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Lafora disease (LD) is a syndrome of progressive myoclonic epilepsy and cumulative neurocognitive deterioration caused by recessively inherited genetic lesions of EPM2A (laforin) or NHLRC1 (malin). Neuropsychiatric symptomatology in LD is thought to be directly downstream of neuronal and astrocytic polyglucosan aggregates, termed Lafora bodies (LBs), which faithfully accumulate in an age-dependent manner in all mouse models of LD. In this study, we applied home-cage monitoring to examine the extent of neurobehavioral deterioration in a model of malin-deficient LD as a means to identify robust preclinical endpoints that may guide the selection of novel genetic treatments. At 6 weeks, ∼6-7 months, and ∼12 months of age, malin-deficient mice ("KO") and wild-type (WT) littermates underwent a standardized home-cage behavioral assessment designed to non-obtrusively appraise features of rest/arousal, consumptive behaviors, risk aversion, and voluntary wheel-running. At all timepoints, and over a range of metrics that we report transparently, WT and KO mice were essentially indistinguishable. In contrast, within WT mice compared across the same timepoints, we identified age-related nocturnal hypoactivity, diminished sucrose preference, and reduced wheel-running. Neuropathological examinations in subsets of the same mice revealed expected age-dependent LB accumulation, gliosis, and microglial activation in cortical and subcortical brain regions. At 12 months of age, despite the burden of neocortical LBs, we did not identify spontaneous seizures during an electroencephalographic (EEG) survey, and KO and WT mice exhibited similar spectral EEG features. However, in an in vitro assay of neocortical function, paroxysmal bursts of network activity (UP states) in KO slices were more prolonged at 3 and 6 months of age, but similar to WT at 12 months. KO mice displayed a distinct response to pentylenetetrazole, with a greater incidence of clonic seizures and a more pronounced postictal suppression of movement, feeding, and drinking behavior. Together, these results highlight the clinicopathologic dissociation in a mouse model of LD, where the accrual of LBs may latently modify cortical circuit function and seizure threshold without clinically meaningful changes in home-cage behavior. Our findings allude to a delay between LB accumulation and neurobehavioral decline in LD: one that may provide a window for treatment, and whose precise duration may be difficult to ascertain within the typical lifespan of a laboratory mouse., (© 2024 Wiley Periodicals LLC.)

Background: Zebrafish has emerged as a potential animal model of acute convulsion for early screening of antiepileptic agents. There is a need for alternative chronic zebrafish models of epilepsy with more correlation to the clinical condition., New Method: Adult zebrafish were repeatedly exposed to subeffective concentrations of pentylenetetrazole (PTZ), until appearance to tonic-clonic seizures, considered as kindled. Valproic acid (VPA) exposure was given during kindling and in kindled fish in 2 different groups. The neurotransmitters level and expression of the genes associated with kindling were studied in the fish brain., Results: There was an increase in seizure severity score at 1.25 mM concentration of PTZ, and 66.66 % of fish achieved kindling after 22 days' exposure. A marked increase in c-fos, crebbpa and crebbpbexpression, and glutamate/GABA level was observed in the brain of kindled fish. VPA inhibited the induction of PTZ-mediated kindling and reduced seizure severity in kindled fish., Comparison With Existing Method: In contrast to an existing adult zebrafish kindling method, the present protocol is of longer duration, with more similarity to clinical epilepsy. Moreover, the induction of kindling involves a simple non-invasive technique without the use of anesthesia. The protocol can be used for evaluation of both antiepileptic and antiepileptogenic agents., Conclusion: Repeated exposure of 1.25 mM PTZ induced kindling in zebrafish, altering the brain neurotransmitter levels and gene expression. Inhibition of kindling induction and decrease in seizures in normal and kindled fish, respectively by VPA validated application of the model for preclinical testing of agents against epilepsy., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The role of PI3K/AKT/mTOR signalling pathway has been widely deciphered in pathogenesis of epilepsy. LY294002 is a selective inhibitor of phosphatidylinositol-3-kinase (PI3K). The present study was designed to explore the anticonvulsant potential of LY294002 in a zebrafish model of pentylenetetrazole (PTZ)-mediated convulsions. Zebrafish larvae at 7dpf (days post fertilization) were pre-incubated with varying concentrations of LY294002, prior to PTZ exposure. The adult zebrafish were also exposed to PTZ after intraperitoneal injection with different concentrations of LY294002, followed by gene expression studies in the brain. The hyperactive responses indicated by total distance travelled and the mean speed of larva was drastically decreased, whereas latency to first clonic-like seizure was increased following LY294002 pre-treatment. Additionally, a marked decrease in c-fos expression was also observed in the larvae group exposed to LY294002 in comparison to control. Furthermore, PTZ evoked seizure severity was considerably decreased, while latency to clonic-like seizure was increased in adult zebrafish group treated with 100 nM of LY294002. Furthermore, the occurrence of tonic-like seizures was also reduced in the adult zebrafish treated with LY294002. The mRNA levels of PIK3CA, PIK3R1, AKT1, mTOR, Rps6 and Rps6kb1 in adult zebrafish brain was significantly reduced as compared to vehicle control group. Our results provided conclusive support for the anticonvulsant potential of LY294002., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Pyrrolone-fused benzosuberene (PBS) compounds were semi-synthesized from α,β,γ-Himachalenes extracted from the essential oil of Cedrus deodara following amino-vinyl-bromide substituted benzosuberenes as intermediates. These PBSs compounds classified as an attractive source of therapeutics. The α-isoform of PI3K which is a pivotal modulator of PI3K/AKT/mTOR signaling pathway, responsible for neurological disorders like epilepsy, found as a potential target molecule against these 17 semi-synthesized PBS compounds using in silico ligand-based pharmacophore mapping and target screening. The compounds screened using binding affinities, ADMET properties, and toxicity that were accessed by in silico docking simulations and pharmacokinetics profiling. Ultimately two compounds viz., PBS-8 and PBS-9 were selected for further in vivo evaluation using a zebrafish (Danio rerio) model of pentylenetetrazol (PTZ)-induced clonic convulsions. Additionally, gene expression studies performed for the genes of the PI3K/AKT/mTOR pathway which further validated our results. In conclusion, these findings suggested that PBS-8 is a promising candidate that could bedeveloped as a potential antiepileptic.

Over the past few years, there has been a tremendous increase in interest of general population toward food-based therapies for management of chronic clinical conditions due to their lesser adverse effects with prolonged use over pharmacotherapies. Foods enriched with omega-3 fatty acids have shown some promising results in case of epilepsy. The present study was envisioned to investigate the effect of early exposure of α-linolenic acid (ALA), an essential omega-3 fatty acid in developing zebrafish (Danio rerio) embryos toward pentylenetetrazol (PTZ)-induced seizure susceptibility. The healthy wild-type zebrafish embryos were incubated in system water or system water containing different ALA concentrations (1-20 µM) till 7 dpf (days post fertilization). Each larva at 7 dpf was placed in 8 mM PTZ solution and seizure event was recorded. ALA incubation at 10 µM and 20 µM concentrations showed a dose-dependent reduction in PTZ-mediated hyperactive responses in larvae indicated by a marked decrease in total distance travelled and speed, as compared to vehicle control. Furthermore, both the treated groups showed increase in the latency to PTZ-induced clonus-like seizures in larvae, as compared to vehicle control. ALA incubated larvae at 10 µM and 20 µM concentrations also showed a significant reduction in c-fos mRNA level. A marked increase in the level of ALA and docosahexaenoic acid was also observed in the larvae incubated at highest effective concentration of ALA. The present study concluded that embryonic exposure of ALA reduced PTZ-induced seizures in zebrafish larva., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The role of neuroinflammatory mediators has been well established in the pathogenesis of temporal lobe epilepsy (TLE) and associated neurobehavioral comorbidities. Mycophenolate mofetil (MMF) is commonly used as an immunosuppressant in organ transplantations. Its neuroprotective effect is well explored in different preclinical and clinical studies. The present study was designed to investigate the effect of MMF in rat model of lithium pilocarpine (LiPc)-induced spontaneous recurrent seizures and its associated neurobehavioral comorbidities. MMF treatment showed a dose-dependent decrease in seizure severity and reduced aggression in epileptic rats. There was marked improvement in spatial and recognition memory functions, along with substantial decrease in depression-like behavior in MMF treated epileptic rats. There was considerable decrease in mossy fiber sprouting in the dentate gyrus and the cornu ammonis 3 regions of the hippocampus, along with reduction in neuronal death in the treated groups. Furthermore, the hippocampal mRNA level of IL-1β, IL-2, PI3K, AKT, HIF-1α, RAPTOR, mTOR, Rps6kb1 and Rps6 was found to be decreased in MMF treated animals. mTOR, S6, pS6 and GFAP protein expression was decreased, whereas NeuN was increased in the rat hippocampus of the treated animals. The results concluded that MMF suppress recurrent seizures, and improves its associated behavioral impairments and cognitive deficit in rat model of TLE. The observed effects of MMF be correlated with the inhibition of IL-2 and IL-1β linked PI3K/AKT/mTOR signaling pathway hyperactivation., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Ethnopharmacological Relevance: Ginkgo biloba L. (Ginkgoaceae) has been widely used in traditional medicine for variety of neurological conditions particularly behavioral and memory impairments., Aim of the Study: The present study was envisaged to explore the effect of a standardized fraction of Ginkgo biloba leaves (GBbf) in rat model of lithium-pilocarpine induced spontaneous recurrent seizures, and associated behavioral impairments and cognitive deficit., Materials and Methods: Rats showing appearance of spontaneous recurrent seizures following lithium pilocarpine (LiPc)-induced status epilepticus (SE) were treated with different doses of GBbf or vehicle for subsequent 4 weeks. The severity of seizures and aggression in rats were scored following treatment with GBbf. Further, open field, forced swim, novel object recognition and Morris water maze tests were conducted. Histopathological, protein levels and gene expression studies were performed in the isolated brains., Results: Treatment with GBbf reduced seizure severity score and aggression in epileptic animals. Improved spatial cognitive functions and recognition memory, along with reduction in anxiety-like behavior were also observed in the treated animals. Histopathological examination by Nissl staining showed reduction in neuronal damage in the hippocampal pyramidal layer. The dentate gyrus and Cornu Ammonis 3 regions of the hippocampus showed reduction in mossy fiber sprouting. GBbf treatment attenuated ribosomal S6 and pS6 proteins, and hippocampal mTOR, Rps6 and Rps6kb1 mRNA levels., Conclusions: The results of present study concluded that GBbf treatment suppressed lithium-pilocarpine induced spontaneous recurrent seizures severity and incidence with improved cognitive functions, reduced anxiety-like behavior and aggression. The effect was found to be due to inhibition of mTOR pathway hyperactivation linked with recurrent seizures., (Copyright © 2017. Published by Elsevier B.V.)

Crocin is a pharmacologically active carotenoid pigment mainly present in the stigmas of Crocus sativus L. (Iridaceae). It has been well explored in experimental animal models of cognitive impairments, depression, anxiety and epilepsy. This study was designed to understand the effect of crocin on pentylenetetrazol (PTZ)-induced kindling development and its associated cognitive deficit in mouse. Crocin treatment at 5, 10 and 20 mg/kg p.o. doses showed a marked reduction in severity of PTZ-induced seizures. There was an increase in novel object preference index and discrimination ratio in the crocin-treated groups in the novel object recognition test. Its treatment also increased percentage spontaneous alternations in T-maze test at all the tested doses. Histopathological examination by Nissl staining showed a reduction in dark neurons in the hippocampal pyramidal layer of crocin-treated animals in contrast to vehicle control, indicating a decrease in neuronal damage. Biochemical estimations showed a significant increase in superoxide dismutase activity and reduced reactive oxygen species (ROS) in the hippocampus of crocin-treated animals. Immunohistochemistry results revealed attenuation in the levels of nuclear factor-κB (NF-κB) and phosphorylated NF-κB in the hippocampal sections of crocin-treated animals. The results of this study concluded that crocin treatment increased seizure threshold, thus inhibiting PTZ-induced kindling development and improving cognitive functions. The effect was found to be due to suppression of seizure-induced ROS generation and its linked NF-κB pathway-associated neuronal damage., (© 2016 Nordic Association for the Publication of BCPT (former Nordic Pharmacological Society).)

The role of phosphatidylinositol 3-kinase linked mammalian target of rapamycin (mTOR) pathway hyperactivation is well established in cancer pathogenesis. Several molecules inhibiting mTOR pathway, leading to inhibition of protein synthesis responsible for angiogenesis of tumor cells have emerged out to be potential anticancers. Similar hyperactivation of mTOR pathway has also reported in epilepsy during latent phase, following precipitating injury causing reorganization of neuronal networks and ultimately leading to induction of seizures. The mTOR inhibitors have also found to attenuate pathological changes in the brain associated with epilepsy, primarily suppression of mossy fiber sprouting. At the same time, a few antiepileptic molecules which have been studied against cancer showed anticancer activity, apart from their principal mechanism of action. These studies suggest mTOR signaling pathway to be a common pathogenic link between cancer and epilepsy. It has been found that, anticancer molecules acting on different molecular targets, that ultimately down regulate the expression of mTOR, can also be used in case of epilepsy to reduce its hyperactivation. There are several unexplored anticancer molecules that act by inhibiting mTOR directly or indirectly available which can be explored as antiepileptic in future. Majority of the molecules which are tested as anticancer do not reach the final phases of clinical trials due to less potency and efficacy, and ultimately a few of them reach the market. Since a lot of experimental/safety studies have already been conducted on such molecules, hence it is worthwhile to test these molecules for other disorders that share common pathogenic pathway like epilepsy, provided their pitfalls have been addressed, as proposed in the present review.

Punica granatum L. (Punicaceae) peel is often considered as a food waste in-spite of its high bioactive metabolite composition. Primarily it is rich in therapeutically active phenolics that act on multiple cellular sites, through diverse mechanisms. Hence, the present study was envisaged to investigate the effect of standardised peel extract of P. granatum against isoproterenol (ISO)-induced myocardial infarction (MI). ISO administration at a dose of 150 mg/kg; s.c., twice at 24 h interval resulted in electrocardiographic abnormalities with increased heart weight and myocardial tissue damage signifying MI. Pretreatment with the extract at 50, 100 and 200 mg/kg; p.o., for 21 days prior to ISO intoxication (30 min prior to intoxication on day 22 and 23) attenuated the observed changes, along with increased myocardial tissue superoxide dismutase activity, reduced glutathione and nitrite levels, and decreased lipid peroxidation. The extract treated groups also showed reduced serum marker enzymes of MI, showing maximum effect at highest tested dose. Immunohistochemical studies revealed increased myocardial expression of nuclear factor erythroid 2-related factor 2 (Nrf2), endothelial nitric oxide synthase (eNOS) and Bcl-2 proteins in the extract treated groups with decreased Bax expression. From the results it can be concluded that the extract pretreatment prevents ISO-induced MI through increased myocardial expression of eNOS, leading to nitric oxide-mediated Nrf2 activation, thus upregulating antioxidant mechanisms, along with inhibition of apoptosis., (Copyright © 2015 Elsevier Inc. All rights reserved.)