The Effects and Mechanisms of Action of TC-G 1008, GPR39 Agonist, in Animal Models of Seizures and Epilepsy

Background and purpose: The G-protein coupled receptor 39 (GPR39) may be activated by zinc ions. Activation of GPR39 was suggested as a novel pharmacological strategy for treating seizures. Experimental approach: We utilized a specific agonist of GPR39, TC-G 1008, and the nonspecific agonist, zinc chloride and a variety of models of acute seizures or a chronic model of epilepsy which were induced in non-genetically modified mice, GPR39 knockout mice or in zebrafish larvae. We examined total serum zinc (by Inductively Coupled Plasma Optical Emission Spectrometry) as well as intracellular zinc ([Zn2+]I) (by Zinpyr-1 staining) concentrations and the expression of selected proteins (by Western blot) which are associated with GPR39 signaling in the hippocampus. Key results: Liquid chromatography tandem mass spectrometry analysis showed that TC-G 1008 is brain penetrant. TC-G 1008 decreased the seizure threshold in the maximal electroshock seizure (MES) threshold test, but it increased the seizure threshold in the 6-Hz induced seizure threshold test. The behavioral effects of TC-G 1008 and MES or 6-Hz seizure were accompanied by alterations in hippocampal [Zn2+]I. TC-G 1008 increased the mean duration of EEG discharges in response to pentylenetetrazole (PTZ) in zebrafish larvae and facilitated the development of PTZ kindling in mice. Using GPR39 knockout mouse line, generated by the CRISPR-Cas-9 method, we showed that GPR39 is a target for TC-G 1008 regarding PTZ-induced epileptogenesis. Conclusion and implications: Our in vivo data obtained using TC-G 1008 generally argue against GPR39 activation as a therapeutic strategy for alleviating seizures/epilepsy.

IL4 Reduces Epileptogenesis Susceptibility Acutely After TBI: The Role of Macrophage/Microglia Polarization

Traumatic brain injury (TBI) is responsible for 5% of all epilepsy cases, which are known as post-traumatic epilepsy. Macrophage/microglia are key players in TBI pathogenesis. They are activated after TBI, transform to inflammatory phenotype (M1) and trigger neuroinflammation, which provokes epileptogenesis. Interleukin-4 (IL-4) is a well-known polarizer of macrophage/microglia to the anti-inflammatory phenotype (M2). We tested the effect of IL-4 on the rate of epileptogenesis, brain expression of inflammatory and anti-inflammatory cytokines, and the lesion size in traumatic rats. Trauma was exerted to temporo-parietal cortex of rats by Controlled Cortical Impact. Thereafter, rats received a single dose (100ng/rat) of IL-4 through intracerebroventricular injection. After 24h, pentylenetetrazole (PTZ) kindling started and development of generalized seizures was recorded. Level of TNF-α, TGF-β, IL-10, and arginase-1 (Arg-1) was measured in the brain by immunoblotting at 6h, 12h, 24h, 48h, and 5 days after TBI. The lesion size and cell survival were determined by staining. Traumatic rats were kindled by 5±1 PTZ injections (significantly less than 11±2 injections of control and sham-operated rats, p<0.001). IL-4 did not change kindling rate in sham-operated rats but inhibited acceleration of kindling rate in traumatic rats (13±1 PTZ injections, p<0.001). IL-4 decreased post-TBI overexpression of TNF-α (6h, p<0.001) whereas upregulated post-TBI expression of TGF-β (48h, p<0.001), IL-10 (24h, p<0.05; 48h, p<0.01), and Arg-1 (24h, p<0.001). IL-4 decreased lesion volume and number of dead neurons. IL-4 suppresses TBI-induced acceleration of epileptogenesis in rats by directing macrophage/microglia to the anti-inflammatory M2 phenotype and inhibition of neuronal death.

Thiamine Alleviates Cognitive Impairment and Epileptogenesis by Relieving Brain Inflammation in PTZ-induced Kindling Rat Model

Epileptogenesis, the process by which the brain becomes epileptic, is related to neuroinflammation, hyperexcitability, and as a result cognitive deficits. Evidence suggests that therapeutic strategies targeting pathologic brain inflammation have emerged as a promising approach that prevents or disease-modifying therapy for epileptogenesis. Therefore, the PTZ kindling model of epilepsy was utilized to assess the neuroprotective role of thiamine in epileptogenesis. Male rats were exposed to PTZ-induced kindling and pretreated with low thiamine (25 mg/kg) or high thiamine (50 mg/kg). Cyclooxygenase (COX-1 and COX-2), interleukin 1-beta (IL-1β), tumor necrosis factor-alpha (TNF-α), and nuclear factor-κB (NF-κB) concentrations in the brain were analyzed using biochemical assays. Cognitive function was evaluated using the passive avoidance test. Thiamine ameliorated epileptogenesis and enhanced the rats' performance in the passive avoidance test. Also, thiamine significantly decreased the level of neuroinflammatory mediators in the brain induced by PTZ. These results provide evidence that thiamine alleviates PTZ-induced neuroinflammation and cognitive impairments.

Anticonvulsant effect of pterostilbene and its influence on the anxiety- and depression-like behavior in the pentetrazol-kindled mice: behavioral, biochemical, and molecular studies

Rationale Pterostilbene is the 3,5-dimethoxy derivative of resveratrol with numerous beneficial effects including neuroprotective properties. Experimental studies revealed its anticonvulsant action in the acute seizure tests. Objectives The purpose of the present study was to evaluate the effect of pterostilbene in the pentetrazol (PTZ)–induced kindling model of epilepsy in mice as well as to assess some possible mechanisms of its anticonvulsant action in this model. Methods Mice were repeatedly treated with pterostilbene (50–200 mg/kg) and its effect on the development of seizure activity in the PTZ kindling was estimated. Influence of pterostilbene on the locomotor activity and anxiety- and depression-like behavior in the PTZ-kindled mice was also assessed. To understand the possible mechanisms of anticonvulsant activity of pterostilbene, γ-aminobutyric acid (GABA) and glutamate concentrations in the prefrontal cortex and hippocampus of the PTZ-kindled mice were measured using LC–MS/MS method. Moreover, mRNA expression of BDNF, TNF-α, IL-1β, IL-6, GABRA1A, and GRIN2B was determined by RT-qPCR technique. Results We found that pterostilbene at a dose of 200 mg/kg considerably reduced seizure activity but did not influence the locomotor activity and depression- and anxiety-like behavior in the PTZ-kindled mice. In the prefrontal cortex and hippocampus, pterostilbene reversed the kindling-induced decrease of GABA concentration. Neither in the prefrontal cortex nor hippocampus pterostilbene affected mRNA expression of IL-1β, IL-6, GABRA1A, and GRIN2B augmented by PTZ kindling. Pterostilbene at a dose of 100 mg/kg significantly decreased BDNF and TNF-α mRNA expression in the hippocampus of the PTZ-kindled mice. Conclusions Although further studies are necessary to understand the mechanism of anticonvulsant properties of pterostilbene, our findings suggest that it might be considered a candidate for a new antiseizure drug.

Role of Standardized Plant Extracts in Controlling Alcohol Withdrawal Syndrome—An Experimental Study

Patients with alcohol use disorder experience alcohol withdrawal syndrome due to the sudden cessation of alcohol. This study was designed to evaluate the protective effect of Ashwagandha and Brahmi on alcohol withdrawal in rats. Thirty rats of either sex were taken and randomly divided into 6 groups (n = 5). Their normal diet was replaced by a modified liquid diet (MLD). Ethanol was added gradually except in the MLD group for a period of 21 days and withdrawn suddenly. Four treatment groups were administered Ashwagandha (3.75 mg of withanolide glycosides per kg body weight), Brahmi (10 mg of bacosides per kg body weight), Ashwagandha + Brahmi (3.75 mg withanolide glycosides + 10 mg bacosides per kg body weight) orally and diazepam (1 mg/kg body weight, i.p.) 45 min before alcohol withdrawal. Rats were assessed for behavioural changes (agitation score and stereotypic behaviour), anxiety and locomotor activity at 2nd and 6th hours of alcohol withdrawal. Pentylenetetrazol (PTZ) kindling seizures were assessed at 6th hour of alcohol withdrawal. Ashwagandha and Brahmi alone and in combination significantly reduced the behavioural changes in alcohol withdrawal rats at 2nd hour and their combination in 6th hour. Ashwagandha and Brahmi suppressed PTZ kindling seizures effectively and improved locomotory activity at 2nd hour and 6th hour of alcohol withdrawal. Reduction in anxiety was significant among Ashwagandha at 2nd hour and the combination group at 2nd and 6th hour. The results were comparable to diazepam. Ashwagandha and Brahmi have beneficial effects in controlling the behavioural changes, anxiety and seizures in alcohol withdrawal symptoms in rats and improved locomotory activity.

Tetrapleura tetraptera (Schumach.) Taub. Fruit Extract Improves Cognitive Behaviour and Some Brain Areas of Pentylenetetrazol-Kindling Rats

Epilepsy is a neuronal disorder that arises from imbalances of some neurotransmitters and manifests as recurrent seizure and cognitive impairment. Most antiepileptic drugs are either expensive, not effec¬tive or are associated with adverse effects warranting efficacious alternatives. This study, therefore, investigated the activity of Tetrapleura tetraptera (Schumach.) Taub fruit extract (TTE) on the behaviour and some brain areas of pentylenetetrazol (PTZ)-kindling rats. Thirty-five male Wistar rats (150-200 g) were assigned into five groups (1-5, n=7): Control (distilled water); TTE (500 mg/kg); PTZ (40 mg/kg); PTZ (40 mg/kg) pre-treated with either sodium valproate (SV, 200 mg/kg) or TTE (500 mg/kg). All treatments were oral, except for the PTZ (intraperitoneally), and carried out 48 hourly, until kindling was also fully achieved (21 days). Subsequently, there was a beam walking behavioural test, deep anaes-thesia and animals’ sacrifice, while whole brains were processed for histology. The results showed that seizure was induced with higher mortality in the PTZ group, and was suppressed with higher quantal protection in the PTZ groups pre-treated with either TTE or SV. There was no difference (p>0.05) in beam walk slips and latency. Simultaneously, the PTZ group showed some degenerative cellular changes in the hippocampus and temporal cortex, with significantly (p<0.05) higher cellular density, except in the cerebellum. These cellular changes were either minimal or not apparent in the PTZ groups pre-treated with either TTE or SV compared with the control group. In conclusion, TTE protected against PTZ - induced seizures and brain histopathology of rats, with results similar to the standard anticonvulsant drug, SV.

Chaihu plus Longgu Muli Decoction Alleviated Brain Injury in Pentylenetetrazole-Kindled Epileptic Mice by Regulating Cyclooxygenase-2/Prostaglandin E2/Multidrug Transporter Pathway

Objective. To evaluate the effect of CLMD administration on epileptic seizures and brain injury in pentylenetetrazole- (PZT-) kindled mice. Methods. The effect of pretreatment with CLMD (5, 10, and 20 ml/kg (mg/kg) by gavage) for seven days on PTZ-induced kindling, duration and grade of kindling-induced seizures, and pathological injury in the cortex and hippocampus was evaluated. Male BALB/c mice with adenosine A1 receptor knockout were subjected to intraperitoneal injection of PTZ (35 mg/kg) once every day until kindling was successfully induced. Quantitative reverse transcription polymerase chain reaction, immunofluorescence, and western blot were performed to assess the mRNA and protein levels of p-glycoprotein (PGP), multidrug resistance-associated protein 1 (MRP1), cyclooxygenase-2 (COX-2), prostaglandin E2 (PGE2), and adenylate kinase (ADK) in the cortex and hippocampus. Results. PTZ successfully induced kindling in mice after 21 days, wherein CLMD showed an obvious dose-dependent antiepileptic effect. High-dose CLMD significantly increased the latency of epileptic seizures, decreased the sustained time of epileptic seizures and the seizure grade, and ameliorated the histopathological changes in the cortex and hippocampus. Furthermore, PTZ kindling induced significantly higher levels of PGP, MRP1, COX-2, PGE2, and ADK, but this effect was inhibited by pretreatment with CLMD in a dose-dependent manner. Conclusion. Pretreatment with CLMD attenuates PTZ-kindled convulsions and brain injury in mice. The mechanism may be related to the cyclooxygenase-2/prostaglandin E2/multidrug transporter pathway.

Immunoreactivity of Muscarinic Acetylcholine M2 and Serotonin 5-HT2B Receptors, Norepinephrine Transporter and Kir Channels in a Model of Epilepsy

Epilepsy is characterized by an imbalance in neurotransmitter activity; an increased excitatory to an inhibitory activity. Acetylcholine (ACh), serotonin, and norepinephrine (NE) may modulate neural activity via several mechanisms, mainly through its receptors/transporter activity and alterations in the extracellular potassium (K+) concentration via K+ ion channels. Seizures may disrupt the regulation of inwardly rectifying K+ (Kir) channels and alter the receptor/transporter activity. However, there are limited data present on the immunoreactivity pattern of these neurotransmitter receptors/transporters and K+ channels in chronic models of epilepsy, which therefore was the aim of this study. Changes in the immunoreactivity of epileptogenesis-related neurotransmitter receptors/transporters (M2, 5-HT2B, and NE transporter) as well as Kir channels (Kir3.1 and Kir6.2) were determined in the cortex, hippocampus and medulla of adult Wistar rats by utilizing a Pentylenetetrazol (PTZ)-kindling chronic epilepsy model. Increased immunoreactivity of the NE transporter, M2, and 5-HT2B receptors was witnessed in the cortex and medulla. While the immunoreactivity of the 5-HT2B receptor was found increased in the cortex and medulla, it was decreased in the hippocampus, with no changes observed in the M2 receptor in this region. Kir3.1 and Kir6.2 staining showed increase immunoreactivity in the cerebral cortex, but channel contrasting findings in the hippocampus and medulla. Our results suggest that seizure kindling may result in significant changes in the neurotransmitter system which may contribute or propagate to future epileptogenesis, brain damage and potentially towards sudden unexpected death in epilepsy (SUDEP). Further studies on the pathogenic role of these changes in neurotransmitter receptors/transporters and K+ channel immunoreactivity may identify newer possible targets to treat seizures or prevent epilepsy-related comorbidities.