memory could possibly be related to its effects on locomotor activity. Agomelatine, thus, could be explored as an adjunct to antiepileptic drugs for seizure control and for alleviating epilepsy-associated depression., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Axonal sprouting and synaptic reorganization are the primary pathophysiological characteristics of epilepsy. Recent studies demonstrated that synaptic adhesion-like molecule 3 (SALM3) is highly expressed in the central nervous system and plays important roles in neurite outgrowth, branching, and axon guidance, mechanisms that are also observed in epilepsy. However, the expression of SALM3 in the epileptic brain and the effect of SALM3 in the pathogenesis of epilepsy remain unclear. The aims of this study were to investigate SALM3 expression in rat models of epilepsy and to explore the functional significance of SALM3 in epilepsy. We demonstrated that SALM3 was expressed at significantly higher levels in epileptic rats compared with controls. Inhibition of SALM3 by SALM3 shRNA inhibited status epilepticus in the acute stage of disease and decreased spontaneous recurrent seizures in the Lithium-pilocarpine model of chronic stages of epilepsy. Consistent with these findings, SALM3 shRNA significantly prolonged the latent period in the PTZ kindling model. Our study suggests that the overexpression of SALM3 might be associated with epileptogenesis and that selectively inhibiting SALM3 may have therapeutic potential in treating epilepsy., (Copyright © 2016 Elsevier Inc. All rights reserved.)

As a promising method for treating intractable epilepsy, the inhibitory effect of low-frequency stimulation (LFS) is well known, although its mechanisms remain unclear. Excessive levels of cerebral glutamate are considered a crucial factor for epilepsy. Therefore, we designed experiments to investigate the crucial parts of the glutamate cycle. We evaluated glutamine synthetase (GS, metabolizes glutamate), glutaminase (synthesizes glutamate), and glutamic acid decarboxylase (GAD, a γ-aminobutyric acid [GABA] synthetase) in different regions of the brain, including the dentate gyrus (DG), CA3, and CA1 subregions of the hippocampus, and the cortex, using western blots, immunohistochemistry, and enzyme activity assays. Additionally, the concentrations of glutamate, GABA, and glutamine (a product of GS) were measured using high-performance liquid chromatography (HPLC) in the same subregions. The results indicated that a transiently promoted glutamate cycle was closely involved in the progression from focal to generalized seizure. Low-frequency stimulation (LFS) delivered to the ventral hippocampus had an antiepileptogenic effect in rats exposed to amygdaloid-kindling stimulation. Simultaneously, LFS could partly reverse the effects of the promoted glutamate cycle, including increased GS function, accelerated glutamate-glutamine cycling, and an unbalanced glutamate/GABA ratio, all of which were induced by amygdaloid kindling in the DG when seizures progressed to stage 4. Moreover, glutamine treatment reversed the antiepileptic effect of LFS with regard to both epileptic severity and susceptibility. Our results suggest that the effects of LFS on the glutamate cycle may contribute to the antiepileptogenic role of LFS in the progression from focal to generalized seizure., (Copyright © 2016 Elsevier Inc. All rights reserved.)

IEM-2062 [1-(6-aminohexylamino)-1-phenylcyclohexyl dihydrochloride], causing a combined block NMDA and AMPA receptors, after chronic oral administration in doses, respectively, 0.3 and 3 mg/kg, induce maximal anticonvulsant effect in the pentylenetetrazol kindling rats because decrease the number of completely kindling rats by 100 %, and also decrease in 2.5-3.3 times the average severity of clonic-tonic kindling seizures. IEM-2062 causes significant anticon- 299 vulsant effects in the widest range of doses, 1-48 mg/kg, which is 24-22 times more than that of memantine (12-20 mg/kg) and sodium valproate (100-200 mg/kg). Sodium valproate and memantine cause significant disturbances of locomotor activity in the «open field» test in doses causing maximal anticonvulsant effect in the kindling rats. At the same time IEM-2062 cause disturbance of locomotor activity only in very high dose of 92 mg/kg, which exceeds in 30.7 times the dose causing the maximum anticonvulsive effect in the kindling rats. Thus, IEM-2062 reduces the severity of kindling seizures in 1.7-1.9 times stronger than sodium valproate and memantine and also by 30.7 times is safer than sodium valproate and memantine.

Background: Oxidative stress plays a key role in the pathogenesis of epilepsy and contributes in underlying epileptogenesis process. Anticonvulsant drugs targeting the oxidative stress domain of epileptogenesis may provide better control of seizure. The present study was carried out to investigate the effect of clinically used anti-epileptic drugs (AEDs) on the course of pentylenetetrazole (PTZ)-induced kindling and oxidative stress markers in mice., Methods: Six mechanistically heterogeneous anticonvulsants: phenobarbital, phenytoin, levetiracetam, pregabalin, topiramate, and felbamate were selected and their redox profiles were determined. Diazepam was used as a drug control for comparison. Kindling was induced by repeated injections of a sub-convulsive dose of PTZ (50 mg/kg, s.c.) on alternate days until seizure score 5 was evoked in the control kindled group. Anticonvulsants were administered daily. Following PTZ kindling, oxidative stress biomarkers were assessed in homogenized whole brain samples and estimated for the levels of nitric oxide, peroxide, malondialdehyde, protein carbonyl, reduced glutathione, and activities of nitric oxide synthase and superoxide dismutase., Results: Biochemical analysis revealed a significant increase in the levels of reactive oxygen species with a parallel decrease in endogenous anti-oxidants in PTZ-kindled control animals. Daily treatment with levetiracetam and felbamate significantly decreased the PTZ-induced seizure score as well as the levels of nitric oxide (p<0.001), nitric oxide synthase activity (p<0.05), peroxide levels (p<0.05), and malondialdehyde (p<0.05). Levetiracetam and felbamate significantly decreased lipid and protein peroxidation whereas topiramate was found to reduce lipid peroxidation only., Conclusions: An AED that produces anticonvulsant effect by the diversified mechanism of action such as levetiracetam, felbamate, and topiramate exhibited superior anti-oxidative stress activity in addition to their anticonvulsant activity.

Background: Epilepsy is one of the most common neurological disorders in humans, and the role of the cerebellum in its physiopathology remains the subject of study. The Purkinje cells (PC), whose axons target the dentate and interpositus nuclei, form the main cerebellar output to forebrain structures involved in epilepsy. Cerebellar atrophy related to loss of PC has been reported in chronic epilepsy although its mechanism remains unclear. Taking into account that an overexpression of β-Catenin has been related with cell death, here we present the signaling of β-Catenin and the type of PC death in cerebellum of rats with seizures induced by the amygdaloid kindling model., Method: Using an immunohistochemistry and western blot assay for β-Catenin, c-Myc, cyclin D3, TUNEL and caspase-3, in rats chronically implanted with electrodes, receiving 0, 3, 15, and 45 electrical stimuli., Results: We found that such rats suffering a major number of stimuli showed the highest amount of marks assessed., Conclusion: We concluded that there is a higher activity of the Wnt/β-Catenin pathway associated with increased number of stimuli may be related with the presence of apoptosis in the cerebellum treated with amygdala kindling. In this way, we suggest this pathway as one of the mechanisms by which cerebellar neurons death in generalized seizures., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

Background: Neuroinflammation, oxidative stress and mitochondrial dysfunction play a significant role to explain the pathophysiology of epilepsy. Neuroinflammation through microglia activation has been documented in epileptogenesis. Compounds which inhibit activation of glial cells have been suggested as one of the treatment approaches for the effective treatment of epilepsy. The present study has been designed to investigate the role of coenzyme Q10 and its interaction with minocycline (microglia inhibitor) against pentylenetetrazol (PTZ) induced kindling epilepsy., Methods: Laca mice received Coenzyme Q10 and minocycline for a period of 29 days. PTZ (40mg/kg ip) injection has been given on alternate days. Various behavioural parameters (kindling score and elevated plus maze), biochemical parameters (lipid peroxidation, superoxide dismutase, reduced glutathione, catalase, nitrite and acetylcholinesterase) and mitochondrial enzyme complex activities of (I, II and IV) were assessed in the discrete areas of the brain., Results: Administration of a subconvulsive dose of PTZ (40mg/kg) repeatedly increased significantly kindling score, oxidative damage and impaired mitochondrial enzyme complex activities (I, II and IV) and pro-inflammatory marker (TNF-α) as compared to naive animals. Coenzyme Q10 (10, 20 and 40mg/kg) and minocycline (50 and 100mg/kg) for a duration of 29days significantly attenuated kindling score, reversed oxidative damage, TNF-α and restored mitochondrial enzyme complex activities (I, II and IV) as compared to control. Further, combinations of CoQ10 (10, 20mg/kg) with minocycline (50 and 100mg/kg) significantly modulate the protective effect of CoQ10 which was significant as compared to their effect per se in PTZ treated animals., Conclusion: The present study suggests the involvement of microglia inhibition in the protective effect of CoQ10 in PTZ induced kindling in mice., (Copyright © 2016 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.)

Voltage-gated sodium channels (VGSCs) play a vital role in controlling neuronal excitability. Nav1.6 is the most abundantly expressed VGSCs subtype in the adult central nervous system and has been found to contribute to facilitate the hyperexcitability of neurons after electrical induction of status epilepticus (SE). To clarify the exact expression patterns of Nav1.6 during epileptogenesis, we examined the expression of Nav1.6 at protein and mRNA levels in two distinct animal models of temporal lobe epilepsy (TLE) including a post-SE model induced by kainic acid (KA) intrahippocampal injection and a kindling model evoked by pentylenetetrazole (PTZ). A prominent, seizure intensity-dependent increase of Nav1.6 expression in reactive astrocytes was observed in ipsilateral hippocampus of post-SE rats, reaching the peak at 21 days after SE, a time point during the latent stage of epileptogenesis. However, Nav1.6 with low expression level was selectively expressed in the hippocampal neurons rather than astrocytes in PTZ-kindled animals. This seizure-related increase of a VGSCs subtype in reactive astrocytes after SE may represent a new mechanism for signal communication between neuron and glia in the course of epileptogenesis, facilitating the neuronal hyperexcitability.

Background: Sleep disruptions are common in epilepsy patients. Our previous study demonstrates that homeostatic factors and circadian rhythm may mediate epilepsy-induced sleep disturbances when epilepsy occurs at different zeitgeber hours. The proinflammatory cytokine, interleukin-1 (IL-1), is a somnogenic cytokine and may also be involved in epileptogenesis; however, few studies emphasize the effect of IL-1 in epilepsy-induced sleep disruption. We herein hypothesized that IL-1 receptor type 1 (IL-1R1) mediates the pathogenesis of epilepsy and epilepsy-induced sleep disturbances. We determined the role of IL-1R1 by using IL-1R1 knockout (IL-1R1 -/- KO) mice., Results: Our results elucidated the decrease of non-rapid eye movement (NREM) sleep during the light period in IL-1R -/- mice and confirmed the somnogenic role of IL-1R1. Rapid electrical amygdala kindling was performed to induce epilepsy at the particular zeitgeber time (ZT) point, ZT13. Our results demonstrated that seizure thresholds induced by kindling stimuli, such as the after-discharge threshold and successful kindling rates, were not altered in IL-1R -/- mice when compared to those obtained from the wildtype mice (IL-1R +/+ mice). This result suggests that IL-1R1 is not involved in kindling-induced epileptogenesis. During sleep, ZT13 kindling stimulation significantly enhanced NREM sleep during the subsequent 6 h (ZT13-18) in wildtype mice, and sleep returned to the baseline the following day. However, the kindling-induced sleep alteration was absent in the IL-1R -/- KO mice., Conclusions: These results indicate that the IL-1 signal mediates epilepsy-induced sleep disturbance, but dose not participate in kindling-induced epileptogenesis.

The ATP-gated P2X7 receptor (P2X7R) is a non-selective cation channel which senses high extracellular ATP concentrations and has been suggested as a target for the treatment of neuroinflammation and neurodegenerative diseases. The use of P2X7R antagonists may therefore be a viable approach for treating CNS pathologies, including epileptic disorders. Recent studies showed anticonvulsant potential of P2X7R antagonists in certain animal models. To extend this work, we tested three CNS-permeable P2X7R blocker (Brilliant Blue G, AFC-5128, JNJ-47965567) and a natural compound derivative (tanshinone IIA sulfonate) in four well-characterized animal seizure models. In the maximal electroshock seizure threshold test and the pentylenetetrazol (PTZ) seizure threshold test in mice, none of the four compounds demonstrated anticonvulsant effects when given alone. Notably, in combination with carbamazepine, both AFC-5128 and JNJ-47965567 increased the threshold in the maximal electroshock seizure test. In the PTZ-kindling model in rats, useful for testing antiepileptogenic activities, Brilliant Blue G and tanshinone exhibited a moderate retarding effect, whereas the potent P2X7R blocker AFC-5128 and JNJ-47965567 showed a significant and long-lasting delay in kindling development. In fully kindled rats, the investigated compounds revealed modest effects to reduce the mean seizure stage. Furthermore, AFC-5128- and JNJ-47965567-treated animals displayed strongly reduced Iba 1 and GFAP immunoreactivity in the hippocampal CA3 region. In summary, our results show that P2X7R antagonists possess no remarkable anticonvulsant effects in the used acute screening tests, but can attenuate chemically-induced kindling. Further studies would be of interest to support the concept that P2X7R signalling plays a crucial role in the pathogenesis of epileptic disorders.

Objective: Anticonvulsive monotherapy fails to be effective in one third of patients with epilepsy resulting in the need for polytherapy regimens. However, with the still limited knowledge, drug choices for polytherapy remain empirical. Here we report experimental data from a chronic epilepsy model for the combination of perampanel and zonisamide, which can render guidance for clinical studies and individual drug choices., Methods: The anticonvulsant effects of the combination of perampanel and zonisamide were evaluated in a rat amygdala kindling model. Furthermore, the potential for motor impairment was evaluated. The type of interaction was quantitatively assessed based on isobolographic analysis., Results: When administered alone, zonisamide dose-dependently increased the afterdischarge threshold in fully kindled rats. Moreover, data confirmed efficacy of perampanel to inhibit seizure initiation and progression with an impact on propagation of activity from the focus. Pronounced threshold increases were observed following administration of a constant zonisamide dosage combined with different doses of perampanel. Isobolographic analysis of drug responses, which is based on individual drug dose-effect data, revealed a synergistic interaction substantiating the high efficacy of the combination. Furthermore, rotarod data indicated that the combination has a favorable tolerability profile when zonisamide is coadministered with low doses of perampanel. Plasma concentration analysis argued against a pharmacokinetic interaction as a basis for the synergism., Significance: The findings clearly indicate a pronounced synergistic anticonvulsant effect for the combination of the noncompetitive α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor antagonist perampanel with zonisamide, which modulates voltage-sensitive sodium channels and T-type calcium currents. Consequently, polytherapy using these two antiepileptic drugs might be efficacious for clinical management of partial-onset seizures. The findings indicate that the impact of dose ratios on tolerability needs be taken into account. With regard to conclusions about the extent of the synergism and its implications further antiepileptic drug combinations need to be evaluated allowing direct comparison., (Wiley Periodicals, Inc. © 2016 International League Against Epilepsy.)

The prognosis of patients exposed to a sub-threshold dose of a proconvulsant is difficult to establish. In this study, we investigated the effect of a single sub-threshold dose of the proconvulsant pilocarpine (PILO) on the progression of seizures that were subsequently induced by daily electrical stimulation (kindling) of the amygdaloid formation. Male Sprague–Dawley rats were each implanted with an electrode in the right basolateral amygdala and an indwelling cannula in the right ventricle. The animals were randomized into groups and were administered one of the following treatments: saline, PILO, saline+L-α-aminoadipic acid (L-AAA; one dosage tested), PILO+L-AAA, or PILO+L-methionine sulfoximine (three dosages tested). Amygdaloid stimulation and electroencephalography were performed once daily. We performed immunohistochemistry and western blot for glial fibrillary acidic protein and glutamine synthetase (GS). We also assayed the enzymic activity of GS in discrete brain regions. An intraperitoneal injection of a sub-threshold PILO dose enhanced the progression of amygdaloid-kindling seizures and was accompanied by an increase in reactive-astrocyte and GS (content and activity) in the hippocampus and piriform cortex. L-AAA and L-methionine sulfoximine, inhibitors of astrocytic and GS function, respectively, abolished the effect of PILO on amygdaloid-kindling seizures. We conclude that one sub-threshold dose of a proconvulsant may enhance the progression of subsequent epilepsy and astrocytic GS may play a role in this phenomenon. Thus, a future therapy for epilepsy could be inhibition of astrocytes and/or GS.

Background: G protein-coupled estrogen receptor 1 (GPER-1) has been demonstrated in several parts of the brain and may play an important role in estrogen downstream signaling pathway. However, the effects of this receptor on epileptic seizure are not clearly known. Therefore, the effects of GPER-1 agonist, G-1, GPER-1 antagonist, G-15 and the main estrogenic hormone, 17β-estradiol were investigated on seizures and brain tissue oxidative damages induced by pentylenetetrazole (PTZ) in rats., Methods: In this study, 30 adult male Wistar albino rats were used. Due to intraperitoneal (ip) injections of a subconvulsant dose of PTZ (35mg/kg) which was repeated 12 times every 48h, chemical kindling occurred and kindling seizure was recorded for 30min. The rats were injected with 17β-estradiol (5μg/kg, ip) or G-1 (5μg/kg, ip), G-15 (5μg/kg, ip), Saline, Ethanol and Dimethyl sulfoxide (DMSO) 30min before each dose of PTZ. Observed seizures were classified between the phase 0-5. Thirty minutes later when the last 12. PTZ administration, all rats were sacrificed and the brain cortex, hippocampus sections were removed and the tissue superoxide dismutase (SOD), malondialdehyde (MDA) and nitric oxide (NO) levels on these tissues were studied., Results: GPER1 agonist, G-1 and estrogenic hormone, 17β-estradiol significantly increased the development of PTZ kindling the seizures. However, GPER1 antagonist, G-15 did not change the development of PTZ kindling the seizures. In the cortex and hippocampus homogenates, the NO levels after G-1 administration had significantly increased (p<0.05) compared to the PTZ groups but SOD activities and MDA levels demonstrated no difference between the groups., Conclusions: This is the first study that explores that GPER-1 receptors have epileptogenic effect on PTZ-induced kindling rat. GPER1 may mediate the epileptogenic effect of estrogens by changing the oxidative or anti-oxidative parameters in the brain., (Copyright © 2015 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.)

The single feature of all malformations in cortical development is the clinical association with epilepsy. It has been proven that Sox-1 expression is essential during neurodevelopment and it is reported that Sox-1 knockout mice present spontaneous generalized seizures. Particularly in cerebellum, Sox-1 plays a key role in the Bergmann´s glia (BG) function, which allows the correct function of the Purkinje cells (PC). The targets of PC are the dentate and interpositus nuclei, which form the main cerebellar efferents involved in the physiopathology of epilepsy. Here we present the Sox-1 expression in cerebellum of rats during electric amygdala-kindling. We obtained seizures and once they had 3, 15 and 45 electric stimuli, the animals were sacrificed; the cerebellum was processed for inmunohistochemistry and Western blot analysis was performed to determine Sox-1 expression. Liquid chromatography was performed to examine gammaaminobutyric acid (GABA) and glutamate concentration. According to the literature, a progressive increase was observed in the electrographic and behavioral parameters. We found that Sox-1 expression in 15 and 45-stimuli groups had a statistically significant decrease as compared with controls, while the 3-stimuli group was similar to the control group. The concentration of glutamate was increased in rats with 45 stimuli. We can conclude that Sox-1 expression decreases as the number of seizures increases, and this is probably due to an altered glutamate regulation by a dysfunctional BG. In this way, we can suggest this mechanism as a one possible explanation of how the cerebellum participates in the pathophysiology of epilepsy.

Synaptic vesicle protein 2A (SV2A) involvement has been reported in the animal models of epilepsy and in human intractable epilepsy. The difference between pharmacosensitive epilepsy and pharmacoresistant epilepsy remains poorly understood. The present study aimed to observe the hippocampus SV2A protein expression in amygdale-kindling pharmacoresistant epileptic rats. The pharmacosensitive epileptic rats served as control. Amygdaloid-kindling model of epilepsy was established in 100 healthy adult male Sprague-Dawley rats. The kindled rat model of epilepsy was used to select pharmacoresistance by testing their seizure response to phenytoin and phenobarbital. The selected pharmacoresistant rats were assigned to a pharmacoresistant epileptic group (PRE group). Another 12 pharmacosensitive epileptic rats (PSE group) served as control. Immunohistochemistry, real-time PCR and Western blotting were used to determine SV2A expression in the hippocampus tissue samples from both the PRE and the PSE rats. Immunohistochemistry staining showed that SV2A was mainly accumulated in the cytoplasm of the neurons, as well as along their dendrites throughout all subfields of the hippocampus. Immunoreactive staining level of SV2A-positive cells was 0.483 ± 0.304 in the PRE group and 0.866 ± 0.090 in the PSE group (P < 0.05). Real-time PCR analysis demonstrated that 2(-ΔΔCt) value of SV2A mRNA was 0.30 ± 0.43 in the PRE group and 0.76 ± 0.18 in the PSE group (P < 0.05). Western blotting analysis obtained the similar findings (0.27 ± 0.21 versus 1.12 ± 0.21, P < 0.05). PRE rats displayed a significant decrease of SV2A in the brain. SV2A may be associated with the pathogenesis of intractable epilepsy of the amygdaloid-kindling rats.

Our previous study showed that the expression of miR-181a in memory impairment group of pentylenetetrazol (PTZ)-induced epileptic rats was up-regulated, but whether miR-181a influenced the cognitive function of PTZ-induced epileptic rats remains unknown. Therefore, we investigated the role of miR-181a in the cognitive function of PTZ-induced epileptic rats. A model of temporal lobe epilepsy (TLE) was induced via PTZ kindling in SD male rats. The epileptic rats were divided into Epilepsy group, Agomir-control group, miR-181a agomir group, 12 rats for each. 12 rats were used as sham group. We found that compared to the sham group, the expression of miR-181a in the Epilepsy group was increased. We also found that escape latency in the 5th day was prolonged and crossing times in the 6th day was reduced via Morris Water Maze test, which may indicate memory impairment. Furthermore, over-expression of miR-181a effectively reduced Bcl-2 protein level and increased apoptosis in hippocampus. Moreover, compared with Agomir-control group, the escape latency of miR-181a agomir group was obviously induced (P<0.05). Our findings suggest that miR-181a may play a role in impairing the cognitive function of PTZ-induced epileptic rats, and miR-181a could decrease the Bcl-2 protein and induce the apoptosis in the hippocampus that might be the way to impair cognitive function.

Previous studies have shown that inhibitors of the Na(+)/Ca(2+) exchanger (NCX) attenuate seizure activity in drug-induced epilepsy models, but the role of NCX in epilepsy is not fully understood. The present study examined the effects of pentylenetetrazole (PTZ)-induced kindling on the mRNA expression of NCX isoforms (NCX1, NCX2 and NCX3) in mouse brain. Chronic administration of PTZ at 40mg/kg resulted in kindling seizure development. It caused decreases in the mRNA levels of NCX1 and NCX2, but not NCX3, in the hippocampus. Changes in NCX isoform expression levels were not observed in the prefrontal cortex or striatum. Acute PTZ at 40mg/kg, which caused little seizure activity, also decreased NCX2, but not NCX1 mRNA levels in the hippocampus. These results suggest that down-regulation of hippocampal NCX1 expression is associated with PTZ-induced kindling seizure development., (Copyright © 2015 Elsevier B.V. All rights reserved.)

JNK-interacting protein 3 (JIP3), also known as JNK stress-activated protein kinase-associated protein 1 (JSAP1), is a scaffold protein mainly involved in the regulation of the pro-apoptotic signaling cascade mediated by c-Jun N-terminal kinase (JNK). Overexpression of JIP3 in neurons in vitro has been reported to lead to accelerated activation of JNK and enhanced apoptosis response to cellular stress. However, the occurrence and the functional significance of stress-induced modulations of JIP3 levels in vivo remain elusive. In this study, we investigated the expression of JIP3 in temporal lobe epilepsy (TLE) and in a kainic acid (KA)-induced mouse model of epileptic seizures, and determined whether down-regulation of JIP3 can decrease susceptibility to seizures and neuron damage induced by KA. We found that JIP3 was markedly increased in TLE patients and a mouse model of epileptic seizures; mice underexpressing JIP3 through lentivirus bearing LV-Letm1-RNAi showed decreased susceptibility, delayed first seizure and decreased seizure duration response to the epileptogenic properties of KA. Subsequently, a decreased activation of JNK following seizure induction was observed in mice underexpressing JIP3, which also exhibited less neuronal apoptosis in the CA3 region of the hippocampus, as assessed three days after KA administration. We also found that mice underexpressing JIP3 exhibited a delayed pentylenetetrazole (PTZ)-induced kindling seizure process., (Copyright © 2015 IBRO. Published by Elsevier Ltd. All rights reserved.)

Aims: Antioxidant compounds have been extensively investigated as a pharmacological alternatives to prevent epileptogenesis. Rosmarinic acid (RA) and caffeic acid (CA) are compounds with antioxidant properties, and RA has been shown to inhibit GABA transaminase activity (in vitro). Our aim was to evaluate the effect of RA and CA on seizures induced by pentylenotetrazole (PTZ) using the kindling model in mice., Main Methods: Male CF-1 mice were treated once every three days during 16days with RA (1, 2 or 4mg/kg; i.p.), or CA (1, 4 or 8mg/kg; i.p.), or positive controls diazepam (1mg/kg; i.p.) or vigabatrin (600mg/kg; p.o.), 30min before PTZ administration (50mg/kg; s.c.). After the last treatment, animals were sacrificed and the cortex was collected to evaluate free radicals (determined by 2',7'-dichlorofluorescein diacetate probe), superoxide dismutase (SOD) and genotoxic activity (Alkaline Comet Assay)., Key Findings: Rosmarinic acid 2mg/kg increased latency and decreased percentage of seizures, only on the 4th day of observation. The other tested doses of RA and CA did not show any effect. Rosmarinic acid 1mg/kg, CA 4mg/kg and CA 8mg/kg decreased free radicals, but no dose altered the levels of enzyme SOD. In the comet assay, RA 4mg/kg and CA 4mg/kg reduced the DNA damage index., Significance: Some doses of rosmarinic acid and CA tested showed neuroprotective action against oxidative and DNA damage produced in the kindling epilepsy model, although they did not produce antiepileptogenic effect in vivo., (Copyright © 2014 Elsevier Inc. All rights reserved.)