Frequency Of Spontaneous Recurrent Seizures Research Articles

Lack of Direct Effects of Neurotrophic Factors in an In Vitro Model of Neuroinflammation.

Neuroinflammation is associated with several neurological disorders including temporal lobe epilepsy. Seizures themselves can induce neuroinflammation. In an in vivo model of epilepsy, the supplementation of brain-derived neurotropic factor (BDNF) and fibroblast growth factor-2 (FGF-2) using a Herpes-based vector reduced epileptogenesis-associated neuroinflammation. The aim of this study was to test whether the attenuation of the neuroinflammation obtained in vivo with BDNF and FGF-2 was direct or secondary to other effects, for example, the reduction in the severity and frequency of spontaneous recurrent seizures. An in vitro model of neuroinflammation induced by lipopolysaccharide (LPS, 100 ng/mL) in a mouse primary mixed glial culture was used. The releases of cytokines and NO were analyzed via ELISA and Griess assay, respectively. The effects of LPS and neurotrophic factors on cell viability were determined by performing an MTT assay. BDNF and FGF-2 were tested alone and co-administered. LPS induced a significant increase in pro-inflammatory cytokines (IL-1β, IL-6, and TNF-α) and NO. BDNF, FGF-2, and their co-administration did not counteract these LPS effects. Our study suggests that the anti-inflammatory effect of BDNF and FGF-2 in vivo in the epilepsy model was indirect and likely due to a reduction in seizure frequency and severity.

Role and mechanism of EphB3 in epileptic seizures and epileptogenesis through Kalirin

BackgroundThe EphB receptor tyrosine kinase family participates in intricate signaling pathways that orchestrate neural networks, guide neuronal axon development, and modulate synaptic plasticity through interactions with surface-bound ephrinB ligands. Additionally, Kalirin, a Rho guanine nucleotide exchange factor, is notably expressed in the postsynaptic membrane of excitatory neurons and plays a role in synaptic morphogenesis. This study postulates that Kalirin may act as a downstream effector of EphB3 in epilepsy. This investigation focuses on understanding the link between EphB3 and epilepsy. Materials and methodsChronic seizure models using LiCl-pilocarpine (LiCl/Pilo) and pentylenetetrazol were developed in rats. Neuronal excitability was gauged through whole-cell patch clamp recordings on rat hippocampal slices. Real-time PCR determined Kalirin's mRNA expression, and Western blotting was employed to quantify EphB3 and Kalirin protein levels. Moreover, dendritic spine density in epileptic rats was evaluated using Golgi staining. ResultsModulation of EphB3 functionality influenced acute seizure severity, latency duration, and frequency of spontaneous recurrent seizures. Golgi staining disclosed an EphB3-driven alteration in dendritic spine density within the hippocampus of epileptic rats, underscoring its pivotal role in the reconfiguration of hippocampal neural circuits. Furthermore, our data propose Kalirin as a prospective downstream mediator of the EphB3 receptor. ConclusionsOur findings elucidate that EphB3 impacts the action potential dynamics in isolated rat hippocampal slices and alters dendritic spine density in the inner molecular layer of epileptic rat hippocampi, likely through Kalirin-mediated pathways. This hints at EphB3's significant role in shaping excitatory circuit loops and recurrent seizure activity via Kalirin.

Anti-epileptic and Neuroprotective Effects of Ultra-low Dose NADPH Oxidase Inhibitor Dextromethorphan on Kainic Acid-induced Chronic Temporal Lobe Epilepsy in Rats.

Neuroinflammation mediated by microglia and oxidative stress play pivotal roles in the development of chronic temporal lobe epilepsy (TLE). We postulated that kainic acid (KA)-Induced status epilepticus triggers microglia-dependent inflammation, leading to neuronal damage, a lowered seizure threshold, and the emergence of spontaneous recurrent seizures (SRS). Extensive evidence from our laboratory suggests that dextromethorphan (DM), even in ultra-low doses, has anti-inflammatory and neuroprotective effects in many animal models of neurodegenerative disease. Our results showed that administration of DM (10 ng/kg per day; subcutaneously via osmotic minipump for 4 weeks) significantly mitigated the residual effects of KA, including the frequency of SRS and seizure susceptibility. In addition, DM-treated rats showed improved cognitive function and reduced hippocampal neuronal loss. We found suppressed microglial activation-mediated neuroinflammation and decreased expression of hippocampal gp91phox and p47phox proteins in KA-induced chronic TLE rats. Notably, even after discontinuation of DM treatment, ultra-low doses of DM continued to confer long-term anti-seizure and neuroprotective effects, which were attributed to the inhibition of microglial NADPH oxidase 2 as revealed by mechanistic studies.

A Ketogenic Diet may Improve Cognitive Function in Rats with Temporal Lobe Epilepsy by Regulating Endoplasmic Reticulum Stress and Synaptic Plasticity.

A ketogenic diet (KD) is often used in the treatment of refractory epilepsy. Many studies have found that it also has a positive impact on cognitive comorbidities, but the specific mechanism remains unclear. In many disease models, endoplasmic reticulum stress (ERS) and synaptic plasticity is considered a new therapeutic target for improving cognitive impairment, and it has become a research focus in recent years. Recently, studies have found that a KD has a certain regulatory effect on both ERS and synaptic plasticity, but this result has not been confirmed in epilepsy. To investigate the effect of a KD on ERS and synaptic plasticity. In this study, a rat model of temporal lobe epilepsy (TLE) induced by lithium chloride-pilocarpine was used. After the model was successfully established, the rats in each group were fed a normal diet or a KD for 28 days, and the effect of a KD on the latency and seizure frequency of spontaneous recurrent seizures (SRSs) was observed via video monitoring. Subsequently, a Morris water maze was used to evaluate the spatial learning and memory abilities of the rats in each group; the ultrastructure of the ER and the synapses of the hippocampus were observed by transmission electron microscopy, and the dendritic spine density of the hippocampus was analysed by Golgi staining. Long-term potentiation (LTP) was used to detect the synaptic plasticity of the rats' hippocampi, and the expression of ERS-related proteins and synapse-related proteins was detected by Western blotting. A KD effectively reduced the frequency of SRSs in rats with TLE and improved their learning and memory impairment. Further investigations found that a KD inhibited the up-regulation of glucose-regulated protein 78, phospho-protein kinase-like ER kinase, phosphorylated α subunit of eukaryotic initiation factor 2, activating transcription factor 4 and C/EBP homologous protein expression in the hippocampi of rats with TLE and protected the ultrastructure of the neuronal ER, suggesting that a KD suppressed excessive ERS induced by epilepsy. Concurrently, we also found that a KD not only improved the synaptic ultrastructure and increased the density of dendritic spines in rats with TLE but also reversed the epilepsy-induced LTP deficit to some extent. More importantly, the expression of postsynaptic density protein 95, synaptotagmin-1 and synaptosomal-associated protein 25 in the hippocampi of rats with epilepsy was significantly increased after KD intervention. The study findings indicate that a KD improves learning and memory impairment in rats with epilepsy, possibly by regulating ERS and synaptic plasticity.

Deep brain stimulation suppresses epileptic seizures in rats via inhibition of adenosine kinase and activation of adenosine A1 receptors.

Deep brain stimulation (DBS) of the anterior nucleus of the thalamus, is an effective therapy for patients with drug-resistant epilepsy, yet, its mechanism of action remains elusive. Adenosine kinase (ADK), a key negative regulator of adenosine, is a potential modulator of epileptogenesis. DBS has been shown to increase adenosine levels, which may suppress seizures via A1 receptors (A1 Rs). We investigated whether DBS could halt disease progression and the potential involvement of adenosine mechanisms. Control group, SE (status epilepticus) group, SE-DBS group, and SE-sham-DBS group were included in this study. One week after a pilocarpine-induced status epilepticus, rats in the SE-DBS group were treated with DBS for 4 weeks. The rats were monitored by video-EEG. ADK and A1 Rs were tested with histochemistry and western blot, respectively. Compared with the SE group and SE-sham-DBS group, DBS could reduce the frequency of spontaneous recurrent seizures (SRS) and the number of interictal epileptic discharges. The DPCPX, an A1 R antagonist, reversed the effect of DBS on interictal epileptic discharges. In addition, DBS inhibited the overexpression of ADK and the downregulation of A1 Rs. The findings indicate that DBS can reduce SRS in epileptic rats via inhibition of ADK and activation of A1 Rs. A1 Rs might be a potential target of DBS for the treatment of epilepsy.

Thalidomide Attenuates Epileptogenesis and Seizures by Decreasing Brain Inflammation in Lithium Pilocarpine Rat Model.

Thalidomide (TAL) has shown potential therapeutic effects in neurological diseases like epilepsy. Both clinical and preclinical studies show that TAL may act as an antiepileptic drug and as a possible treatment against disease development. However, the evidence for these effects is limited. Therefore, the antiepileptogenic and anti-inflammatory effects of TAL were evaluated herein. Sprague Dawley male rats were randomly allocated to one of five groups (n = 18 per group): control (C); status epilepticus (SE); SE-TAL (25 mg/kg); SE-TAL (50 mg/kg); and SE-topiramate (TOP; 60mg/kg). The lithium-pilocarpine model was used, and one day after SE induction the rats received pharmacological treatment for one week. The brain was obtained, and the hippocampus was micro-dissected 8, 18, and 28 days after SE. TNF-α, IL-6, and IL-1β concentrations were quantified. TOP and TAL (50 mg/kg) increased the latency to the first of many spontaneous recurrent seizures (SRS) and decreased SRS frequency, as well as decreasing TNF-α and IL-1β concentrations in the hippocampus. In conclusion, the results showed that both TAL (50 mg/kg) and TOP have anti-ictogenic and antiepileptogenic effects, possibly by decreasing neuroinflammation.

Seizure-induced overexpression of NPY induces epileptic tolerance in a mouse model of spontaneous recurrent seizures.

Epileptic seizures result in pronounced over-expression of neuropeptide Y (NPY). In vivo and in vitro studies revealed that NPY exerts potent anticonvulsive actions through presynaptic Y2 receptors by suppressing glutamate release from principal neurons. We now investigated whether seizure-induced over-expression of NPY contributes to epileptic tolerance induced by preceding seizures. We used a previously established animal model based on selective inhibition of GABA release from parvalbumin (PV)-containing interneurons in the subiculum in mice. The animals present spontaneous recurrent seizures (SRS) and clusters of interictal spikes (IS). The frequency of SRS declined after five to six weeks, indicating development of seizure tolerance. In interneurons of the subiculum and sector CA1, SRS induced over-expression of NPY that persisted there for a prolonged time despite of a later decrease in SRS frequency. In contrast to NPY, somatostatin was not overexpressed in the respective axon terminals. Contrary to interneurons, NPY was only transiently expressed in mossy fibers. To demonstrate a protective function of endogenous, over-expressed NPY, we injected the selective NPY-Y2 receptor antagonist JNJ 5207787 simultaneously challenging the mice by a low dose of pentylenetetrazol (PTZ, 30 or 40 mg/kg, i.p.). In control mice, neither PTZ nor PTZ plus JNJ 5207787 induced convulsions. In mice with silenced GABA/PV neurons, PTZ alone only modestly enhanced EEG activity. When we injected JNJ 5207787 together with PTZ (either dose) the number of seizures, however, became significantly increased. In addition, in the epileptic mice CB1 receptor immunoreactivity was reduced in terminal areas of basket cells pointing to reduced presynaptic inhibition of GABA release from these neurons. Our experiments demonstrate that SRS result in overexpression of NPY in hippocampal interneurons. NPY overexpression persists for several weeks and may be related to later decreasing SRS frequency. Injection of the Y2 receptor antagonist JNJ 5207787 prevents this protective action of NPY only when release of the peptide is triggered by injection of PTZ and induces pronounced convulsions. Thus, over-expressed NPY released “on demand” by seizures may help terminating acute seizures and may prevent from recurrent epileptic activity.

Conditional knockout of ASK1 in microglia/macrophages attenuates epileptic seizures and long-term neurobehavioural comorbidities by modulating the inflammatory responses of microglia/macrophages

BackgroundApoptosis signal-regulating kinase 1 (ASK1) not only causes neuronal programmed cell death via the mitochondrial pathway but also is an essential component of the signalling cascade during microglial activation. We hypothesize that ASK1 selective deletion modulates inflammatory responses in microglia/macrophages(Mi/Mϕ) and attenuates seizure severity and long-term cognitive impairments in an epileptic mouse model.MethodsMi/Mϕ-specific ASK1 conditional knockout (ASK1 cKO) mice were obtained for experiments by mating ASK1flox/flox mice with CX3CR1creER mice with tamoxifen induction. Epileptic seizures were induced by intrahippocampal injection of kainic acid (KA). ASK1 expression and distribution were detected by western blotting and immunofluorescence staining. Seizures were monitored for 24 h per day with video recordings. Cognition, social and stress related activities were assessed with the Y maze test and the three-chamber social novelty preference test. The heterogeneous Mi/Mϕ status and inflammatory profiles were assessed with immunofluorescence staining and real-time polymerase chain reaction (q-PCR). Immunofluorescence staining was used to detect the proportion of Mi/Mϕ in contact with apoptotic neurons, as well as neuronal damage.ResultsASK1 was highly expressed in Mi/Mϕ during the acute phase of epilepsy. Conditional knockout of ASK1 in Mi/Mϕ markedly reduced the frequency of seizures in the acute phase and the frequency of spontaneous recurrent seizures (SRSs) in the chronic phase. In addition, ASK1 conditional knockout mice displayed long-term neurobehavioral improvements during the Y maze test and the three-chamber social novelty preference test. ASK1 selective knockout mitigated neuroinflammation, as evidenced by lower levels of Iba1+/CD16+ proinflammatory Mi/Mϕ. Conditional knockout of ASK1 increased Mi/Mϕ proportion in contact with apoptotic neurons. Neuronal loss was partially restored by ASK1 selective knockout.ConclusionConditional knockout of ASK1 in Mi/Mϕ reduced seizure severity, neurobehavioral impairments, and histological damage, at least via inhibiting proinflammatory microglia/macrophages responses. ASK1 in microglia/macrophages is a potential therapeutic target for inflammatory responses in epilepsy.

Corticotropin Releasing Factor Mediates KCa3.1 Inhibition, Hyperexcitability, and Seizures in Acquired Epilepsy.

Temporal lobe epilepsy (TLE), the most common focal seizure disorder in adults, can be instigated in experimental animals by convulsant-induced status epilepticus (SE). Principal hippocampal neurons from SE-experienced epileptic male rats (post-SE neurons) display markedly augmented spike output compared with neurons from nonepileptic animals (non-SE neurons). This enhanced firing results from a cAMP-dependent protein kinase A-mediated inhibition of KCa3.1, a subclass of Ca2+-gated K+ channels generating the slow afterhyperpolarizing Ca2+-gated K+ current (IsAHP). The inhibition of KCa3.1 in post-SE neurons leads to a marked reduction in amplitude of the IsAHP that evolves during repetitive firing, as well as in amplitude of the associated Ca2+-dependent component of the slow afterhyperpolarization potential (KCa-sAHP). Here we show that KCa3.1 inhibition in post-SE neurons is induced by corticotropin releasing factor (CRF) through its Type 1 receptor (CRF1R). Acute application of CRF1R antagonists restores KCa3.1 activity in post-SE neurons, normalizing KCa-sAHP/IsAHP amplitudes and neuronal spike output, without affecting these variables in non-SE neurons. Moreover, pharmacological antagonism of CRF1Rs in vivo reduces the frequency of spontaneous recurrent seizures in post-SE chronically epileptic rats. These findings may provide a new vista for treating TLE.SIGNIFICANCE STATEMENT Epilepsy, a common neurologic disorder, often develops following a brain insult. Identifying key cellular mechanisms underlying acquired epilepsy is critical for developing effective antiepileptic therapies. In an experimental model of acquired epilepsy, principal hippocampal neurons manifest hyperexcitability because of downregulation of KCa3.1, a subtype of Ca2+-gated K+ ion channels. We show that KCa3.1 downregulation is mediated by corticotropin releasing factor (CRF) acting through its Type 1 receptor (CRF1R). Congruently, acute application of selective CRF1R antagonists restores KCa3.1 channel activity, leading to normalization of neuronal excitability. In the same model, injection of a CRF1R antagonist to epileptic animals markedly decreases the frequency of electrographic seizures. Therefore, targeting CRF1Rs may provide a new strategy in the treatment of acquired epilepsy.

Myo-Inositol Limits Kainic Acid-Induced Epileptogenesis in Rats.

Epilepsy is a severe neurological disease characterized by spontaneous recurrent seizures (SRS). A complex pathophysiological process referred to as epileptogenesis transforms a normal brain into an epileptic one. Prevention of epileptogenesis is a subject of intensive research. Currently, there are no clinically approved drugs that can act as preventive medication. Our previous studies have revealed highly promising antiepileptogenic properties of a compound–myo-inositol (MI) and the present research broadens previous results and demonstrates the long-term disease-modifying effect of this drug, as well as the amelioration of cognitive comorbidities. For the first time, we show that long-term treatment with MI: (i) decreases the frequency and duration of electrographic SRS in the hippocampus; (ii) has an ameliorating effect on spatial learning and memory deficit associated with epileptogenesis, and (iii) attenuates cell loss in the hippocampus. MI treatment also alters the expression of the glial fibrillary acidic protein, LRRC8A subunit of volume-regulated anion channels, and protein tyrosine phosphatase receptor type R, all expected to counteract the epileptogenesis. All these effects are still present even 4 weeks after MI treatment ceased. This suggests that MI may exert multiple actions on various epileptogenesis-associated changes in the brain and, therefore, could be considered as a candidate target for prevention of epileptogenesis.

Spontaneous recurrent seizures in an intra-amygdala kainate microinjection model of temporal lobe epilepsy are differentially sensitive to antiseizure drugs

The discovery and development of novel antiseizure drugs (ASDs) that are effective in controlling pharmacoresistant spontaneous recurrent seizures (SRSs) continues to represent a significant unmet clinical need. The Epilepsy Therapy Screening Program (ETSP) has undertaken efforts to address this need by adopting animal models that represent the salient features of human pharmacoresistant epilepsy and employing these models for preclinical testing of investigational ASDs. One such model that has garnered increased interest in recent years is the mouse variant of the Intra-Amygdala Kainate (IAK) microinjection model of mesial temporal lobe epilepsy (MTLE). In establishing a version of this model, several methodological variables were evaluated for their effect(s) on pertinent quantitative endpoints. Although administration of a benzodiazepine 40 min after kainate (KA) induced status epilepticus (SE) is commonly used to improve survival, data presented here demonstrates similar outcomes (mortality, hippocampal damage, latency periods, and 90-day SRS natural history) between mice given midazolam and those that were not. Using a version of this model that did not interrupt SE with a benzodiazepine, a 90-day natural history study was performed and survival, latency periods, SRS frequencies and durations, and SRS clustering data were quantified. Finally, an important step towards model adoption is to assess the sensitivities or resistances of SRSs to a panel of approved and clinically used ASDs. Accordingly, the following ASDs were evaluated for their effects on SRSs in these mice: phenytoin (20 mg/kg, b.i.d.), carbamazepine (30 mg/kg, t.i.d.), valproate (240 mg/kg, t.i.d.), diazepam (4 mg/kg, b.i.d.), and phenobarbital (25 and 50 mg/kg, b.i.d.). Valproate, diazepam, and phenobarbital significantly attenuated SRS frequency relative to vehicle controls at doses devoid of observable adverse behavioral effects. Only diazepam significantly increased seizure freedom. Neither phenytoin nor carbamazepine significantly altered SRS frequency or freedom under these experimental conditions. These data demonstrate that SRSs in this IAK model of MTLE are pharmacoresistant to two representative sodium channel-inhibiting ASDs (phenytoin and carbamazepine) and partially sensitive to GABA receptor modulating ASDs (diazepam and phenobarbital) or a mixed-mechanism ASD (valproate). Accordingly, this model is being incorporated into the NINDS-funded ETSP testing platform for treatment resistant epilepsy.

Anxious Profile Influences Behavioral and Immunohistological Findings in the Pilocarpine Model of Epilepsy.

Anxiety and epilepsy have a complex bidirectional relationship, where a depressive/anxious condition is a factor that can trigger seizures which in turn can aggravate the depressive/anxious condition. In addition, brain structures such as the hippocampus and amygdala might have a critical relevance in both epilepsy and anxiety. The aim of the present work was to investigate the influence of different anxious profiles to epileptogenesis. Initially, animals were screened through the elevated plus-maze anxiety test, and then seizure development was evaluated using the pilocarpine model of epilepsy. There were no differences in the susceptibility to status epilepticus, mortality rate or frequency of spontaneous recurrent seizures between animals characterized as anxious as compared to the non-anxious animals. Next, we evaluated immunohistological patterns related to seizures and anxiety in various related brain areas. Despite a decrease in the density of neuropeptide Y and parvalbumin expression in epileptic animals, those presenting greater neuropeptide Y immunoreactivity in various brain regions, also showed higher spontaneous recurrent seizures frequency. Differences on the anxious profile showed to interfere with some of these findings in some regions. In addition, animals that were injected with pilocarpine, but did not develop status epilepticus, had behavioral and neuroanatomical alterations as compared to control animals, indicating its importance as an additional tool for investigating the heterogeneity of the epileptogenic response after an initial insult. This study allowed to better understand the association between anxiety and temporal lobe epilepsy and might allow for therapeutic targets to be developed to minimize the negative impacts associated with it.

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.

Anakinra Reduces Epileptogenesis, Provides Neuroprotection, and Attenuates Behavioral Impairments in Rats in the Lithium-Pilocarpine Model of Epilepsy.

Temporal lobe epilepsy is a widespread chronic disorder that manifests as spontaneous seizures and is often characterized by refractoriness to drug treatment. Temporal lobe epilepsy can be caused by a primary brain injury; therefore, the prevention of epileptogenesis after a primary event is considered one of the best treatment options. However, a preventive treatment for epilepsy still does not exist. Neuroinflammation is directly involved in epileptogenesis and neurodegeneration, leading to the epileptic condition and cognitive decline. In the present study, we aimed to clarify the effect of treatment with a recombinant form of the Interleukin-1 receptor antagonist (anakinra) on epileptogenesis and behavioral impairments in rats using the lithium–pilocarpine model. We found that anakinra administration during the latent phase of the model significantly suppressed the duration and frequency of spontaneous recurrent seizures in the chronic phase. Moreover, anakinra administration prevented some behavioral impairments, including motor hyperactivity and disturbances in social interactions, during both the latent and chronic periods. Histological analysis revealed that anakinra administration decreased neuronal loss in the CA1 and CA3 areas of the hippocampus but did not prevent astro- and microgliosis. The treatment increased the expression level of the solute carrier family 1 member 2 gene (Slc1a2, encoding excitatory amino acid transporter 2 (EAAT2)) in the hippocampus, potentially leading to a neuroprotective effect. However, the increased gene expression of proinflammatory cytokine genes (Interleukin-1β (Il1b) and tumor necrosis factor α (Tnfa)) and astroglial marker genes (glial fibrillary acidic protein (Gfap) and inositol 1,4,5-trisphosphate receptor type 2 (Itpr2)) in experimental rats was not affected by anakinra treatment. Thus, our data demonstrate that the administration of anakinra during epileptogenesis has some beneficial disease-modifying effects.

No Synergistic Effect of Silibinin and Morin in a Kainic Acid-Induced Epileptic Mouse Model.

Temporal lobe epilepsy (TLE) is the most common form of localization-related epilepsy, with the highest prevalence rate in adulthood. Recently, we reported the beneficial effects of the individual treatment with flavonoids such as silibinin and morin in kainic acid (KA)-treated mouse model for TLE. In this study, we investigated whether there is a synergistic effect of co-treatment with silibinin and morin on the susceptibility to seizure, the frequency of spontaneous recurrent seizures (SRSs), and granule cell dispersion in the dentate gyrus, which could be partially controlled by treatment with each flavonoid in the animal model for TLE. Unfortunately, we did not observe any synergistic effect against the susceptibility of seizure and SRS induced by KA treatment. However, the combination of these flavonoids showed similar antiepileptic effects compared with treatment with each one individually. Therefore, although silibinin and morin are not suitable for combination therapy, our results still suggest that these flavonoids can be used as potent therapeutic compounds for preventing epileptic seizures.

Proof-of-concept that network pharmacology is effective to modify development of acquired temporal lobe epilepsy

Epilepsy is a complex network phenomenon that, as yet, cannot be prevented or cured. We recently proposed network-based approaches to prevent epileptogenesis. For proof of concept we combined two drugs (levetiracetam and topiramate) for which in silico analysis of drug-protein interaction networks indicated a synergistic effect on a large functional network of epilepsy-relevant proteins. Using the intrahippocampal kainate mouse model of temporal lobe epilepsy, the drug combination was administered during the latent period before onset of spontaneous recurrent seizures (SRS). When SRS were periodically recorded by video-EEG monitoring after termination of treatment, a significant decrease in incidence and frequency of SRS was determined, indicating antiepileptogenic efficacy. Such efficacy was not observed following single drug treatment. Furthermore, a combination of levetiracetam and phenobarbital, for which in silico analysis of drug-protein interaction networks did not indicate any significant drug-drug interaction, was not effective to modify development of epilepsy. Surprisingly, the promising antiepileptogenic effect of the levetiracetam/topiramate combination was obtained in the absence of any significant neuroprotective or anti-inflammatory effects as indicated by multimodal brain imaging and histopathology. High throughput RNA-sequencing (RNA-seq) of the ipsilateral hippocampus of mice treated with the levetiracetam/topiramate combination showed that several genes that have been linked previously to epileptogenesis, were significantly differentially expressed, providing interesting entry points for future mechanistic studies. Overall, we have discovered a novel combination treatment with promise for prevention of epilepsy.

Consequences of housing conditions and interindividual diversity in rodent models of acquired epilepsy.

Consequences of housing conditions and interindividual diversity in rodent models of acquired epilepsy.

Low-dose intranasal insulin improves cognitive function and suppresses the development of epilepsy

Intranasal insulin exerts neuroprotective effects in a variety of neurological diseases. Whether intranasal insulin affects epileptic activity and whether it has neuroprotective effects in epileptic diseases is however still unknown. In this study we show that low-dose intranasal insulin inhibited kainic acid (KA)- or pentylenetetrazole (PTZ)-induced acute seizures and reduced epileptic discharge activities in mice, potentially by alleviating the increase in seizure-induced glutamate in the hippocampus. Meanwhile, intranasal insulin increased GABA levels and the activities of hippocampal theta, which may affect the excitability of the hippocampus. In chronic KA-induced epilepsy, low-dose intranasal insulin reduces the frequency of spontaneous recurrent seizures and epileptic discharges, while it increases theta energy and thereby improves spatial memory. Larger doses of intranasal insulin increased the frequency of seizures but did not aggravate cognitive impairment, suggesting that the frequency of seizures may not be related to impaired cognitive function. Overall, our findings show that low-dose intranasal insulin inhibits epileptic events and improves cognitive impairment in epileptic mice, suggesting that learning and memory can be improved by intranasal insulin. However, larger doses might increase the risk of epileptic seizures.

Disease Modification by Combinatorial Single Vector Gene Therapy: A Preclinical Translational Study in Epilepsy

Gene therapy has been suggested as a plausible novel approach to achieve seizure control in patients with focal epilepsy that do not adequately respond to pharmacological treatment. We investigated the seizure-suppressant potential of combinatorial neuropeptide Y and Y2 receptor single vector gene therapy based on adeno-associated virus serotype 1 (AAV1) in rats. First, a dose-response study in the systemic kainate-induced acute seizure model was performed, whereby the 1012 genomic particles (gp)/mL titer of the vector was selected as an optimal concentration. Second, an efficacy study was performed in the intrahippocampal kainate chronic model of spontaneous recurrent seizures (SRSs), designed to reflect a likely clinical scenario, with magnetic resonance image (MRI)-guided focal unilateral administration of the vector in the hippocampus during the chronic stage of the disease. The efficacy study demonstrated a favorable outcome of the gene therapy, with a 31% responder rate (more than 50% reduction in SRS frequency) and 13% seizure-freedom rate, whereas no such effects were observed in the control animals. The inter-SRS and SRS cluster intervals were also significantly prolonged in the treated group compared to controls. In addition, the SRS duration was significantly reduced in the treated group but not in the controls. This study establishes the SRS-suppressant ability of the single vector combinatorial neuropeptide Y/Y2 receptor gene therapy in a clinically relevant chronic model of epilepsy.

Anti-inflammatory treatment with a soluble epoxide hydrolase inhibitor attenuates seizures and epilepsy-associated depression in the LiCl-pilocarpine post-status epilepticus rat model

PurposeThis study aimed to investigate whether 1-trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl) urea (TPPU), a soluble epoxide hydrolase inhibitor with anti-inflammatory effects, could alleviate spontaneous recurrent seizures (SRS) and epilepsy-associated depressive behaviours in the lithium chloride (LiCl)-pilocarpine-induced post-status epilepticus (SE) rat model. MethodsThe rats were intraperitoneally (IP) injected with LiCl (127 mg/kg) and pilocarpine (40 mg/kg) to induce SE. A video surveillance system was used to monitor SRS in the post-SE model for 6 weeks (from the onset of the 2nd week to the end of the 7th week after SE induction). TPPU (0.1 mg/kg/d) was intragastrically given for 4 weeks from the 21st day after SE induction in the SRS + 0.1 TPPU group. The SRS + PEG 400 group was given the vehicle (40% polyethylene glycol 400) instead, and the control group was given LiCl and PEG 400 but not pilocarpine. The sucrose preference test (SPT) and forced swim test (FST) were conducted to evaluate the depression-like behaviours of rats. Immunofluorescent staining, enzyme-linked immunosorbent assay, and western blot analysis were performed to measure astrocytic and microglial gliosis, neuronal loss, and levels of soluble epoxide hydrolase (sEH), cytokines [tumour necrosis factor alpha (TNF-α), interleukin (IL)-1β, and IL-6], and cyclic adenosine monophosphate (cAMP)-response element binding protein (CREB). ResultsThe frequency of SRS was significantly decreased at 6 weeks and 7 weeks after SE induction in the 0.1TPP U group compared with the SRS + PEG 400 group. The immobility time (IMT) evaluated by FST was significantly decreased, whereas the climbing time (CMT) was increased, and the sucrose preference rate (SPR) evaluated by SPT was in an increasing trend. The levels of sEH, TNF-α, IL-1β, and IL-6 in the hippocampus (Hip) and prefrontal cortex (PFC) were all significantly increased in the SRS + PEG 400 group compared with the control group; neuronal loss, astrogliosis, and microglial activation were also observed. The astrocytic and microglial activation and levels of the pro-inflammatory cytokines in the Hip and PFC were significantly attenuated in the TPPU group compared with the SRS + PEG 400 group; moreover, neuronal loss and the decreased CREB expression were significantly alleviated as well. ConclusionTPPU treatment after SE attenuates SRS and epilepsy-associated depressive behaviours in the LiCl-pilocarpine induced post-SE rat model, and it also exerts anti-inflammatory effects in the brain. Our findings suggest a new therapeutic approach for epilepsy and its comorbidities, especially depression.