Pentylenetetrazole-Induced Seizures Cause Short-Term Changes in the Phenotype of Microglial and Astroglial Cells in the Hippocampus and Temporal Cortex of Young Male Wistar Rats.

Astrocytes and microglia can adopt two distinct phenotypes in various pathological processes: neurotoxic A1/M1 and neuroprotective A2/M2. Recent evidence suggests that these cells play a significant role in epileptogenesis. The objective of this study was to characterize the phenotype of astrocytes and microglial cells in the hippocampus and temporal cortex of young male Wistar rats at 3 h, 1, 3, and 7 days after pentylenetetrazole-induced seizures. RT-qPCR was employed to examine the expression of glial genes (Gfap, Aif1, Slc1a1, Slc1a2, Slc1a3, Itpr2, Gdnf, Bdnf, Fgf2, Tgfb, Il1b, Tnf, Il1rn, Lcn2, S100a10, Nlrp3, Arg1). The most notable alterations in the expression of glial genes were observed on the first day following seizures in the temporal cortex. An increase in the expression of the Gfap, Slc1a2, Slc1a1, Il1b, Tnfa, Bdnf, and Fgf2 genes, and the A2 astrocyte condition marker S100a10, was observed. An increase in the expression of the Gfap and Slc1a2 genes was observed in the hippocampus on the first day after seizures. However, in contrast to the changes observed in the cortex, the changes in the hippocampus were opposite for the Il1rn, Bdnf, Tgfb, and Arg1 genes. Nevertheless, the alterations in GFAP and EAAT2 protein levels were not corroborated by Western blot analysis. Conversely, a more comprehensive immunohistochemical analysis confirmed an augmentation in the number of GFAP-positive cells in the hippocampus 1 day after seizures. Based on the presented evidence, we can conclude that a single convulsive seizure episode in 3-week-old rats results in transient astroglial activation and polarization to a neuroprotective phenotype (A2).

Cenobamate suppresses seizures without inducing cell death in neonatal rats.

GABA modulators such as phenobarbital (PB) and sodium channel blockers such as phenytoin (PHT) have long been the mainstay of pharmacotherapy for the epilepsies. In the context of neonatal seizures, both PB and PHT display incomplete clinical efficacy. Moreover, in animal models, neonatal exposure to these medications result in neurodegeneration raising concerns about safety. Cenobamate, a more recently approved medication, displays unique pharmacology as it is both a positive allosteric modulator of GABA-A receptors, and a voltage-gated sodium channel blocker. While cenobamate is approved for adult use, its efficacy and safety profile against neonatal seizures is poorly understood. To address this gap, we assessed the efficacy and safety of cenobamate in immature rodents. Postnatal day (P)7 rat pups were pretreated with cenobamate and challenged with the chemoconvulsant pentylenetetrazole (PTZ) to screen for anti-seizure effects. In a separate experiment, P7 rats were treated with cenobamate, and brains were processed to assess induction of cell death. Cenobamate displays dose-dependent anti-seizure efficacy in neonatal rats. Unlike PHB and PHT, it does not induce neurotoxicity in P7 rats. Thus, cenobamate may be effective at treating neonatal seizures while avoiding unwanted neurotoxic side effects such as cell death.

Effects of baicalin pre-treatment on pentylenetetrazole-induced seizures: Insights from zebrafish larvae locomotor behavior and neuronal calcium imaging.

Natural compounds are increasingly being studied for their potential neuroprotective effects against inflammatory neurological diseases. Epilepsy is a common neurological disease associated with inflammatory processes, and around 30% of people with epilepsy do not respond to traditional treatments. Some flavonoids, when taken along with antiseizure medications can help reduce the likelihood of drug-resistant epilepsy. Baicalin, a plant-based compound, has been shown to possess pharmacological properties such as anti-inflammatory, neuroprotective, anticonvulsant, and antioxidant activities. In this study, we tested the effect of baicalin on an established model of pharmacologically induced seizure in zebrafish using measures of both locomotor behavior and calcium imaging of neuronal activity. The results of our study showed that, at the tested concentration, and contrary to other studies in rodents, baicalin did not have an anti-seizure effect in zebrafish larvae. However, given its known properties, other concentrations and approaches should be explored to determine if it could potentially have other beneficial effects, either alone or when administered in combination with classic antiseizure medications.

Dynamic changes in seizure state and anxiety-like behaviors during pentylenetetrazole kindling in rats.

INTRODUCTION: Excessive anxiety is a mental disorder, and its treatment involves the use of benzodiazepines, a class of drugs that enhance the effects of the neurotransmitter gamma-aminobutyric acid (GABA) at the GABAA receptor. Anxiety disorders are frequent comorbidities in patients with epilepsy, and it has been speculated that anxiety disorders and epileptic seizures share common neurobiological mechanisms. However, conflicting results regarding anxiolytic and anxiogenic effects have been reported in animal models of epilepsy induced by pentylenetetrazole (PTZ) injections, and the causes of this discrepancy are unknown. We hypothesized that anxiety-like behaviors would change dynamically according to the changes in epilepsy susceptibility that occur during the PTZ kindling process. Therefore, we attempted to change anxiety-like behaviors bidirectionally depending on the number of PTZ injections. METHODS: Adult male rats were injected with PTZ 20 times every other day, and stages of seizure onset were classified according to the Racine staging system. Anxiety-like behaviors were measured after 10 and 20 injections. The control group was injected with an equal volume of saline solution. Anxiety-like behaviors were investigated using the open-field, light/dark transition, elevated plus maze, and social interaction tests. RESULTS: Bimodal changes in seizure stage were observed in response to PTZ kindling. The increase in the seizure stage was transiently suppressed after 10 injections, and this decrease in epileptic sensitivity disappeared after 20 injections. However, none of the rats reached a fully kindled state after 20 PTZ injections. After 10 PTZ injections, anxiety-like behaviors decreased compared with those of the control group in the open field, light/dark transition, and elevated plus-maze tests. The anxiolytic effects correlated with the seizure stage in individual rats. After 20 PTZ injections, anxiety-like behaviors returned to control levels. CONCLUSION: PTZ kindling induced bimodal changes in the seizure stage. Anxiety-like behaviors decreased with transient decrease in epileptic sensitivity and returned to control levels with the disappearance of these states. These findings suggest a common neurobiological mechanism underlying anxiety disorders and epileptic seizures. In addition, the discrepancy in the previous studies, in which anxiety levels increase or decrease in PTZ-kindled animals, may be due to examination at different phases of the kindling process.

Synthesis, Theoretical Study, and Anticonvulsant Evaluation of N-Arylenaminones.

N-Arylenaminones are highly versatile compounds which can be synthesized in relatively simple ways. In this work we explored the synthesis of the four monosubstituted N-(4-R-phenyl)enaminones 3 a (R=NO2), 3 b (R=F), 3 c (R=H), and 3 d (R=OMe) with the goal of determining the influence of the substituents' electronic effects on tautomer stability and biological activity. These compounds were analyzed by means of Density Functional Theory calculations (DFT), to evaluate the relative stability of the possible tautomers. We found that the enaminone structure is the most stable with respect to the ketoimine and iminoenol forms. In addition, all four compounds display anticonvulsant activity, with 3 d being the one that mostly increased latency and mostly decreased the number of convulsions with respect to the control group. The suggested mechanism of action involves blockage of the voltage-dependent Na+ channels, considering that these molecules meet the structural characteristics needed to block the receptor, as is the case of the positive control molecules phenytoin (PHT) and valproic acid (VPA).

Evaluation of thiopyrano[2,3-d]thiazole derivatives as potential anticonvulsant agents.

Anticonvulsant drug discovery has achieved significant progress; however, pharmacotherapy of epilepsy continues to be a challenge for modern medicine and pharmacy. To expand the chemical space of heterocycles as potential antiepileptic agents, herein we report on the synthesis and evaluation of anticonvulsant properties of a series of thiopyrano[2,3-d]thiazoles. The studied heterocycles are characterized by satisfactory drug-likeness and pharmacokinetics properties, calculated in silico using SwissADME. The anticonvulsant activity of thiopyrano[2,3-d]thiazole derivatives was evaluated in vivo using the subcutaneous pentylenetetrazole test. Three hits, that is, compounds 12, 14, and 16, that caused a pronounced anticonvulsant effect were identified. Derivatives 12, 14, and 16 positively affected the latent period of onset of clonic seizures, number of seizures, mortality rate, and duration of the seizure period of animals under experimental conditions. The anticonvulsant properties of compound 14 were equivalent to the effect of the reference drug, sodium valproate. All hit compounds are characterized by satisfying toxicity properties in the human lymphocytes and HEK293 cell line. The most active hit 14 possesses a potential affinity with the GABAA receptor in the molecular docking study and forms a stable complex in the molecular dynamics experiments equal to diazepam. Preliminary SAR results were obtained and discussed based on screening data.

Oral Administration of Probiotic Bifidobacterium breve Ameliorates Tonic-Clonic Seizure in a Pentylenetetrazole-Induced Kindling Mouse Model via Integrin-Linked Kinase Signaling.

Epilepsy is a chronic neurological disorder characterized by recurrent seizures that affects over 70 million people worldwide. Although many antiepileptic drugs that block seizures are available, they have little effect on preventing and curing epilepsy, and their side effects sometimes lead to serious morbidity. Therefore, prophylactic agents with anticonvulsant properties and no adverse effects need to be identified. Recent studies on probiotic administration have reported a variety of beneficial effects on the central nervous system via the microbiota-gut-brain axis. In this study, we investigated the effects of the oral administration of Bifidobacterium breve strain A1 [MCC1274] (B. breve A1) on tonic-clonic seizure in a pentylenetetrazole (PTZ)-induced kindling mouse (KD mouse) model. We found that the oral administration of B. breve A1 every other day for 15 days significantly reduced the seizure score, which gradually increased with repetitive injections of PTZ in KD mice. The administration of B. breve A1, but not saline, to KD mice significantly increased the level of Akt Ser473 phosphorylation (p-Akt) in the hippocampus; this increase was maintained for a minimum of 24 h after PTZ administration. Treatment of B. breve A1-administered KD mice with the selective inhibitor of integrin-linked kinase (ILK) Cpd22 significantly increased the seizure score and blocked the antiepileptic effect of B. breve A1. Moreover, Cpd22 blocked the B. breve A1-induced increase in hippocampal p-Akt levels. These results suggest that the ILK-induced phosphorylation of Akt Ser473 in the hippocampus might be involved in the antiepileptic effect of B. breve A1.

The Effect of Adenosine Signaling on Memory Impairment Induced by Pentylenetetrazole in Zebrafish.

Epilepsy is characterized by the manifestation of spontaneous and recurrent seizures. The high prevalence of comorbidities associated with epilepsy, such as cognitive dysfunction, affects the patients quality of life. Adenosine signaling modulation might be an effective alternative to control seizures and epilepsy-associated comorbidities. This study aimed to verify the role of adenosine modulation on the seizure development and cognitive impairment induced by pentylenetetrazole (PTZ) in zebrafish. At first, animals were submitted to a training session in the inhibitory avoidance test and, after 10 min, they received an intraperitoneal injection of valproate, adenosine A1 receptor agonist cyclopentyladenosine (CPA), adenosine A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), adenosine A2A receptor antagonist ZM 241385, adenosine deaminase inhibitor erythro-9-(2-hydroxy-3-nony1)-adenine hydrochloride (EHNA) or the nucleoside transporter inhibitor dipyridamole. Thirty min after the intraperitoneal injection, the animals were exposed to 7.5 mM PTZ for 10 min, where they were evaluated for latency to reach the seizure stages (I, II, and III). Finally, 24 h after the training session, the animals were submitted to the inhibitory avoidance test to verify their cognitive performance during the test session. Valproate, CPA, and EHNA showed antiseizure effects and prevented the memory impairment induced by PTZ exposure. DPCPX, ZM 241385, and dipyridamole pretreatments caused no changes in seizure development; however, these drugs prevented memory impairment without altering locomotion. Our results reinforce the antiseizure effects of adenosine signaling and support the idea that the involvement of adenosine in memory processes may be a target for preventive strategies against cognitive impairment associated with epilepsy.

Possible Interaction of Opioidergic and Nitrergic Pathways in the Anticonvulsant Effect of Ivermectin on Pentylenetetrazole-Induced Clonic Seizures in Mice.

Ivermectin (IVM) is an antiparasitic drug that primarily works by the activation of GABAA receptors. The potential pharmacological pathways behind the anti-convulsant effect of IVM haven't yet been identified. In this study, intravenous injection of pentylenetetrazole (PTZ)-induced clonic seizure in mice was investigated in order to assess the possible influence of IVM on clonic seizure threshold (CST). We also look at the function of the Opioidergic and nitrergic pathways in IVM anticonvulsant action on clonic seizure threshold. IVM (0.5, 1, 5, and 10 mg/kg, i.p.) raised the PTZ-induced CST, according to our findings. Furthermore, the ineffective dose of nitric oxide synthase inhibitors (L-NAME 10 mg/kg, i.p.), and (7-NI 30 mg/kg, i.p.) or opioidergic system agonist (morphine 0.25 mg/kg, i.p.) were able to amplify the anticonvulsive action of IVM (0.2 mg/kg, i.p.). Moreover, the anticonvulsant effect of IVM was reversed by an opioid receptor antagonist (naltrexone 1 mg/kg, i.p.). Furthermore, the combination of the ineffective dose of morphine as an opioid receptor agonist with either L-NAME (2 mg/kg, i.p.) or 7-NI (10 mg/kg, i.p.) and with an ineffective dose of IVM (0.2 mg/kg, i.p.) had a significant anticonvulsant effect. Taken together, IVM has anticonvulsant activity against PTZ-induced clonic seizures in mice, which may be mediated at least in part through the interaction of the opioidergic system and the nitric oxide pathway.

Effects of Diclofenac Sodium on Seizure Activity in Rats with Pentylenetetrazole-Induced Convulsions.

Epilepsy is a disease which affects between 1 and 2% of the population, and a large proportion of these people do not react to currently available anticonvulsant medications, indicating the need for further research into novel pharmacological therapies. Numerous studies have demonstrated that oxidative stress and inflammation occur during epilepsy and may contribute to its development and progression, indicating higher levels of oxidative and inflammatory parameters in experimental models and clinical patients. This research aimed to assess the impact of diclofenac sodium, a nonsteroidal anti-inflammatory medicine, on seizure and levels of oxidative stress and inflammatory biomarkers in a rat model of epilepsy triggered by pentylenetetrazole (PTZ). 60 rats were randomly allocated to one of two groups: electroencephalography (EEG) recordings or behavioral evaluation. Rats received diclofenac sodium at three various doses (25, 50, and 75 mg/kg) intraperitoneally (IP) or a placebo, followed by intraperitoneal (IP) pentylenetetrazole, a powerful seizure-inducing medication. To investigate if diclofenac sodium had antiseizure properties, seizure activity in rats was evaluated using EEG recordings, the Racine convulsion scale (RCS) behaviour score, the duration of the first myoclonic jerk (FMJ), and the levels of MDA, TNF-α, and SOD. The average percentage of EEG spike waves decreased from 76.8% (placebo) to 64.1% (25 mg/kg diclofenac), 55.9% (50 mg/kg diclofenac), and 37.8% (75 mg/kg diclofenac). FMJ had increased from a mean of 58.8 s (placebo), to 93.6 s (25 mg/kg diclofenac), 185.8 s (50 mg/kg diclofenac) and 231.7 s (75 mg/kg diclofenac). RCS scores decreased from a mean score of 5.6 (placebo), to 3.75 (25 mg/kg diclofenac), 2.8 (50 mg/kg diclofenac) and 1.75 (75 mg/kg diclofenac). MDA levels reduced from 14.2 ng/gr (placebo) to 9.6 ng/gr (25 mg/kg diclofenac), 8.4 ng/gr (50 mg/kg diclofenac) and 5.1 ng/gr (75 mg/kg diclofenac). Likely, TNF-α levels decreased from 67.9 ng/gr (placebo) to 48.1 ng/gr (25 mg/kg diclofenac), 33.5 ng/gr (50 mg/kg diclofenac) and 21.3 ng/gr (75 mg/kg diclofenac). SOD levels, however, enhanced from 0.048 U/mg (placebo) to 0.055 U/mg (25 mg/kg diclofenac), 0.14 U/mg (50 mg/kg diclofenac), and 0.18 U/mg (75 mg/kg diclofenac). Diclofenac sodium (25, 50, and 75 mg/kg i.p.) effectively lowered the spike percentages and RCS scores linked with PTZ-induced epilepsy in rats, as well as significantly decreased MDA, TNF-α, IL-1ß, PGE2 and increased SOD levels. Probably as a result of its anti-oxidative and anti-inflammatory effects, diclofenac sodium dramatically lowered seizure activity at both doses compared to placebo control. Each of these results were significant, with p-values of < 0.01, < 0.05. Therefore, the therapeutic application diclofenac sodium as a potential anticonvulsant should be investigated further.

Nicotinic acetylcholine receptors and epilepsy.

Despite recent advances in understanding the causes of epilepsy, especially the genetic, comprehending the biological mechanisms that lead to the epileptic phenotype remains difficult. A paradigmatic case is constituted by the epilepsies caused by altered neuronal nicotinic acetylcholine receptors (nAChRs), which exert complex physiological functions in mature as well as developing brain. The ascending cholinergic projections exert potent control of forebrain excitability, and wide evidence implicates nAChR dysregulation as both cause and effect of epileptiform activity. First, tonic-clonic seizures are triggered by administration of high doses of nicotinic agonists, whereas non-convulsive doses have kindling effects. Second, sleep-related epilepsy can be caused by mutations on genes encoding nAChR subunits widely expressed in the forebrain (CHRNA4, CHRNB2, CHRNA2). Third, in animal models of acquired epilepsy, complex time-dependent alterations in cholinergic innervation are observed following repeated seizures. Heteromeric nAChRs are central players in epileptogenesis. Evidence is wide for autosomal dominant sleep-related hypermotor epilepsy (ADSHE). Studies of ADSHE-linked nAChR subunits in expression systems suggest that the epileptogenic process is promoted by overactive receptors. Investigation in animal models of ADSHE indicates that expression of mutant nAChRs can lead to lifelong hyperexcitability by altering i) the function of GABAergic populations in the mature neocortex and thalamus, ii) synaptic architecture during synaptogenesis. Understanding the balance of the epileptogenic effects in adult and developing networks is essential to plan rational therapy at different ages. Combining this knowledge with a deeper understanding of the functional and pharmacological properties of individual mutations will advance precision and personalized medicine in nAChR-dependent epilepsy.

Antibiotics modulate frequency and early generation of epileptic seizures in zebrafish.

Antibiotics have been used for decades to treat various bacterial infections. Apart from bactericidal activities, their potential side effects have not been much studied or evaluated. Neurotoxicity is a major concern in the case of ß-lactam and fluoroquinolone families, which can result in convulsions or seizures. Here, we proposed a hypothesis to check whether antibiotic treatment can conclusively enhance anxiety-like behaviours and how seizure behavioural profile gets modulated in pentylenetetrazole (PTZ)-treated zebrafish. Zebrafish were treated with selected antibiotics such as 25 mg/L Penicillin G (PG) and Ciprofloxacin (CPFX), for 7 days and thereafter exposed to PTZ (7.5 mM) for 20 min. The data indicate that PG and CPFX-treated groups exhibited anxiety-like or stressed behavioural phenotypes in the novel tank test (6 min), and also, they were found to promote hyperactivity. Early onset of PTZ-induced seizure-like behavioural scores, the heightened intensity of seizure and reduced latency in different scores were found in PG and CPFX-administered groups. This study substantiates that PG and CPFX as potential seizure modulators in zebrafish. The zebrafish is a well-established and still expanding model organism in many fields. Here, we again reinforce zebrafish as a prominent model to investigate seizure-like neuro-behavioural entities and confirm that chronic antibiotic use has negative consequences that can exacerbate the circumstances of vertebrate species exhibiting seizure-related reactions.

Dietary flavonoids-rich Citrus reticulata peel extract interacts with CREB signaling to suppress seizures and linked neurobehavioral impairments in a kindling mouse model.

Objectives: The citrus fruits peel contains a variety of bioactive metabolites that have shown multiple therapeutic effects. However, despite having substantial ethnomedicinal value, citrus peels remained underexplored and regarded as bio-waste. This present study was planned to investigate the effect of a characterized peel extract of Citrus reticulata c.v. (CRE) in pentylenetetrazole (PTZ)-induced kindling and associated cognitive and behavioral impairments in a mouse model.Methods: The kindled animals were treated daily with CRE (100 and 200 mg/kg) and challenged with a sub-effective dose of PTZ every 5th day to record the severity of seizures. In the end, different tests were performed to record behavioral and cognitive performance.Results: CRE-treated kindled animals showed a significant suppression in seizure severity following 20 days of the treatment. In the T-maze test, the extract treatment resulted in a marked increase in the spontaneous alternations, whereas it showed no change in anxiety behavior of kindled animals in the elevated plus-maze test. In both forced swim and tail suspension tests, CRE treatment demonstrated a considerable reduction in immobility time. However, no change in overall locomotion was observed in the open field test among all the groups. An increase in the hippocampal Creb and Bdnf expression and decreased glutamate-to-GABA ratio were observed in the CRE-treated kindled animals.Discussion: The results showed that CRE treatment suppresses epileptic seizures and associated cognitive deficits and depression-like behavior in kindled mice. The gene expression findings supported that the observed protective effects of CRE be due to its interaction with CREB signaling.

Impact of noscapine on halting the progression of pentylenetetrazole induced kindling epilepsy in mice.

Epilepsy is caused by an excessive recurrent excitatory neuronal firing, characterized by motor, psychomotor, and sensory impairments. Current therapies fail to produce 100% outcomes because of the complexity of the disease, poor diagnosis, and upsurge to drug-resistant epilepsy. The study repurposed the drug 'noscapine' mainly known for its anti-tussive properties. For the management of epilepsy and its associated secondary complications. To confirm the effect of noscapine, adult mice were injected with pentylenetetrazole (PTZ) (35 mg/kg i.p.) on an alternate day for 29 days to induce epilepsy. Animals were pretreated with noscapine in three doses (5, 10, and 20 mg/kg i.p.) for 33 days. Various behavioural assessments like the open field test, Morris water maze, and tail suspension test were performed to observe animals' locomotor activity, spatial memory, and anxiety-depressive behaviour. On the 34th day, animals were sacrificed, and brains were removed for biochemical estimations. Prolonged PTZ treatment reduced locomotor, learning activity, and increased anxiety-depressive behaviour, which was further confirmed by reduced antioxidant levels such as reduced glutathione (GSH), superoxide dismutase (SOD), and catalase because of increased oxido-nitrosative stress, that is, malondialdehyde (MDA) and nitrite in the brain. In comparison, noscapine pretreatment attenuated PTZ-induced behavioural and biochemical changes in the animals. The results indicate that noscapine ameliorates the oxido-nitrosative stress. However, studies indicate that oxido-nitrosative stress is a significant concern for the GABAergic neurons and promotes the disease progression. Further studies are required to explore the molecular mechanism of noscapine, which might be a practical approach as a newer antiepileptic agent.

The protective effect of inhibiting mitochondrial fission on the juvenile rat brain following PTZ kindling through inhibiting the BCL2L13/LC3 mitophagy pathway.

Maintaining the balance of mitochondrial fission and mitochondrial autophagy on seizures is helpful to find a solution to control seizures and reduce brain injuries. The present study is to investigate the protective effect of inhibiting mitochondrial fission on brain injury in juvenile rat epilepsy induced by pentatetrazol (PTZ) by inhibiting the BCL2L13/LC3-mediated mitophagy pathway. PTZ was injected (40 mg/kg) to induce kindling once every other day, for a total of 15 times. In the PTZ + DMSO (DMSO), PTZ + Mdivi-1 (Mdivi-1), and PTZ + WY14643 (WY14643) groups, rats were pretreated with DMSO, Mdivi-1 and WY14643 for half an hour prior to PTZ injection. The seizure attacks of young rats were observed for 30 min after model establishment. The Morris water maze (MWM) was used to test the cognition of experimental rats. After the test, the numbers of NeuN(+) neurons and GFAP(+) astrocytes were observed and counted by immunofluorescence (IF). The protein expression levels of Drp1, BCL2L13, LC3 and caspase 3 in the hippocampus of young rats were detected by immunohistochemistry (IHC) and Western blotting (WB). Compared with the PTZ and DMSO groups, the seizure latency in the Mdivi-1 group was longer (P < 0.01), and the severity degree and frequency of seizures were lower (P < 0.01). The MWM test showed that the incubation periods of crossing the platform in the Mdivi-1 group was significantly shorter. The number of platform crossings, the platform stay time, and the ratio of residence time/total stay time were significantly increased in the Mdivi-1 group (P < 0.01). The IF results showed that the number of NeuN(+) neurons in the Mdivi-1 group was greater, while the number of GFAP(+) astrocytes was lower. IHC and WB showed that the average optical density (AOD) and relative protein expression levels of Drp1, BCL2L13, LC3 and caspase 3 in the hippocampi of rats in the Mdivi-1 group were higher (P < 0.05). The above results in the WY14643 group were opposite to those in the Mdivi-1 group. Inhibition of mitochondrial fission could reduce seizure attacks, protect injured neurons, and improve cognition following PTZ-induced epilepsy by inhibiting mitochondrial autophagy mediated by the BCL2L13/LC3 mitophagy pathway.

The blockade of transient receptor potential ankyrin 1 (TRPA1) protects against PTZ-induced seizure.

Treatment of epilepsy remains a major problem as some epileptic patients do not respond to the current therapeutics. Transient receptor potential ankyrin 1 (TRPA1) belongs to the TRP channels and has diverse physiological functions in the body. Considering its physiological properties, we aimed to evaluate its role in two experimental models of epilepsy, including pentylenetetrazol (PTZ)-induced acute seizure and PTZ-evoked kindling. Furthermore, the TRPA1 protein levels were assessed in the cerebral cortex, hippocampus, and cerebellum after seizure induction. Three groups of Wistar rats received acute intraperitoneal injection of pentylenetetrazol (PTZ, 85 mg/kg). The groups received intraventricular injections of vehicle (dimethyl sulfoxide, Tween 80, and sterile 0.9% saline), valproate (30 µg/rat), or HC030031 (TRPA1 antagonist, 14 µg/rat) before PTZ injection. In the PTZ-induced kindling model, PTZ was administrated 35 mg/kg every other day for 24 days. PTZ gradually provoked seizure-related behaviors. After experiments, the TRPA1 levels in the brain were assessed using western blot. The results showed that HC030031 reduced the median of seizure scores and S5 duration while increasing S2 and S5 latencies in acute and kindling models. The anticonvulsant effect of HC030031 was comparable with valproate as a standard anticonvulsant drug. Furthermore, induction of seizure, either acute or kindling, enhanced TRPA1 levels in the cerebral cortex, hippocampus, and cerebellum that were prevented by HC030031 or valproate administration. The results of this study showed that HC030031 as a TRPA1 receptor antagonist promoted a significant anticonvulsant effect comparable with valproate. Both drugs prevented TRPA1 upregulation during seizures. These findings imply that TRPA1 is a potential target in treating epilepsy.

In Silico Analysis, Anticonvulsant Activity, and Toxicity Evaluation of Schisandrin B in Zebrafish Larvae and Mice.

The aim of this study is to evaluate the anticonvulsant potential of schisandrin B, a main ingredient of Schisandra chinensis extracts. Schisandrin B showed anticonvulsant activity in the zebrafish larva pentylenetetrazole acute seizure assay but did not alter seizure thresholds in the intravenous pentylenetetrazole test in mice. Schisandrin B crosses the blood-brain barrier, which we confirmed in our in silico and in vivo analyses; however, the low level of its unbound fraction in the mouse brain tissue may explain the observed lack of anticonvulsant activity. Molecular docking revealed that the anticonvulsant activity of the compound in larval zebrafish might have been due to its binding to a benzodiazepine site within the GABAA receptor and/or the inhibition of the glutamate NMDA receptor. Although schisandrin B showed a beneficial anticonvulsant effect, toxicological studies revealed that it caused serious developmental impairment in zebrafish larvae, underscoring its teratogenic properties. Further detailed studies are needed to precisely identify the properties, pharmacological effects, and safety of schisandrin B.

Pentylenetetrazole-Induced Seizures Are Increased after Kindling, Exhibiting Vitamin-Responsive Correlations to the Post-Seizures Behavior, Amino Acids Metabolism and Key Metabolic Regulators in the Rat Brain.

Epilepsy is characterized by recurrent seizures due to a perturbed balance between glutamate and GABA neurotransmission. Our goal is to reveal the molecular mechanisms of the changes upon repeated challenges of this balance, suggesting knowledge-based neuroprotection. To address this goal, a set of metabolic indicators in the post-seizure rat brain cortex is compared before and after pharmacological kindling with pentylenetetrazole (PTZ). Vitamins B1 and B6 supporting energy and neurotransmitter metabolism are studied as neuroprotectors. PTZ kindling increases the seizure severity (1.3 fold, p < 0.01), elevating post-seizure rearings (1.5 fold, p = 0.03) and steps out of the walls (2 fold, p = 0.01). In the kindled vs. non-kindled rats, the post-seizure p53 level is increased 1.3 fold (p = 0.03), reciprocating a 1.4-fold (p = 0.02) decrease in the activity of 2-oxoglutarate dehydrogenase complex (OGDHC) controlling the glutamate degradation. Further, decreased expression of deacylases SIRT3 (1.4 fold, p = 0.01) and SIRT5 (1.5 fold, p = 0.01) reciprocates increased acetylation of 15 kDa proteins 1.5 fold (p < 0.01). Finally, the kindling abrogates the stress response to multiple saline injections in the control animals, manifested in the increased activities of the pyruvate dehydrogenase complex, malic enzyme, glutamine synthetase and decreased malate dehydrogenase activity. Post-seizure animals demonstrate correlations of p53 expression to the levels of glutamate (r = 0.79, p = 0.05). The correlations of the seizure severity and duration to the levels of GABA (r = 0.59, p = 0.05) and glutamate dehydrogenase activity (r = 0.58, p = 0.02), respectively, are substituted by the correlation of the seizure latency with the OGDHC activity (r = 0.69, p < 0.01) after the vitamins administration, testifying to the vitamins-dependent impact of the kindling on glutamate/GABA metabolism. The vitamins also abrogate the correlations of behavioral parameters with seizure duration (r 0.53-0.59, p < 0.03). Thus, increased seizures and modified post-seizure behavior in rats after PTZ kindling are associated with multiple changes in the vitamin-dependent brain metabolism of amino acids, linked to key metabolic regulators: p53, OGDHC, SIRT3 and SIRT5.

Pharmacological Analysis of GABAA Receptor and Sigma1R Chaperone Interaction: Research Report I-Investigation of the Anxiolytic, Anticonvulsant and Hypnotic Effects of Allosteric GABAA Receptors' Ligands.

Two groups of facts have been established in previous drug development studies of the non-benzodiazepine anxiolytic fabomotizole. First, fabomotizole prevents stress-induced decrease in binding ability of the GABAA receptor's benzodiazepine site. Second, fabomotizole is a Sigma1R chaperone agonist, and exposure to Sigma1R antagonists blocks its anxiolytic effect. To prove our main hypothesis of Sigma1R involvement in GABAA receptor-dependent pharmacological effects, we performed a series of experiments on BALB/c and ICR mice using Sigma1R ligands to study anxiolytic effects of benzodiazepine tranquilizers diazepam (1 mg/kg i.p.) and phenazepam (0.1 mg/kg i.p.) in the elevated plus maze test, the anticonvulsant effects of diazepam (1 mg/kg i.p.) in the pentylenetetrazole-induced seizure model, and the hypnotic effects of pentobarbital (50 mg/kg i.p.). Sigma1R antagonists BD-1047 (1, 10, and 20 mg/kg i.p.), NE-100 (1 and 3 mg/kg i.p.), and Sigma1R agonist PRE-084 (1, 5, and 20 mg/kg i.p.) were used in the experiments. Sigma1R antagonists have been found to attenuate while Sigma1R agonists can enhance GABAARs-dependent pharmacological effects.

Anticonvulsant Profile of Selected Medium-Chain Fatty Acids (MCFAs) Co-Administered with Metformin in Mice in Acute and Chronic Treatment.

In contrast to the other components of the medium-chain triglycerides ketogenic diet (MCT KD), i.e., caprylic acid (CA8), a comprehensive evaluation of caproic (CA6) and lauric acids' (CA12) properties in standard chemical and electrical seizure tests in mice has not yet been performed. We investigated their effects in maximal electroshock seizure threshold (MEST), 6 Hz seizure threshold and intravenous (i.v.) pentylenetetrazole (PTZ) seizure tests. Since ketone body production can be regulated by the activation of 5'AMP-activated protein kinase (AMPK), we hypothesized that metformin (an AMPK activator) enhance ketogenesis and would act synergistically with the fatty acids to inhibit convulsions. We assessed the effects of acute and chronic co-treatment with metformin and CA6/CA8 on seizures. CA6 and CA12 (p.o.) increased seizure threshold in the 6 Hz seizure test. CA6 at the highest tested dose (30 mmol/kg) developed toxicity in several mice, impaired motor performance and induced ketoacidosis. Acute and chronic co-treatment with metformin and CA6/CA8 did not affect seizure thresholds. Moreover, we observed the pro-convulsive effect of the acute co-administration of CA8 (5 mmol/kg) and metformin (100 mg/kg). Since this co-treatment was pro-convulsive, the safety profile and risk/benefit ratio of MCT KD and metformin concomitant therapy in epileptic patients should be further evaluated.