Pilocarpine-induced Epilepsy Model Research Articles

Contribution of prefrontal cortex and ventral hippocampus to anxiety in young epileptic mice

Children with epilepsy are particularly vulnerable to anxiety disorders, where these disorders are frequently underdiagnosed and untreated. Despite the high prevalence of anxiety in epilepsy, the underlying neurobiological mechanisms are not fully understood. The medial prefrontal cortex (mPFC) and ventral hippocampus (vHPC) are key brain regions implicated in the genesis and modulation of anxiety, and their interactions play a crucial role in emotional processing including anxiety. We utilized a pilocarpine-induced epilepsy model in young mice (7 weeks old) to assess anxiety-like behaviors using the open field test (OFT), light/dark box, and elevated plus maze (EPM). Local field potential (LFP) recordings were conducted to examine theta power and coherence between the mPFC and vHPC. LFP recordings revealed significantly altered theta power variation in both the mPFC and vHPC during exposure to anxiogenic contexts, suggesting the involvement of these regions in anxiety in the young epileptic mice. Notably, theta-frequency synchrony between the mPFC and vHPC was not significantly altered in the young epileptic mice, indicating that altered theta power rather than inter-regional synchrony may underlie anxiety behaviors in young epileptic mice. Furthermore, we demonstrated that chemogenetic inhibition of excitatory neurons in the mPFC and vHPC reduced anxiety levels in young epileptic mice. Altogether, our findings highlight the critical contributions of mPFC and vHPC to the pathogenesis of comorbid anxiety in epilepsy. These findings underscore the potential therapeutic significance of modulating the activity in these two regions as means to alleviate anxiety in a youth epilepsy population.

The m6A reader YTHDC2 promotes the pathophysiology of temporal lobe epilepsy by modulating SLC7A11-dependent glutamate dysregulation in astrocytes.

Rationale: Epilepsy affects over 70 million people globally, with temporal lobe epilepsy with hippocampal sclerosis (TLE-HS) often progressing to a drug-resistant state. Recent research has highlighted the role of reactive astrocytes and glutamate dysregulation in epilepsy pathophysiology. This study aims to investigate the involvement of astrocytic xCT, a glutamate-cystine antiporter, and its regulation by the m6A reader protein YTHDC2 in TLE-HS. Methods: A pilocarpine-induced epilepsy model in mice was used to study the role of xCT in reactive astrocytes. The expression of xCT and its regulation by YTHDC2 were assessed through various molecular and cellular techniques. Quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting were used to measure mRNA and protein levels of xCT and YTHDC2, respectively; immunofluorescence was utilized to visualize their localization and expression in astrocytes. In vivo glutamate measurements were conducted using microdialysis to monitor extracellular glutamate levels in the hippocampus. RNA immunoprecipitation-qPCR (RIP-qPCR) was performed to investigate the binding of YTHDC2 to SLC7A11 mRNA, while methylated RNA immunoprecipitation-qPCR (MeRIP-qPCR) was performed to quantify m6A modifications on SLC7A11 mRNA. A dual-luciferase reporter assay was conducted to assess the effect of m6A modifications on SLC7A11 mRNA translation, and polysome profiling was employed to evaluate the translational efficiency of SLC7A11 mRNA. Inhibition experiments involved shRNA-mediated knockdown of SLC7A11 (commonly known as xCT) and YTHDC2 expression in astrocytes. Video-electroencephalogram (EEG) recordings were used to monitor seizure activity in mice. Results: The xCT transporter in reactive astrocytes significantly contributes to elevated extracellular glutamate levels, enhancing neuronal excitability and seizure activity. Increased xCT expression is influenced by the m6A reader protein YTHDC2, which regulates its expression through m6A methylation. Inhibition of xCT or YTHDC2 in astrocytes reduces glutamate levels and effectively controls seizures in a mouse model. Specifically, mice with SLC7A11- or YTHDC2-knockdown astrocytes showed decreased glutamate concentration in the hippocampus and reduced frequency and duration of epileptic seizures. Conclusions: This study highlights the therapeutic potential of targeting YTHDC2 and xCT in reactive astrocytes to mitigate epilepsy. The findings provide a novel perspective on the mechanisms of glutamate dysregulation and their implications in seizure pathophysiology, suggesting that modulation of YTHDC2 and xCT could be a promising strategy for treating TLE.

Beneficial effect of moringa stenopetala (bak.f) cuf. on lithium–pilocarpine-induced temporal lobe epilepsy in experimental animals

Background: Moringa stenopetala is traditionally used for the treatment of epilepsy in southern Ethiopia. Additionally, the plant material is used for the treatment of various ailments such as pain, diabetes, hypertension, and hyperlipemia. Aim of the Study: The aim of the study was to investigate the effect of M. stenopetala crude extract on the development of lithium–pilocarpine-induced temporal lobe epilepsy model in rats. Materials and Methods: Male Sprague-Dawley rats weighing 200–225 g were randomly divided into nine groups. Groups I, V, and VI served as normal control, positive control, and negative control, respectively. While Groups II–IV were treated with three different doses (400, 600, and 800 mg/kg) levels of crude extract in pre- and postinduction of status epilepticus (SE). On the other hand, Groups VII–IX were treated with the same three dose levels but after induction of SE only. All the rats, except those in normal control, were subjected to lithium (3 meq/kg) and pilocarpine (35 mg/kg) for the induction of SE. In addition, the elevated plusmaze and infrared actimeter models were used to evaluate anxiolytic and antidepressant efficacy in SE animals. Results: The rats treated with the crude extract of M. stenopetala significantly increased the latency period to the first motor seizure (58.1 min) and SE (80.4 min) compared to the control group; those developed seizure and SE at 13.3 and 35.4 min, respectively. The chronic spontaneous seizure was less severity just below spontaneous recurrent seizure at Pinel and Rovner scale 6 in rats treated with extract pre- and postinduction. The crude extract also showed significant dose-dependent anxiolytic activity (P < 0.001) and improved locomotor activity (P < 0.001). The histopathological studies of hippocampus formation revealed that the extract prevented damages compared to control animals. Conclusion: The current findings suggest that a crude extract of M. stenopetala has anticonvulsant activity in a lithium–pilocarpine-induced epilepsy model M. stenopetala also reduced depression and anxiety associated with epilepsy that indicates that it is having antidepressant and anxiolytic activity.

Intravenous infusion of bone marrow mononuclear cells promotes functional recovery and improves impaired cognitive function via inhibition of Rho guanine nucleotide triphosphatases and inflammatory signals in a model of chronic epilepsy.

Temporal lobe epilepsy is the most common form of intractable epilepsy in adults. More than 30% of individuals with epilepsy have persistent seizures and have drug-resistant epilepsy. Based on our previous findings, treatment with bone marrow mononuclear cells (BMMC) could interfere with early and chronic phase epilepsy in rats and in clinical settings. In this pilocarpine-induced epilepsy model, animals were randomly assigned to two groups: control (Con) and epileptic pre-treatment (Ep-pre-t). The latter had status epilepticus (SE) induced through pilocarpine intraperitoneal injection. Later, seizure frequency was assessed using a video-monitoring system. Ep-pre-t was further divided into epileptic treated with saline (Ep-Veh) and epileptic treated with BMMC (Ep-BMMC) after an intravenous treatment with BMMC was done on day 22 after SE. Analysis of neurobehavioral parameters revealed that Ep-BMMC had significantly lower frequency of spontaneous recurrent seizures (SRS) in comparison to Ep-pre-t and Ep-Veh groups. Hippocampus-dependent spatial and non-spatial learning and memory were markedly impaired in epileptic rats, a deficit that was robustly recovered by treatment with BMMC. Moreover, long-term potentiation-induced synaptic remodeling present in epileptic rats was restored by BMMC. In addition, BMMC was able to reduce abnormal mossy fiber sprouting in the dentate gyrus. Molecular analysis in hippocampal tissue revealed that BMMC treatment down-regulates the release of inflammatory cytokine tumor necrosis factor-α (TNF-α) and Allograft inflammatory factor-1 (AIF-1) as well as the Rho subfamily of small GTPases [Ras homolog gene family member A (RhoA) and Ras-related C3 botulinum toxin substrate 1 (Rac)]. Collectively, delayed BMMC treatment showed positive effects when intravenously infused into chronic epileptic rats.

The Antagonism of 5-HT6 Receptor Attenuates Current-Induced Spikes and Improves Long-Term Potentiation via the Regulation of M-Currents in a Pilocarpine-Induced Epilepsy Model.

Recent studies have documented that reduced M-current promotes epileptogenesis and attenuates synaptic remodeling. Neurite growth is closely related to the level of 5-HT6 receptor (5-HT6R) in the central nervous system. However, little research is available regarding the relation between 5-HT6R and M-current and the role of 5-HT6R in M-current regulation. Herein, we found that the expression of 5-HT6R was notably increased and the expression of KNCQ2/3, the main components of the M channel, was decreased in a time-dependent manner in pilocarpine-induced chronic epileptic hippocampus. Interestingly, antagonism of 5-HT6R by SB271046 upregulated the expression of KCNQ2 but not KCNQ3. SB271046 greatly alleviated excitatory/inhibitory imbalance and improved the impaired LTP in the chronic epileptic hippocampus. Further mechanism exploration revealed that the above effects of SB271046 can be reversed by the M-channel inhibitor XE991, which also confirmed that SB271046 can indeed improve abnormal M current. These data indicate that the antagonism of 5-HT6R may decrease the excitability of hippocampal pyramidal neurons in chronic epileptic rats and improve the impaired long-term potentiation by upregulating the expression of KCNQ2 in the M-channel.

Pinellia Total Alkaloids Modulate the GABAergic System in Hippocampal Formation on Pilocarpine-Induced Epileptic Rats.

To investigate the neuromodulatory effect of pinellia total alkaloids (PTA) on the gamma-aminobutyric acidergic (GABAergic) system in epileptic rats, and preliminarily evaluate the anti-epileptic effect of PTA. Ninety-one male Sprague-Dawley rats were randomized to a control group (n=17) or an epileptic group (n=74) using computer-generated random numbers. Status epilepticus (SE) was induced with pilocarpine in the epileptic group. Epileptic rats that survived SE were randomly divided into 4 groups, namely an epilepsy group (n=13), a topiramate (TPM, 60 mg/kg) group (n=12), a high-dose PTA (800 mg/kg) group (n=12), and a low-dose PTA (400 mg/kg) group (n=10). Treatments were given intragastrically once daily for 14 days. The control group and epilepsy group received normal saline. Spontaneous recurrent seizures (SRSs) were monitored 8-h daily for 7 days after treatment. Then, the hippocampal formation tissues were collected. GABA level was measured using enzyme-linked immunosorbent assay. Protein and mRNA expression levels of glutamate decarboxylase 65 (GAD65), GABA transporter-1 (GAT-1), GABA transaminase (GABA-T), and GABAA receptor (GABAAR) α4, α5, γ2 and δ subunits were measured using Western-blotting analysis and quantitative polymerase chain reaction. PTA lowered the incidence and frequency of SRS (both doses vs. the TPM group, P>0.05). Compared with the epilepsy group, PTA increased the levels of GABA (both doses P<0.01) and GAD65 (mRNA, 800 mg/kg, P<0.01), and suppressed the levels of GAT-1 (mRNA, 800 mg/kg, P<0.01; 400 mg/kg, P<0.05), GABA-T (mRNA, both doses P<0.01), and GABAAR δ subunit (protein, 800 mg/kg, P<0.05) and γ2 subunit (protein, both doses P<0.01). PTA upregulated the low-expressed mRNA levels of GABAAR α5 subunit (400 mg/kg, P<0.01), δ subunit (800 mg/kg, P<0.05), and γ2 subunit (400 mg/kg, P<0.05). PTA regulated the GABAergic system through modulating GABA levels and the expression levels of GAD65, GAT-1, GABA-T, and GABAAR α4, α5, γ2 and δ subunits. PTA may exert anti-epileptic effects on the pilocarpine-induced epilepsy model.

Pilocarpine/ascorbic acid interaction in the immature brain: Electrophysiological and oxidative effects in well-nourished and malnourished rats

Ascorbic acid (AA) administration has been associated with neuroprotection against oxidative stress, although at high doses it can facilitate oxidation and acts like a proconvulsing drug. The pilocarpine-induced epilepsy model has been widely studied. However, less is known about the effects of sub-convulsive doses of pilocarpine on brain activity in immature animals under normal or deficient nutritional conditions. Herein, we investigated the effects of chronic pilocarpine administration in a sub-convulsive dose, with or without AA, on the excitability-related phenomenon denominated as cortical spreading depression (CSD) and levels of lipid peroxidation-induced malondialdehyde in well-nourished and malnourished rats. At postnatal days 7–28, rats received no gavage treatment (naïve group), saline (vehicle group), 45 mg/kg/d of pilocarpine and/or 120 mg/kg/d of AA. CSD propagation and malondialdehyde levels were analyzed at 34–40 days. The pilocarpine group presented with lower CSD velocities, while AA groups exhibited higher CSD velocities and augmented malondialdehyde levels compared with controls. The co-administration of AA partially antagonized the pilocarpine CSD effects, but did not revert it to control levels. Malnutrition increased CSD amplitude and velocity in comparison to the well-nourished condition. The electrocorticogram (ECoG) amplitude increased after CSD (ECoG potentiation) when compared with the baseline amplitude before CSD. However, no intergroup difference was observed in this CSD-related ECoG potentiation. The results support the hypothesis of a pilocarpine/ascorbic acid interaction in the immature rat brain and might help further the understanding of this interaction on neuronal electrical activity and oxidative stress.

Clustering of spontaneous recurrent seizures separated by long seizure-free periods: An extended video-EEG monitoring study of a pilocarpine mouse model.

Seizure clustering is a common and significant phenomenon in patients with epilepsy. The clustering of spontaneous recurrent seizures (SRSs) in animal models of epilepsy, including mouse pilocarpine models, has been reported. However, most studies have analyzed seizures for a short duration after the induction of status epilepticus (SE). In this study, we investigated the detailed characteristics of seizure clustering in the chronic stage of a mouse pilocarpine-induced epilepsy model for an extended duration by continuous 24/7 video-EEG monitoring. A seizure cluster was defined as the occurrence of one or more seizures per day for at least three consecutive days and at least five seizures during the cluster period. We analyzed the cluster duration, seizure-free period, cluster interval, and numbers of seizures within and outside the seizure clusters. The video-EEG monitoring began 84.5±33.7 days after the induction of SE and continued for 53.7±20.4 days. Every mouse displayed seizure clusters, and 97.0% of the seizures occurred within a cluster period. The seizure clusters were followed by long seizure-free periods of 16.3±6.8 days, showing a cyclic pattern. The SRSs also occurred in a grouped pattern within a day. We demonstrate that almost all seizures occur in clusters with a cyclic pattern in the chronic stage of a mouse pilocarpine-induced epilepsy model. The seizure-free periods between clusters were long. These findings should be considered when performing in vivo studies using this animal model. Furthermore, this model might be appropriate for studying the unrevealed mechanism of ictogenesis.

Structural and Functional Alterations at Pre-Epileptic Stage Are Closely Associated with Epileptogenesis in Pilocarpine-induced Epilepsy Model.

Pilocarpine-induced rat epilepsy model is an established animal model that mimics medial temporal lobe epilepsy in humans. The purpose of this study was to investigate neuroimaging abnormalities in various stages of epileptogenesis and to correlate them with seizure severity in pilocarpine-induced rat epilepsy model. Fifty male Sprague-Dawley rats were subject to continuous video and electroencephalographic monitoring after inducing status epilepticus (SE) and seizure severity was estimated by frequency and total durations of class 3 to 5 spontaneous recurrent seizures (SRS) by modified Racine's classification. The 7.0 Tesla magnetic resonance imaging (MRI) with high resolution flurodeoxyglucose positron emission tomography (FDG-PET) was performed at 3 hours, 1, 3, 7 days and 4 weeks after the initial insult. The initial SRS was observed 9.7±1.3 days after the pilocarpine injection. MRI revealed an abnormal T2 signal change with swelling in both hippocampi and amygdala in acute (day 1 after injection) and latent phases (days 3 and 7), in association with PET hypometabolism in these areas. Interestingly, the mean frequency of class 3 to 5 SRS was positively correlated with abnormal T2 signals in hippocampal area at 3 days. SRS duration became longer with more decreased glucose metabolism in both hippocampi and amygdala at 7 days after pilocarpine injection. This study indicates that development and severity of SRS at chronic phase could be closely related with structural and functional changes in hippocampus during the latent period, a pre-epileptic stage.

Altered MT1 and MT2 melatonin receptors expression in the hippocampus of pilocarpine-induced epileptic rats.

Clinical and experimental findings show that melatonin may be used as an adjuvant to the treatment of epilepsy-related complications by alleviates sleep disturbances, circadian alterations and attenuates seizures alone or in combination with AEDs. In addition, it has been observed that there is a circadian component on seizures, which cause changes in circadian system and in melatonin production. Nevertheless, the dynamic changes of the melatoninergic system, especially with regard to its membrane receptors (MT1 and MT2) in the natural course of TLE remain largely unknown. The aim of this study was to evaluate the 24-hour profile of MT1 and MT2 mRNA and protein expression in the hippocampus of rats submitted to the pilocarpine-induced epilepsy model analyzing the influence of the circadian rhythm in the expression pattern during the acute, silent, and chronic phases. Melatonin receptor MT1 and MT2 mRNA expression levels were increased in the hippocampus of rats few hours after SE, with MT1 returning to normal levels and MT2 reducing during the silent phase. During the chronic phase, mRNA expression levels of both receptors return to levels close to control, however, presenting a different daily profile, showing that there is a circadian change during the chronic phase. Also, during the acute and silent phase it was possible to verify MT1 label only in CA2 hippocampal region with an increased expression only in the dark period of the acute phase. The MT2 receptor was present in all hippocampal regions, however, it was reduced in the acute phase and it was found in astrocytes. In chronic animals, there is a reduction in the presence of both receptors especially in regions where there is a typical damage derived from epilepsy. Therefore, we conclude that SE induced by pilocarpine is able to change melatonin receptor MT1 and MT2 protein and mRNA expression levels in the hippocampus of rats few hours after SE as well as in silent and chronic phases.

Purslane protects against the reproductive toxicity of carbamazepine treatment in pilocarpine-induced epilepsy model

ObjectiveTo investigate the protective effect of purslane with carbamazepine treatment. MethodsMale albino rats were modulated by pilocarpine to be epileptic. Both the normal and epileptic rats were treated with carbamazepine, purslane or carbamazepine plus purslane, with separate non-treated control groups for both normal and epileptic rats. ResultsThe data from the current study showed amelioration in amino acids and electrolytes in the epileptic rats treated with purslane and carbamazepine, with this amelioration occurring without decreasing the fertility hormones (testosterone, dehydroepiandrosterone, luteinizing hormone and follicle stimulating hormone). Purslane treatments also prevented the increase in estradiol. The decreased epileptic hyperexcitability with purslane was evidenced by decreased glial fibrillary acidic protein and lipid peroxidation. ConclusionsNatural products like purslane could be used with the highly repetitive drugs like carbamazepine to reduce or prevent its side-effects.

Bring CLARITY to Temporal Lobe Epilepsy: 3D Visualization of p-Tau(Ser262) and 14-3-3 Zeta

CLARITY is one new technology which allows the brain tissue become transparent. It has successfully been combined with immunofluorescence staining to achieve the 3D visualization of some molecules or neuronal cells in some disease brains. The temporal lobe epilepsy (TLE) is one neuronal disease which is characterized by the sprouting of the massy fibers (MSF). Previous study has showed that MSF could be affected by the phosphorylation at site-262 of microtubule association protein Tau (p-tau (Ser262)). However, in TLE little useful information was reported concerning the 3D architecture of p-tau (Ser262) and its relationship with 14-3-3 zeta that regulated the phosphorylation of Tau in AD disease. In this paper, pilocarpine-induced epilepsy model was established and identified by Timm-staining. Double immunofluorescent staining results showed that the development of TLE gave rise to the colocalization of p-tau (Ser262) and 14-3-3 zeta protein in CA1 and CA3 zone in hippocampi. The mm-thick brain sections were passively clarified, and 3D reconstruction imaging of the immunofluorescent staining showed that p-tau (Ser262) was diverse cluster-like shape. These results proved that CLARITY could be used to study TLE, in which the 3D morphologic changes of p-tau (Ser262) and the role of 14-3-3 zeta in the regulation of Tau needed to be further investigated.

Activation of LILRB2 signal pathway in temporal lobe epilepsy patients and in a pilocarpine induced epilepsy model

Temporal lobe epilepsy (TLE) is a frequent form of focal intractable epilepsy in adults, but the specific mechanism underlying the epileptogenesis of TLE is still unknown. Human leukocyte immunoglobulin-like receptor B2 (LILRB2) (the murine homolog gene called paired immunoglobulin-like receptor B, or PirB), participates in the process of synaptic plasticity and neurite growth in the central nervous system (CNS), suggesting a potential role of LILRB2 in epilepsy. However, the expression pattern of LILRB2 and the downstream molecular signal in intractable TLE remains poorly understood. In the present study, western blotting and immunohistochemistry results showed that LILRB2 expression was upregulated in the temporal neocortex of patients with TLE. Moreover, protein levels of LILRB2 negatively correlated with the frequency of seizures in TLE patients. In the pilocarpine-induced C57BL/6 mouse model, PirB upregulation in the hippocampus began 12h after status epilepticus (SE), reached a peak at 7days and then maintained a significantly high level until day 60. Similarly, we found a remarkable increase in PirB expression at 1day, 7days and30days post-SE in the temporal cortex. Double-labeled immunofluorescence showed that LILRB2/PirB were highly expressed in neurons and astrocytes but not microglia. In addition, protein levels of POSH, SHROOM3, ROCK1 and ROCK2, the important downstream factors of the LILRB2 pathway, were significantly increased in the epileptic foci of TLE patients and located on the NeuN-positive neurons and GFAP-positive astrocytes. Taken together, our results indicate that LILRB2/PirB may be involved in the process of TLE.

Expression of SHANK3 in the Temporal Neocortex of Patients with Intractable Temporal Epilepsy and Epilepsy Rat Models.

SH3 and multiple ankyrin (ANK) repeat domain 3 (SHANK3) is a synaptic scaffolding protein enriched in the postsynaptic density of excitatory synapses. SHANK3 plays an important role in the formation and maturation of excitatory synapses. In the brain, SHANK3 directly or indirectly interacts with various synaptic molecules including N-methyl-D-aspartate receptor, the metabotropic glutamate receptor (mGluR), and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor. Previous studies have shown that Autism spectrum disorder is a result of mutations of the main SHANK3 isoforms, which may be due to deficit in excitatory synaptic transmission and plasticity. Recently, accumulating evidence has demonstrated that overexpression of SHANK3 could induce seizures in vivo. However, little is known about the role of SHANK3 in refractory temporal lobe epilepsy (TLE). Therefore, we investigated the expression pattern of SHANK3 in patients with intractable temporal lobe epilepsy and in pilocarpine-induced models of epilepsy. Immunofluorescence, immunohistochemistry, and western blot analysis were used to locate and determine the expression of SHANK3 in the temporal neocortex of patients with epilepsy, and in the hippocampus and temporal lobe cortex of rats in a pilocarpine-induced epilepsy model. Double-labeled immunofluorescence showed that SHANK3 was mainly expressed in neurons. Western blot analysis confirmed that SHANK3 expression was increased in the neocortex of TLE patients and rats. These results indicate that SHANK3 participates in the pathology of epilepsy.

Pilocarpine-induced epilepsy alters the expression and daily variation of the nuclear receptor RORα in the hippocampus of rats.

It is widely known that there is an increase in the inflammatory responses and oxidative stress in temporal lobe epilepsy (TLE). Further, the seizures follow a circadian rhythmicity. Retinoic acid receptor-related orphan receptor alpha (RORα) is related to anti-inflammatory and antioxidant enzyme expression and is part of the machinery of the biological clock and circadian rhythms. However, the participation of RORα in this neurological disorder has not been studied. The aim of this study was to evaluate the RORα mRNA and protein content profiles in the hippocampus of rats submitted to a pilocarpine-induced epilepsy model at different time points throughout the 24-h light-dark cycle analyzing the influence of the circadian rhythm in the expression pattern during the acute, silent, and chronic phases of the experimental model. Real-time PCR and immunohistochemistry results showed that RORα mRNA and protein expressions were globally reduced in both acute and silent phases of the pilocarpine model. However, 60days after the pilocarpine-induced status epilepticus (chronic phase), the mRNA expression was similar to the control except for the time point 3h after the lights were turned off, and no differences were found in immunohistochemistry. Our results indicate that the status epilepticus induced by pilocarpine is able to change the expression and daily variation of RORα in the rat hippocampal area during the acute and silent phases. These findings enhance our understanding of the circadian pattern present in seizures as well as facilitate strategies for the treatment of seizures.

The progressive changes of filamentous actin cytoskeleton in the hippocampal neurons after pilocarpine-induced status epilepticus

In a previous study, we reported a persistent reduction of F-actin puncta but a compensating increase in puncta size in the mouse hippocampus at 2 months after pilocarpine-induced status epilepticus (Epilepsy Res. 108 (2014), 379–389). However, the F-actin changes during the period of epileptogenesis remain unknown. This study was designed to examine the temporal and spatial changes of F-actin during the period of epileptogenesis in a pilocarpine-induced epilepsy model, primarily by the histological and TUNEL evaluation of cell loss, phalloidin detection of F-actin, and immunohistochemical analysis of glial reactions. The results demonstrated that F-actin continued to decrease after pilocarpine treatment, which was consistent in its time course with hippocampal neuronal death. Within different hippocampal subfields, the spatial changes of F-actin exhibited similar features. First, the F-actin puncta decreased in number. Thereafter, F-actin was transiently aggregated in dendritic shafts and neuronal cell bodies and eventually was completely lost in the degenerated neurons. The progressive changes of F-actin in the degenerating neurons reported in this study may help to elucidate a cytoskeletal mechanism that may link to the delayed cell loss that occurs during epileptogenesis.

Neurofibromin Regulates Seizure Attacks in the Rat Pilocarpine-Induced Model of Epilepsy.

Studies have shown that neurofibromin (NF1) restricts GABA release at inhibitory synapses and regulates dendritic spine formation, which may play an important role in temporal lobe epilepsy (TLE). NF1 expression was detected by double-label immunofluorescence, immunohistochemistry, and western blot analysis in the brains of pilocarpine-induced epilepsy model rats at 6h, 24h, 72h, 7days, 14days, 30days, and 60days after kindling. NF1 was localized primarily in the nucleus and cytoplasm of neurons. NF1 protein levels significantly increased in the chronic phase (from 7days until 60days) in this epileptic rat model. After NF1 expression was knocked down by specific siRNA, the effects of kindling with pilocarpine were evaluated on the 7th day after kindling. The onset latencies of pilocarpine-induced seizures were elevated, and the seizure frequency and duration were reduced in these rats. Our study demonstrates that NF1 promoted seizure attacks in rats with pilocarpine-induced epilepsy.

Change of MicroRNA-134, CREB and p-CREB expression in epileptic rat.

To To investigate the changes of MicroRNA-134, CREB and p-CREB expression in epileptic rat brains in order to elucidate the molecular mechanisms of epilepsy, providing new ideas for clinical treatment. Sixty-four Spraque-Dawley (SD) rats were divided into groups randomly, including control group, six hours after seizure group, 24-hour group, three-day group, one-week group, two-week group, four-week group, and eight-week group. All groups were placed under a pilocarpine-induced epilepsy model except the control group, and all rats were decapitated in different points of time. Brain specimens were taken for quantitative PCR experiments, immunohistochemistry and Western blot experiments. The results of the epilepsy model groups and the control group were compared. There were no significant differences between the six hours after seizure group, the 24-hour group and the control group about the MicroRNA-134 levels. MicroRNA-134 in the hippocampus tissue of the three-day group significantly reduced compared with the control group; same result was observed with the one-week, two-week, four-week and eight-week groups. The CREB and p-CREB levels in the three-day group's rat hippocampus significantly increased compared with the control group; and the high levels of CREB and p-CREB were constantly maintained in the one-week, two-week, four-week and eight-week groups. The MicroRNA-134 level of the epileptic rat hippocampus is significantly lower than normal after three days, and continues to maintain a low level; while CREB and p-CREB levels are rsignificantly increased after three days, and continue to remain at a high level. MicroRNA-134 plays a role in inhibiting synaptic plasticity by inhibiting CREB and p-CREB expressions.

Berberine exerts an anticonvulsant effect and ameliorates memory impairment and oxidative stress in a pilocarpine-induced epilepsy model in the rat.

Though new antiepileptic drugs are emerging, approximately a third of epileptic patients still suffer from recurrent convulsions and cognitive dysfunction. Therefore, we tested whether berberine (Ber), a vegetable drug, has an anticonvulsant property and attenuates memory impairment in a pilocarpine (Pilo)-induced epilepsy model in rats. The rats were injected with 400 mg/kg Pilo to induce convulsions, and Ber 25, 50, and 100 mg/kg were administrated by the intragastric route once daily 7 days before Pilo injection until the experiment was over. Convulsions were observed after Pilo injection. For the rats that developed status epilepticus (SE), malondialdehyde, glutathione levels, superoxide dismutase, and catalase activity in the hippocampus were measured 24 hours after SE. The rats received the Morris water-maze test 2 weeks after SE, and then were killed for fluoro-jade B staining to detect the degenerating neurons. We found Ber delayed latency to the first seizure and the time to develop SE in a dose-dependent manner. Malondialdehyde levels were decreased, while glutathione and catalase activity were strengthened in Ber-injected SE rats. In the Morris water-maze test, Ber decreased escape latency compared to saline-treated SE rats. Additionally, Ber reduced the number of fluoro-jade B-positive cells in the hippocampal CA1 region. Our data suggest that Ber exerts anticonvulsant and neuroprotective effects on Pilo-induced epilepsy in rats. Simultaneously, Ber attenuates memory impairment. The beneficial effect may be partly due to mitigation of the oxidative stress burden.

Expression and activity of thimet oligopeptidase (TOP) are modified in the hippocampus of subjects with temporal lobe epilepsy (TLE)

Thimet oligopeptidase (TOP) is a metalloprotease that has been associated with peptide processing in several nervous system structures, and its substrates include several peptides such as bradykinin, amyloid beta (Aβ), and major histocompatibility complex (MHC) class I molecules. As shown previously by our research group, patients with temporal lobe epilepsy (TLE) have a high level of kinin receptors as well as kallikrein, a kinin-releasing enzyme, in the hippocampus. In this study, we evaluated the expression, distribution, and activity of TOP in the hippocampus of patients with TLE and autopsy-control tissues, through reverse-transcription polymerase chain reaction (RT-PCR), enzymatic activity, Western blot, and immunohistochemistry. In addition, hippocampi of rats were analyzed using the pilocarpine-induced epilepsy model. Animals were grouped according to the epilepsy phases defined in the model as acute, silent, and chronic. Increased TOP mRNA expression, decreased protein levels and enzymatic activity were observed in tissues of patients, compared to control samples. In addition, decreased TOP distribution was also visualized by immunohistochemistry. Similar results were observed in tissues of rats during the acute phase of epilepsy model. However, increased TOP mRNA expression and no changes in immunoreactivity were found in the silent phase, whereas increased TOP mRNA expression and increased enzymatic activity were observed in the chronic phase. The results show that these alterations could be related to a failure in the mechanisms involved in clearance of inflammatory peptides in the hippocampus, suggesting an accumulation of potentially harmful substances in nervous tissue such as Aβ, bradykinin, and antigenic peptides. These accumulations could be related to hippocampal inflammation observed in TLE subjects.