Kainic Model Research Articles

BASF inks joint development deal for Thyssenkrupp's dehydrogenation technology

Although the available therapeutic agents alleviate the symptoms in patients with temporal lobe epilepsy (TLE), these antiepileptic drugs do not provide adequate control of seizures in 30–40 % of patients. This study was conducted to evaluate anti-epileptic effects of simultaneous inhibition of dipeptidyl peptidase-4 and P2 × 7 purinoceptors in Kainate treated rats.Brilliant Blue G)BBG(, linagliptin)lin(and lin + BBG were administrated 30 min prior to induction of the intrahippocampal kainate model of epilepsy in male Wistar rats. In the case of valproic acid group, the animals intraperitoneally received valproic acid for 7 consecutive days prior to induction of the model. We carried out histological evaluations, monitoring of behavior, recording of intracranial electroencepholography (IEEG), and determination of astrogliosis and DNA fragmentation using ELISA methods.Our results showed that BBG and lin combination therapy had better effects on decrease in astrogliosis, DNA fragmentation and cognitive disturbances than ones whereas its effects on neuronal survival and seizure severity was similar to only BBG or lin. Likewise, the effects of lin + BBG on decrease in DNA fragmentation and cognitive disturbances were better than valproic acid group.Our findings suggest that simultaneous inhibition of dipeptidyl peptidase-4 and P2 × 7 purinoceptors might more efficiently provide protection against progression of the kainate-induced TLE in rats.

Combination therapy with dipeptidyl peptidase-4 and P2X7 purinoceptor inhibitors gives rise to antiepileptic effects in rats

Objective(s)Although the available therapeutic agents alleviate the symptoms in patients with temporal lobe epilepsy (TLE), these antiepileptic drugs do not provide adequate control of seizures in 30–40 % of patients. This study was conducted to evaluate anti-epileptic effects of simultaneous inhibition of dipeptidyl peptidase-4 and P2 × 7 purinoceptors in Kainate treated rats. Materials and MethodsBrilliant Blue G)BBG(, linagliptin)lin(and lin + BBG were administrated 30 min prior to induction of the intrahippocampal kainate model of epilepsy in male Wistar rats. In the case of valproic acid group, the animals intraperitoneally received valproic acid for 7 consecutive days prior to induction of the model. We carried out histological evaluations, monitoring of behavior, recording of intracranial electroencepholography (IEEG), and determination of astrogliosis and DNA fragmentation using ELISA methods. ResultsOur results showed that BBG and lin combination therapy had better effects on decrease in astrogliosis, DNA fragmentation and cognitive disturbances than ones whereas its effects on neuronal survival and seizure severity was similar to only BBG or lin. Likewise, the effects of lin + BBG on decrease in DNA fragmentation and cognitive disturbances were better than valproic acid group. ConclusionOur findings suggest that simultaneous inhibition of dipeptidyl peptidase-4 and P2 × 7 purinoceptors might more efficiently provide protection against progression of the kainate-induced TLE in rats.

Neuroprotective and anticonvulsant effects of sinomenine in kainate rat model of temporal lobe epilepsy: Involvement of oxidative stress, inflammation and pyroptosis

Oxidative stress, inflammation and pyroptosis are three of the most important mechanisms in the pathophysiology of temporal lobe epilepsy (TLE). Most people with TLE are refractory to the existing drugs. Sinomenine has shown neuroprotective effects through counteracting oxidative stress, inflammation and pyroptosis. In this study, we evaluated the effect of sinomenine on seizure behavior, oxidative stress, inflammation and pyroptosis markers in addition to its neuroprotective potential in intrahippocampal kainate-induced rat model of TLE. For this purpose, male rats (n = 60) were randomly divided into five groups, i.e., sham, kainate (lesion) with an intrahippocampal injection of kainate, kainate groups receiving sinomenine at doses of 30 or 50 mg/kg, and kainate group receiving valproic acid at a dose of 200 mg/kg (as the positive control). Our obtained data showed that sinomenine administration at a dose of 50 mg/kg can significantly decreases severity of seizures and incidence of status epilepticus (SE), hippocampal aberrant MFS and DNA fragmentation and prevents reduction of neuronal density. It also significantly restored level of ROS, MDA, HO-1 and SOD but its effect on GSH level was not significant. Additionally, sinomenine at a dose of 50 mg/kg partially counteracted the increase of NF-κB, TLR 4, TNFα, GFAP and caspase 1. These results suggest that sinomenine has anticonvulsant and neuroprotective effects by reducing hippocampal oxidative stress, inflammation, pyroptosis and apoptosis in intrahippocampal kainate model of TLE.

Neuronal network remodeling and Wnt pathway dysregulation in the intra-hippocampal kainate mouse model of temporal lobe epilepsy.

Mouse models of mesial temporal lobe epilepsy recapitulate aspects of human epilepsy, which is characterized by neuronal network remodeling in the hippocampal dentate gyrus. Observational studies suggest that this remodeling is associated with altered Wnt pathway signaling, although this has not been experimentally examined. We used the well-characterized mouse intrahippocampal kainate model of temporal lobe epilepsy to examine associations between hippocampal neurogenesis and altered Wnt signaling after seizure induction. Tissue was analyzed using immunohistochemistry and confocal microscopy, and gene expression analysis was performed by RT-qPCR on RNA extracted from anatomically micro-dissected dentate gyri. Seizures increased neurogenesis and dendritic arborization of newborn hippocampal dentate granule cells in peri-ictal regions, and decreased neurogenesis in the ictal zone, 2-weeks after kainate injection. Interestingly, administration of the novel canonical Wnt pathway inhibitor XAV939 daily for 2-weeks after kainate injection further increased dendritic arborization in peri-ictal regions after seizure, without an effect on baseline neurogenesis in control animals. Transcriptome analysis of dentate gyri demonstrated significant canonical Wnt gene dysregulation in kainate-injected mice across all regions for Wnt3, 5a and 9a. Intriguingly, certain Wnt genes demonstrated differential patterns of dysregulation between the ictal and peri-ictal zones, most notably Wnt5B, 7B and DKK-1. Together, these results demonstrate regional variation in Wnt pathway dysregulation early after seizure induction, and surprisingly, suggest that some Wnt-mediated effects might actually temper aberrant neurogenesis after seizures. The Wnt pathway may therefore provide suitable targets for novel therapies that prevent network remodeling and the development of epileptic foci in high-risk patients.

The impact of postsynaptic density 95 blocking peptide (Tat-NR2B9c) and an iNOS inhibitor (1400W) on proteomic profile of the hippocampus in C57BL/6J mouse model of kainate-induced epileptogenesis.

Antiepileptogenic agents that prevent the development of epilepsy following a brain insult remain the holy grail of epilepsy therapeutics. We have employed a label-free proteomic approach that allows quantification of large numbers of brain-expressed proteins in a single analysis in the mouse (male C57BL/6J) kainate (KA) model of epileptogenesis. In addition, we have incorporated two putative antiepileptogenic drugs, postsynaptic density protein-95 blocking peptide (PSD95BP or Tat-NR2B9c) and a highly selective inducible nitric oxide synthase inhibitor, 1400W, to give an insight into how such agents might ameliorate epileptogenesis. The test drugs were administered after the induction of status epilepticus (SE) and the animals were euthanized at 7days, their hippocampi removed, and subjected to LC-MS/MS analysis. A total of 2,579 proteins were identified; their normalized abundance was compared between treatment groups using ANOVA, with correction for multiple testing by false discovery rate. Significantly altered proteins were subjected to gene ontology and KEGG pathway enrichment analyses. KA-induced SE was most robustly associated with an alteration in the abundance of proteins involved in neuroinflammation, including heat shock protein beta-1 (HSP27), glial fibrillary acidic protein, and CD44 antigen. Treatment with PSD95BP or 1400W moderated the abundance of several of these proteins plus that of secretogranin and Src substrate cortactin. Pathway analysis identified the glutamatergic synapse as a key target for both drugs. Our observations require validation in a larger-scale investigation, with candidate proteins explored in more detail. Nevertheless, this study has identified several mechanisms by which epilepsy might develop and several targets for novel drug development. OPEN PRACTICES: This article has been awarded Open Data. All materials and data are publicly accessible as supporting information. Learn more about the Open Practices badges from the Center for Open Science: https://osf.io/tvyxz/wiki.

Partial depletion of septohippocampal cholinergic cells reduces seizure susceptibility, but does not mitigate hippocampal neurodegeneration in the kainate model of epilepsy

The brain cholinergic system may undergo structural and functional alterations both in human epilepsy and in respective animal models, but the causal relationships between these alterations and epilepsy remain to be established. In this study, we attempted to examine how the inhibition of epilepsy-related cholinergic plasticity may be reflected in seizure susceptibility and/or in the development of chronic epilepsy and its neurological consequences. For this purpose, adult Wistar rats received intrahippocampal injections of low doses of 192-IgG-saporin (SAP) to produce a moderate, but significant loss of septohippocampal cholinergic cells and to suppress their plasticity. Then, animals were treated with kainic acid to induce status epilepticus, which leads to the development of chronic epilepsy later in life. It was found that SAP-pretreatment was associated with longer latency to the onset of status epilepticus and with reduced mortality rate, suggesting that increased activity of septal cholinergic cells may potentiate seizures. Interestingly, months later, a greater percentage of rats with intact septohippocampal cholinergic connections showed spontaneous seizures, when compared to SAP-pretreated rats. Treatment with kainic acid produced death of 40–50% of hippocampal neurons and this effect was not ameliorated by prior cholinergic depletion. Moreover, the kainate induced cognitive deficits were detected in both SAP-pretreated and sham-pretreated groups. These data suggest that seizure-induced plasticity of cholinergic cells may indeed enhance seizure susceptibility and contribute to epileptogenic processes. They do not support the hypothesis that epilepsy-related hypertrophy of cholinergic neurons may potentiate hippocampal cell loss and respective behavioral impairments.

Ripple-related firing of identified deep CA1 pyramidal cells in chronic temporal lobe epilepsy in mice.

SummaryObjectiveTemporal lobe epilepsy (TLE) is often associated with memory deficits. Reactivation of memory traces in the hippocampus occurs during sharp‐wave ripples (SWRs; 140‐250 Hz). To better understand the mechanisms underlying high‐frequency oscillations and cognitive comorbidities in epilepsy, we evaluated how rigorously identified deep CA1 pyramidal cells (dPCs) discharge during SWRs in control and TLE mice.MethodsWe used the unilateral intraamygdala kainate model of TLE in video–electroencephalography (EEG) verified chronically epileptic adult mice. Local field potential and single‐cell recordings were performed using juxtacellular recordings from awake control and TLE mice resting on a spherical treadmill, followed by post hoc identification of the recorded cells.ResultsHippocampal SWRs in TLE mice occurred with increased intraripple frequency compared to control mice. The frequency of SWR events was decreased, whereas the overall frequency of SWRs, interictal epileptiform discharges, and high‐frequency ripples (250‐500 Hz) together was not altered. CA1 dPCs in TLE mice showed significantly increased firing during ripples as well as between the ripple events. The strength of ripple modulation of dPC discharges increased in TLE without alteration of the preferred phase of firing during the ripple waves.SignificanceThese juxtacellular electrophysiology data obtained from identified CA1 dPCs from chronically epileptic mice are in general agreement with recent findings indicating distortion of normal firing patterns during offline SWRs as a mechanism underlying deficits in memory consolidation in epilepsy. Because the primary seizure focus in our experiments was in the amygdala and we recorded from the CA1 region, these results are also in agreement with the presence of altered high‐frequency oscillations in areas of secondary seizure spread.

Effect of carbamazepine on spontaneous recurrent seizures recorded from the dentate gyrus in rats with kainate-induced epilepsy.

Animal models of chronic epilepsy with spontaneous recurrent seizures (SRSs) may be useful in the discovery and mechanistic analyses of antiseizure drugs (ASDs). Carbamazepine (CBZ), a widely used ASD with a well-defined mechanism, was analyzed in this proof-of-principle study to determine how a traditional ASD affects the properties of SRSs. The effects of CBZ on electrographic SRSs recorded from the dentate gyrus were studied in freely behaving rats using a repeated, low-dose kainate model of acquired epilepsy with a repeated-measures, crossover protocol. Almost all seizure durations were >20seconds. Both seizure likelihood and duration appeared to be similar between 1 and 8hours after individual CBZ injections. CBZ-induced decreases in seizure frequency were not significant at 10mg/kg; however, at 30mg/kg, seizure frequency was significantly reduced for convulsive but not nonconvulsive seizures. At 100mg/kg, CBZ strongly suppressed both convulsive and nonconvulsive seizures. Although CBZ had a dose-dependent effect on seizure frequency, CBZ did not affect seizure duration at any dose. The preceding interictal interval did not affect seizure duration; however, at 30mg/kg CBZ, nearly all seizures were nonconvulsive when the interictal interval was <30minutes (ie, during clusters). Increased doses of CBZ (10-100mg/kg) suppressed the frequency but not the duration of convulsive and nonconvulsive seizures in the repeated, low-dose kainate model. The repeated-measures, crossover protocol, which requires relatively few animals and compensates for progressive increases in seizure frequency during epileptogenesis after status epilepticus, allowed quantitative analyses of clinically relevant and translatable properties of SRSs.

Human and rodent temporal lobe epilepsy is characterized by changes in O-GlcNAc homeostasis that can be reversed to dampen epileptiform activity

Temporal Lobe Epilepsy (TLE) is frequently associated with changes in protein composition and post-translational modifications (PTM) that exacerbate the disorder. O-linked-β-N-acetyl glucosamine (O-GlcNAc) is a PTM occurring at serine/threonine residues that is derived from and closely associated with metabolic substrates. The enzymes O-GlcNActransferase (OGT) and O-GlcNAcase (OGA) mediate the addition and removal, respectively, of the O-GlcNAc modification. The goal of this study was to characterize OGT/OGA and protein O-GlcNAcylation in the epileptic hippocampus and to determine and whether direct manipulation of these proteins and PTM's alter epileptiform activity. We observed reduced global and protein specific O-GlcNAcylation and OGT expression in the kainate rat model of TLE and in human TLE hippocampal tissue. Inhibiting OGA with Thiamet-G elevated protein O-GlcNAcylation, and decreased both seizure duration and epileptic spike events, suggesting that OGA may be a therapeutic target for seizure control. These findings suggest that loss of O-GlcNAc homeostasis in the kainate model and in human TLE can be reversed via targeting of O-GlcNAc related pathways.

Neuronal Adenosine A2A Receptors Are Critical Mediators of Neurodegeneration Triggered by Convulsions.

Neurodegeneration is a process transversal to neuropsychiatric diseases and the understanding of its mechanisms should allow devising strategies to prevent this irreversible step in brain diseases. Neurodegeneration caused by seizures is a critical step in the aggravation of temporal lobe epilepsy, but its mechanisms remain undetermined. Convulsions trigger an elevation of extracellular adenosine and upregulate adenosine A2A receptors (A2AR), which have been associated with the control of neurodegenerative diseases. Using the rat and mouse kainate model of temporal lobe epilepsy, we now tested whether A2AR control convulsions-induced hippocampal neurodegeneration. The pharmacological or genetic blockade of A2AR did not affect kainate-induced convulsions but dampened the subsequent neurotoxicity. This neurotoxicity began with a rapid A2AR upregulation within glutamatergic synapses (within 2 h), through local translation of synaptic A2AR mRNA. This bolstered A2AR-mediated facilitation of glutamate release and of long-term potentiation (LTP) in CA1 synapses (4 h), triggered a subsequent synaptotoxicity, heralded by decreased synaptic plasticity and loss of synaptic markers coupled to calpain activation (12 h), that predated overt neuronal loss (24 h). All modifications were prevented by the deletion of A2AR selectively in forebrain neurons. This shows that synaptic A2AR critically control synaptic excitotoxicity, which underlies the development of convulsions-induced neurodegeneration.

Selective Vulnerability to Transcellular Pathway of Blood-Brain Barrier in Chronic Stage of the Kainate Model of Temporal Lobe Epilepsy in Rats

Selective Vulnerability to Transcellular Pathway of Blood-Brain Barrier in Chronic Stage of the Kainate Model of Temporal Lobe Epilepsy in Rats

Curcumin in epilepsy disorders.

Curcumin, a principal curcuminoid present in turmeric, has an antioxidant, anti-inflammatory and neuroprotective properties. Preclinical studies have indicated its beneficial effect for the treatment of epilepsy disorders. The molecule has an anti-seizure potential in preclinical studies, including chemical and electrical models of acute and chronic epilepsy. Curcumin also possesses an anti-epileptogenic activity as it reduces spontaneous recurrent seizures severity in a kainate model of temporal lobe epilepsy. The antioxidant and anti-inflammatory nature of curcumin might be responsible for its observed anti-seizure effects; nevertheless, the exact mechanism is not yet clear. The poor availability of curcumin to the brain limits its use in clinics. The application of nanoliposome and liposome technologies has been tested to enhance its brain availability and penetrability. Unfortunately, there are no randomized, double-blinded controlled clinical trials validating the use of curcumin in epilepsy. The present article analyzes different preclinical evidence illustrating the effect of curcumin in seizure models. The review encourages carrying out clinical trials in this important area of research. In conclusion, curcumin might be beneficial in patients with epilepsy disorders, if its bioavailability issues are resolved.

Electrophysiological Evidence for the Development of a Self-Sustained Large-Scale Epileptic Network in the Kainate Mouse Model of Temporal Lobe Epilepsy.

Most research on focal epilepsy focuses on mechanisms of seizure generation in the primary epileptic focus (EF). However, neurological deficits that are not directly linked to seizure activity and that may persist after focus removal are frequent. The recruitment of remote brain regions of an epileptic network (EN) is recognized as a possible cause, but a profound lack of experimental evidence exists concerning their recruitment and the type of pathological activities they exhibit. We studied the development of epileptic activities at the large-scale in male mice of the kainate model of unilateral temporal lobe epilepsy using high-density surface EEG and multiple-site intracortical recordings. We show that, along with focal spikes and fast ripples that remain localized to the injected hippocampus (i.e., the EF), a subpopulation of spikes that propagate across the brain progressively emerges even before the expression of seizures. The spatiotemporal propagation of these generalized spikes (GSs) is highly stable within and across animals, defining a large-scale EN comprising both hippocampal regions and frontal cortices. Interestingly, GSs are often concomitant with muscular twitches. In addition, while fast ripples are, as expected, highly frequent in the EF, they also emerge in remote cortical regions and in particular in frontal regions where GSs propagate. Finally, we demonstrate that these remote interictal activities are dependent on the focus in the early phase of the disease but continue to be expressed after focus silencing at later stages. Our results provide evidence that neuronal networks outside the initial focus are progressively altered during epileptogenesis.SIGNIFICANCE STATEMENT It has long been held that the epileptic focus is responsible for triggering seizures and driving interictal activities. However, focal epilepsies are associated with heterogeneous symptoms, calling into question the concept of a strictly focal disease. Using the mouse model of hippocampal sclerosis, this work demonstrates that focal epilepsy leads to the development of pathological activities specific to the epileptic condition, notably fast ripples, that appear outside of the primary epileptic focus. Whereas these activities are dependent on the focus early in the disease, focus silencing fails to control them in the chronic stage. Thus, dynamical changes specific to the epileptic condition are built up outside of the epileptic focus along with disease progression, which provides supporting evidence for network alterations in focal epilepsy.

The pedunculopontine and laterodorsal tegmental nuclei in the kainate model of epilepsy

Prior studies showed that epilepsy can be associated with reorganization of the septohippocampal cholinergic fiber system. Using the kainate model of epilepsy, we wished to further examine the structural integrity of the mesopontine tegmental nuclei (pedunculopontine, PPN, and laterodorsal, LDT), which provide the cholinergic input to the thalamus. It was found that the total numbers of the PPN and LDT cells immunoreactive to the vesicular acetylcholine transporter did not differ between control and epileptic rats. However, the cholinergic cells had enlarged perikarya in epileptic rats. We further examined the effects of epilepsy on the distribution pattern of cholinergic fiber varicosities in the parafascicular nucleus, one of the principal thalamic targets of PPN projections. The density of cholinergic varicosities, represented by two distinct populations, was increased in epileptic rats. These data provide the first morphological evidence for structural alterations in mesopontine cholinergic neurons in experimental epilepsy. They suggest dysfunctional cholinergic transmission in the brainstem-thalamic pathway, which may partly account for various epilepsy-related neurological disturbances.

Selective Vulnerability to Transcellular Pathway of Blood-Brain Barrier in Chronic Stage of the Kainate Model of Temporal Lobe Epilepsy in Rats

1. Abstract 1.1. Background: The blood-brain barrier (BBB) integrity is severely affected in many epilepsy syndromes including temporal lobe epilepsy (TLE). 1.2. Aim: In the present study, we investigated the effects of acute and chronic seizures induced by kainic acid (KA) on the transport pathways of BBB in rats. 1.3. Methods: Electroencephalography (EEG) was recorded to evaluate seizure activity. Immunohistochemistry for claudin-5, a tight junction protein, caveolin-1, and the glial fibrillary acidic protein (GFAP), a marker of astrocyte activation, was performed. Electron microscopy was used to ultrastructurally assess the presence and route of extravasation of horseradish peroxidase (HRP), a permeability marker, in barrier type of brain capillary endothelial cells. 1.4. Results: The immunoreactivity of claudin-5 and caveolin-1 in hippocampus increased by both acute and chronic seizures (p 1.5. Conclusion: This study provides immunohistochemical and ultrastructural evidence of BBB disruption through a selective vulnerability of the transcellular transport via an increase in caveolar vesicles in the endothelial cells brain capillaries rather than activation of paracellular pathway in the KA-induced rat model of TLE. 2. Keywords: Blood-Brain Barrier; Caveolin-1; Claudin-5; Electron Microscopy; Kainic Acid; Temporal Lobe Epilepsy

Glial source of nitric oxide in epileptogenesis: A target for disease modification in epilepsy.

Epileptogenesis is the process of developing an epileptic condition and/or its progression once it is established. The molecules that initiate, promote, and propagate remarkable changes in the brain during epileptogenesis are emerging as targets for prevention/treatment of epilepsy. Epileptogenesis is a continuous process that follows immediately after status epilepticus (SE) in animal models of acquired temporal lobe epilepsy (TLE). Both SE and epileptogenesis are potential therapeutic targets for the discovery of anticonvulsants and antiepileptogenic or disease-modifying agents. For translational studies, SE targets are appropriate for screening anticonvulsive drugs prior to their advancement as therapeutic agents, while targets of epileptogenesis are relevant for identification and development of therapeutic agents that can either prevent or modify the disease or its onset. The acute seizure models do not reveal antiepileptogenic properties of anticonvulsive drugs. This review highlights the important components of epileptogenesis and the long-term impact of intervening one of these components, nitric oxide (NO), in rat and mouse kainate models of TLE. NO is a putative pleotropic gaseous neurotransmitter and an important contributor of nitro-oxidative stress that coexists with neuroinflammation and epileptogenesis. The long-term impact of inhibiting the glial source of NO during early epileptogenesis in the rat model of TLE is reviewed. The importance of sex as a biological variable in disease modification strategies in epilepsy is also briefly discussed.

Losartan suppresses the kainate-induced changes of angiotensin AT1 receptor expression in a model of comorbid hypertension and epilepsy

AimsExperimental and clinical studies have demonstrated that components of renin-angiotensin system are elevated in the hippocampus in epileptogenic conditions. In the present work, we explored the changes in the expression of angiotensin II receptor, type 1 (AT1 receptor) in limbic structures, as well as the effect of the AT1 receptor antagonist losartan in a model of comorbid hypertension and epilepsy. Main methodsThe expression of AT1 receptors was compared between spontaneously hypertensive rats (SHRs) and Wistar rats by using immunohistochemistry in the kainate (KA) model of temporal lobe epilepsy (TLE). The effect of losartan was studied on AT1 receptor expression in epileptic rats that were treated for a period of 4weeks after status epilepticus. Key findingsThe naive and epileptic SHRs were characterized by stronger protein expression of AT1 receptor than normotensive Wistar rats in the CA1, CA3a, CA3b, CA3c field and the hilus of the dentate gyrus of the dorsal hippocampus but fewer cells were immunostained in the piriform cortex. Increased AT1 immunostaining was observed in the basolateral amygdala of epileptic SHRs but not of epileptic Wistar rats. Losartan exerted stronger and structure-dependent suppression of AT1 receptor expression in SHRs compared to Wistar rats. SignificanceOur results confirm the important role of AT1 receptor in epilepsy and suggest that the AT1receptor antagonists could be used as a therapeutic strategy for treatment of comorbid hypertension and epilepsy.

Role of the Fyn-PKCδ signaling in SE-induced neuroinflammation and epileptogenesis in experimental models of temporal lobe epilepsy

Status epilepticus (SE) induces neuroinflammation and epileptogenesis, but the mechanisms are not yet fully delineated. The Fyn, a non-receptor Src family tyrosine kinase (SFK), and its immediate downstream target, PKCδ are emerging as potential mediators of neuroinflammation. In order to first determine the role of Fyn kinase signaling in SE, we tested the efficacy of a SFK inhibitor, saracatinib (25mg/kg, oral) in C57BL/6J mouse kainate model of acute seizures. Saracatinib pretreatment dampened SE severity and completely prevented mortality. We further utilized fyn−/− and fyn+/+ mice (wildtype control for the fyn−/− mice on same genetic background), and the rat kainate model, treated with saracatinib post-SE, to validate the role of Fyn/SFK in SE and epileptogenesis. We observed significant reduction in SE severity, epileptiform spikes, and electrographic non-convulsive seizures in fyn−/− mice when compared to fyn+/+ mice. Interestingly, significant reductions in phosphorylated pSrc-416 and PKCδ (pPKCδ-507) and naive PKCδ were observed in fyn−/− mice as compared to fyn+/+ mice suggesting that PKCδ signaling is a downstream mediator of Fyn in SE and epileptogenesis. Notably, fyn−/− mice also showed a reduction in key proinflammatory mediators TNF-α, IL-1β, and iNOS mRNA expression; serum IL-6 and IL-12 levels; and nitro-oxidative stress markers such as 4-HNE, gp91phox, and 3-NT in the hippocampus. Immunohistochemistry revealed a significant increase in reactive microgliosis and neurodegeneration in the hippocampus and hilus of dentate gyrus in fyn+/+ mice in contrast to fyn−/− mice. Interestingly, we did not observe upregulation of Fyn in pyramidal neurons of the hippocampus during post-SE in fyn+/+ mice, but it was upregulated in hilar neurons of the dentate gyrus when compared to naïve control. In reactive microglia, both Fyn and PKCδ were persistently upregulated during post-SE suggesting that Fyn-PKCδ may drive neuroinflammation during epileptogenesis. Since disabling the Fyn kinase prior to SE, either by treating with saracatinib or fyn gene knockout, suppressed seizures and the subsequent epileptogenic events, we further tested whether Fyn/SFK inhibition during post-SE modifies epileptogenesis. Telemetry-implanted, SE-induced, rats were treated with saracatinib and continuously monitored for a month. At 2h post-diazepam, the saracatinib (25mg/kg) or the vehicle was administered orally and repeated twice daily for first three days followed by a single dose/day for the next four days. The saracatinib post-treatment prevented epileptogenesis in >50% of the rats and significantly reduced spontaneous seizures and epileptiform spikes in the rest (one animal did not respond) when compared to the vehicle treated group, which had >24 seizures in a month. Collectively, the findings suggest that Fyn/SFK is a potential mediator of epileptogenesis and a therapeutic target to prevent/treat seizures and epileptogenesis.

Serotonin depletion increases seizure susceptibility and worsens neuropathological outcomes in kainate model of epilepsy.

Serotonin is implicated in the regulation of seizures, but whether or not it can potentiate the effects of epileptogenic factors is not fully established. Using the kainic acid model of epilepsy in rats, we tested the effects of serotonin depletion on (1) susceptibility to acute seizures, (2) development of spontaneous recurrent seizures and (3) behavioral and neuroanatomical sequelae of kainic acid treatment. Serotonin was depleted by pretreating rats with p-chlorophenylalanine. In different groups, kainic acid was injected at 3 different doses: 6.5mg/kg, 9.0mg/kg or 12.5mg/kg. A single dose of 6.5mg/kg of kainic acid reliably induced status epilepticus in p-chlorophenylalanine-pretreated rats, but not in saline-pretreated rats. The neuroexcitatory effects of kainic acid in the p-chlorophenylalanine-pretreated rats, but not in saline-pretreated rats, were associated with the presence of tonic-clonic convulsions and high lethality. Compared to controls, a greater portion of serotonin-depleted rats showed spontaneous recurrent seizures after kainic acid injections. Loss of hippocampal neurons and spatial memory deficits associated with kainic acid treatment were exacerbated by prior depletion of serotonin. The present findings are of particular importance because they suggest that low serotonin activity may represent one of the major risk factors for epilepsy and, thus, offer potentially relevant targets for prevention of epileptogenesis.

Agomelatine protects against neuronal damage without preventing epileptogenesis in the kainate model of temporal lobe epilepsy

Recent studies about the novel antidepressant agomelatine, which is a mixed MT1 and MT2 melatonin receptor agonist and 5HT2C serotonin receptor antagonist possessing an anticonvulsant and neuroprotective action, suggest that it may have potential to contribute against epileptogenesis and epilepsy-induced memory impairment. In order to ascertain whether protection of some brain structures could suppress epileptogenesis, in the present study, we evaluated the effect of chronic post-status treatment with agomelatine on epileptogenesis, behavioral and neuronal damage induced by kainate acid (KA) status epilepticus (SE). Agomelatine/vehicle treatment (40mg/kg, i.p.) started one hour after SE and continued up to 10weeks in Wistar rats. Latency for onset of spontaneous motor seizures (SMS) and their frequency was detected by a 24-h video-recording. Locomotor activity, anxiety and hippocampus-dependent spatial memory in open field (OF), elevated plus maze (EPM), light-dark test (LDT) and radial arm maze (RAM) test, respectively, were evaluated during the last two weeks after SE. Agomelatine significantly decreased the latency for onset of SMS and increased the seizure frequency during the 2nd and the 3rd week of treatment. The MT1 and MT2 receptor agonist and serotonin 5HT2C receptor antagonist exacerbated the KA-induced hyperlocomotion and impulsive behavior and it was unable to prevent spatial memory impairment of epileptic rats. However, agomelatine induced a neuroprotection in the dorsal hippocampus, specifically in the CA1, septal CA2 and partially in the CA3c region, the hilus of the dentate gyrus, piriform cortex and septo-temporal and temporal basolateral amygdala. Our findings suggest that the beneficial impact against SE-induced neuronal loss exerted by agomelatine is not crucial for the suppression of epileptogenesis and its deleterious consequences in KA model of temporal lobe epilepsy.