KA-induced Seizures Research Articles

Melatonin Pretreatment Protects Against Status epilepticus, Glutamate Transport, and Oxidative Stress Induced by Kainic Acid in Zebrafish.

Status epilepticus (SE) develops from abnormal electrical discharges, resulting in neuronal damage. Current treatments include antiepileptic drugs. However, the most common drugs used to treat seizures may sometimes be ineffective and have many side effects. Melatonin is an endogenous physiological hormone that is considered an alternative treatment for neurological disorders because of its free radical scavenging property. Thus, this study aimed to determine the effects of melatonin pretreatment on SE by inducing glutamatergic hyperstimulation in zebrafish. Seizures were induced in zebrafish using kainic acid (KA), a glutamate analog, and the seizure intensity was recorded for 60min. Melatonin treatment for 7days showed a decrease in seizure intensity (28%), latency to reach score 5 (14min), and duration of SE (29%). In addition, melatonin treatment attenuated glutamate transporter levels, which significantly decreased in the zebrafish brain after 12h of KA-induced seizures. Melatonin treatment reduced the increase in oxidative stress by reactive oxygen species formation through thiobarbituric acid reactive substances and 2',7'-dichiorofluorescin, induced by KA-seizure. An imbalance of antioxidant enzyme activities such as superoxide dismutase and catalase was influenced by melatonin and KA-induced seizures. Our study indicates that melatonin promotes a neuroprotective response against the epileptic profile in zebrafish. These effects could be related to the modulation of glutamatergic neurotransmission, recovery of glutamate uptake, and oxidative stress parameters in the zebrafish brain.

Pretreatment with Methylene Blue Protects Against Acute Seizure and Oxidative Stress in a Kainic Acid-Induced Status Epilepticus Model.

BackgroundThe aim of the present study was to investigate the potential anticonvulsant effect of methylene blue (MB) in a kainic acid (KA)-induced status epilepticus (SE) model. The effects of MB on levels of oxidative stress and glutamate (Glu) also were explored.Material/MethodsSixty C57BL/6 mice were randomly divided into 5 equal-sized groups: (1) controls; (2) KA; (3) MB 0.5 mg/kg+KA; (4) MB 1 mg/kg+KA; and (5) vehicle+KA. The SE model was established by intra-amygdala microinjection of KA. Behavioral observations and simultaneous electroencephalographic records of the seizures in different groups were analyzed to determine the potential anticonvulsant effect of MB. The influences of MB on oxidative stress markers and glutamate were also detected to explore the possible mechanism.ResultsMB afforded clear protection against KA-induced acute seizure, as measured by the delayed latency of onset of generalized seizures and SE, decreased percentage of SE, and increased survival rate in mice with acute epilepsy. MB markedly increased the latency to first onset of epileptiform activity and decreased the average duration of epileptiform events, as well as the percentage of time during which the epileptiform activity occurred. Administration of MB prevented KA-induced deterioration of oxidative stress markers and Glu.ConclusionsMB is protective against acute seizure in SE. This beneficial effect may be at least partially related to its potent antioxidant ability and influence on Glu level.

Exendin-4 Pretreatment Attenuates Kainic Acid-Induced Hippocampal Neuronal Death.

Exendin-4 (Ex-4) is a glucagon-like peptide-1 receptor (GLP-1R) agonist that protects against brain injury. However, little is known about the effect of Ex-4 on kainic acid (KA)-induced seizures and hippocampal cell death. Therefore, this study evaluated the neuroprotective effects of Ex-4 pretreatment in a mouse model of KA-induced seizures. Three days before KA treatment, mice were intraperitoneally injected with Ex-4. We found that Ex-4 pretreatment reversed KA-induced reduction of GLP-1R expression in the hippocampus and attenuated KA-induced seizure score, hippocampal neuronal death, and neuroinflammation. Ex-4 pretreatment also dramatically reduced hippocampal lipocalin-2 protein in KA-treated mice. Furthermore, immunohistochemical studies showed that Ex-4 pretreatment significantly alleviated blood–brain barrier leakage. Finally, Ex-4 pretreatment stimulated hippocampal expression of phosphorylated cyclic adenosine monophosphate (cAMP) response element-binding protein (p-CREB), a known target of GLP-1/GLP-1R signaling. These findings indicate that Ex-4 pretreatment may protect against KA-induced neuronal damage by regulating GLP-1R/CREB-mediated signaling pathways.

Repeated exposure to microcystin-leucine-arginine potentiates excitotoxicity induced by a low dose of kainate

Microcystin-leucine-arginine (MLCR) is a cyanobacterial toxin, and has been demonstrated to cause neurotoxicity. In addition, MCLR has been identified as an inhibitor of protein phosphatase (PP)1 and PP2A, which are known to regulate the phosphorylation of various molecules related to synaptic excitability. Thus, in the present study, we examined whether MCLR exposure affects seizures induced by a low dose of kainic acid (KA; 0.05 μg, i.c.v.) administration. KA-induced seizure occurrence and seizure score significantly increased after repeated exposure to MCLR (2.5 or 5.0 μg/kg, i.p., once a day for 10 days), but not after acute MCLR exposure (2.5 or 5.0 μg/kg, i.p., 2 h and 30 min prior to KA administration), and hippocampal neuronal loss was consistently facilitated by repeated exposure to MCLR. In addition, repeated MCLR significantly elevated the membrane expression of kainate receptor GluK2 subunits, p-pan-protein kinase C (PKC), and p-extracellular signal-related kinase (ERK) at 1 h after KA. However, KA-induced membrane expression of Ca2+/calmodulin-dependent kinase II (CaMKII) was significantly reduced by repeated MCLR exposure. Consistent with the enhanced seizures and neurodegeneration, MCLR exposure significantly potentiated KA-induced oxidative stress and microglial activation, which was accompanied by increased expression of p-ERK and p-PKCδ in the hippocampus. The combined results suggest that repeated MCLR exposure potentiates KA-induced excitotoxicity in the hippocampus by increasing membrane GluK2 expression and enhancing oxidative stress and neuroinflammation through the modulation of p-CaMKII, p-PKC, and p-ERK.

Everolimus inhibits PI3K/Akt/mTOR and NF-kB/IL-6 signaling and protects seizure-induced brain injury in rats

BackgroundEpilepsy is a common chronic neurological disease caused by the over-synchronization of neurons leading to brain dysfunction. Recurrent seizures can lead to cognitive and behavioral deficits, and irreversible brain damage. While the PI3K/Akt/mTOR pathway regulates various physiological processes of neurons and glia, it may also lead to abnormal neuronal signal transduction under pathological conditions, including that of epilepsy. Everolimus (Eve), an mTOR inhibitor, may modulate neuronal excitability and therefore exert protection against epilepsy. Therefore, this study aimed to investigate the neuroprotective effect of Everolimus on seizure-induced brain injury and its regulation of the PI3K/Akt/mTOR and NF-kB/IL-6 signaling pathway. Kainic acid (KA) 15 mg/kg was used to induce seizures and Everolimus (1, 2, 5 mg/kg) was administered as a pretreatment. Hippocampal tissue was extracted 24 h post-seizure. ResultsThe protein and mRNA expression levels of PI3K、p-AKt、p-mTOR、NF-kB and IL-6 as well as neuronal apoptosis and microglia activation, significantly increased after KA-induced seizures, however, these effects were inhibited by Everolimus treatment. Furthermore, pretreatment with Everolimus decreased seizure scores and increased seizure latency. ConclusionsEverolimus can decrease the PI3K/Akt/mTOR and NF-kB/IL-6 signaling pathway, reduce neuronal apoptosis and microglia activation, and attenuate seizure susceptibility and intensity, thus having a protective effect on seizure-induced brain damage.

MiR-128 regulates epilepsy sensitivity in mice by suppressing SNAP-25 and SYT1 expression in the hippocampus

Epilepsy is accompanied by abnormal neurotransmission, and microRNAs, as versatile players in the modulation of gene expression, are important in epilepsy pathology. Here, we found that miR-128 expression was elevated in the acute seizure phase and decreased during the recurrent seizure phase after status epilepticus in mice. Both SNAP-25 and SYT1 are regulated by miR-128 in vitro and in vivo. Overexpressing miR-128 in cultured neurons decreased neurotransmitter released by suppressing SNAP-25 and SYT1 expression. Anti-miR-128 injection before kainic acid (KA) injection increased the sensitivity of mice to KA-induced seizures, while overexpressing miR-128 at the latent and recurrent phases had a neuroprotective effect in KA-induced seizures. Our study shows for the first time that miR-128, a key regulator of neurotransmission, plays an important role in epilepsy pathology and that miR-128 might be a potential candidate molecular target for epilepsy therapy.

Effect of thiamazole on kainic acid-induced seizures in mice.

Kainic acid (KA) induced epileptic seizures in mice is a commonly used experimental model of epilepsy. Previous studies have suggested the roles of various neurotransmitters and oxidative stress in KA-induced seizures. An important role of hypothyroidism has also been suggested in epilepsy. Thiamazole (TZ) is an anti-hyperthyroid drug with antioxidant property. This study reports the effect of TZ on KA-induced epileptic seizures in mice, produced by intraperitoneal (IP) injection of KA (18 mg/kg). Prior to KA injection, the animals were treated with TZ (12.5, 25 and 50 mg/kg IP). Our results showed that in KA alone group, about half of the animals developed seizures. Pre-treatment of mice with TZ significantly increased the frequency of seizures in dose-dependent manner. Administration of TZ significantly reduced the latency time and aggravated the severity of seizures. TZ also increased the mortality in KA-treated mice. Striatal dopamine and serotonin levels were markedly increased in KA alone treated mice, which were not significantly affected by TZ treatment. Among the indices of oxidative stress, we observed a significant reduction in cerebral vitamin E whereas the levels of cerebral malondialdehyde and conjugated dienes were significantly increased in animals with high severity of seizures. In conclusion, TZ potentiated the frequency and severity of experimental seizure in mice. There is a possibility of altered metabolism of KA in presence of TZ that might have potentiated the toxicity of KA. These findings suggest a caution while administering anti-hyperthyroid drugs in epileptic seizures.

Silymarin Inhibits Glutamate Release and Prevents against Kainic Acid-Induced Excitotoxic Injury in Rats.

Silymarin, a polyphenoic flavonoid derived from the seeds of milk thistle (Silybum marianum), exhibits neuroprotective effects. In this study, we used a model of rat cerebrocortical synaptosomes to investigate whether silymarin affects the release of glutamate, an essential neurotransmitter involved in excitotoxicity. Its possible neuroprotective effect on a rat model of kainic acid (KA)-induced excitotoxicity was also investigated. In rat cortical synaptosomes, silymarin reduced glutamate release and calcium elevation evoked by the K+ channel blocker 4-aminopyridine but did not affect glutamate release caused by the Na+ channel activator veratridine or the synaptosomal membrane potential. Decreased glutamate release by silymarin was prevented by removal of extracellular calcium and blocking of N- and P/Q-type Ca2+ channel or extracellular signal-regulated kinase 1/2 (ERK1/2) but not by blocking of intracellular Ca2+ release. Immunoblotting assay results revealed that silymarin reduced 4-aminopyridine-induced phosphorylation of ERK1/2. Moreover, systemic treatment of rats with silymarin (50 or 100 mg/kg) 30 min before systemic KA (15 mg/kg) administration attenuated KA-induced seizures, glutamate concentration elevation, neuronal damage, glial activation, and heat shock protein 70 expression as well as upregulated KA-induced decrease in Akt phosphorylation in the rat hippocampus. Taken together, the present study demonstrated that silymarin depressed synaptosomal glutamate release by suppressing voltage-dependent Ca2+ entry and ERK1/2 activity and effectively prevented KA-induced in vivo excitotoxicity.

Antiepileptic effects of exogenous β-hydroxybutyrate on kainic acid-induced epilepsy.

The aim of the present study was to explore the potential anticonvulsant effects of β-hydroxybutyrate (BHB) in a kainic acid (KA)-induced rat epilepsy model. The KA-induced rat seizure model was established and BHB was administrated intraperitoneally at a dose of 4 mmol/kg 30 min prior to KA injection. Hippocampal tissues were then obtained 1, 3 and 7 days following KA administration, following which the expression levels of neuron-specific enolase (NSE) and glial fibrillary acidic protein (GFAP) were measured using a double immunofluorescence labeling method. In addition, the contents of glutathione (GSH), γ-aminobutyric acid (GABA) and ATP were measured using ELISA. Pretreatment with BHB markedly increased the expression of NSE after KA injection compared with that in the normal saline (NS) + KA group, suggesting that the application of BHB could alleviate neuronal damage in rats. The protective effect of BHB may be associated with suppressed inflammatory responses, which was indicated by the observed inhibition of GFAP expression in rats in the BHB + KA group compared with that in the NS + KA group. It was also found that GSH and GABA contents were notably increased after the rats were pretreated with BHB compared with those in the NS + KA group. To conclude, the application of exogenous BHB can serve as a novel therapeutic agent for epilepsy.

Xenon Exerts Neuroprotective Effects on Kainic Acid-Induced Acute Generalized Seizures in Rats via Increased Autophagy

Xenon has been shown to have neuroprotective effects and is clinically used as a favorable safe inhalation anesthetic. We previously confirmed the neuroprotective effects of xenon treatment in epileptic animals. However, the mechanism underlying these protective effects remains unclear. We aimed to assess the effects of xenon inhalation on autophagy in neuronal injury induced by acute generalized seizures. Kainic acid (KA) was injected into the lateral ventricle of male Sprague–Dawley rats to induce acute generalized seizures. Next, the rats were treated via inhalation of a 70% xenon/21% oxygen/9% nitrogen mixture for 60 min immediately after KA administration. The control group was treated via inhalation of a 79% nitrogen/21% oxygen mixture. Subsequently, two inhibitors (3-methyladenine or bafilomycin A1) or an autophagy inducer (rapamycin) were administered, respectively, before KA and xenon administration to determine the role of autophagy in the protective effects of xenon. The levels of apoptosis, neuronal injury, and autophagy were determined in all the rats. Xenon inhalation significantly attenuated the severity of the seizure-induced neuronal injury. Increased autophagy accompanied this inhibitive effect. Autophagy inhibition eliminated these xenon neuroprotective effects. A simulation of autophagy using rapamycin recapitulated xenon’s protective effects on KA-induced acute generalized seizures in the rats. These findings confirmed that xenon exerts strong neuroprotective effects in KA-induced acute generalized seizures. Further, they indicate that increased autophagy may underlie the protective effects of xenon. Therefore, xenon and autophagy inducers may be useful clinical options for their neuroprotective effects in epileptic seizures.

CircHivep2 contributes to microglia activation and inflammation via miR-181a-5p/SOCS2 signalling in mice with kainic acid-induced epileptic seizures.

Epilepsy is a chronic brain disease characterized by recurrent seizures. Circular RNA (circRNA) is a novel family of endogenous non‐coding RNAs that have been proposed to regulate gene expression. However, there is a lack of data on the role of circRNA in epilepsy. In this study, the circRNA profiles were evaluated by microarray analysis. In total, 627 circRNAs were up‐regulated, whereas 892 were down‐regulated in the hippocampus in mice with kainic acid (KA)‐induced epileptic seizures compared with control. The expression of circHivep2 was significantly down‐regulated in hippocampus tissues of mice with KA‐induced epileptic seizures and BV‐2 microglia cells upon KA treatment. Bioinformatics analysis predicted that circHivep2 interacts with miR‐181a‐5p to regulate SOCS2 expression, which was validated using a dual‐luciferase reporter assay. Moreover, overexpression of circHivep2 significantly inhibited KA‐induced microglial activation and the expression of inflammatory factors in vitro, which was blocked by miR‐181a‐5p, whereas circHivep2 knockdown further induced microglia cell activation and the release of pro‐inflammatory proteins in BV‐2 microglia cells after KA treatment. The application of circHivep2+ exosomes derived from adipose‐derived stem cells (ADSCs) exerted significant beneficial effects on the behavioural seizure scores of mice with KA‐induced epilepsy compared to control exosomes. The circHivep2+ exosomes also inhibited microglial activation, the expression of inflammatory factors, and the miR‐181a‐5p/SOCS2 axis in vivo. Our results suggest that circHivep2 regulates microglia activation in the progression of epilepsy by interfering with miR‐181a‐5p to promote SOCS2 expression, indicating that circHivep2 may serve as a therapeutic tool to prevent the development of epilepsy.

Adipose-derived stem cell transplantation improves learning and memory via releasing neurotrophins in rat model of temporal lobe epilepsy

Epilepsy is a common neurological disease and its most common type is temporal lobe epilepsy (TLE). Novel therapeutics is needed as many TLE patients are resistant to treatments like anticonvulsants or temporal lobectomy. Stem cell therapy has great promise in regeneration medicine. In the current study, we tried to investigate the potential protective effects of adipose-derived stem cell (ADSC) transplantation in epileptic rats. Epilepsy model was established by intra-hippocampal injection of kainic acid (KA) in rats. ADSCs were isolated, differentiated and transplanted into hippocampus of KA rats. There were three groups of rats: normal control group receiving saline injection and no transplantation, KA + sham group receiving KA injection and sham transplantation surgery and KA + transplantation group receiving KA injection and ADSC transplantation. We found that ADSCs were highly positive for CD44, CD90, CD29 and CD105, and neural differentiation induced the expression of neuronal markers like Tuj1, MAP2, NeuN and PSD-95. EEG recording showed that KA induced seizure activity while ADSC transplantation inhibited seizure activity. In training session of Morris water maze task, KA injection impaired the learning capacity of rats while ADSC transplantation restored the learning capacity at 2-week or 2-month post transplantation. In probe session of Morris water maze task, KA injection impaired the memory of rats while ADSC transplantation restored the memory at 2-week or 2-month post transplantation. Transplanted ADSCs released BDNF, NT3 and NT4. Pro-apoptotic BAX was reduced while anti-apoptotic BCL-2 and BCL-xL were increased in hippocampus post ADSC transplantation. Our study suggests that ADSC transplantation inhibits KA-induced seizures and improves the learning and memory function of epileptic rats.

Kainic Acid Activates TRPV1 via a Phospholipase C/PIP2-Dependent Mechanism in Vitro.

Kainic acid (KA) is an excitotoxic glutamate analogue produced by a marine seaweed. It elicits neuronal excitotoxicity leading to epilepsy in rodents. Activation of transient receptor potential vanilloid subfamily 1 (TRPV1), a nonselective cation channel protein, by capsaicin, prevents KA-induced seizures in a mouse model of temporal lobe epilepsy. However, the precise mechanism behind this protective effect of capsaicin remains unclear. In order to analyze the direct effect of KA on TRPV1, we evaluated the ability of KA to activate TRPV1 and analyzed its binding to TRPV1 using a molecular modeling approach. In vitro, KA activates a Ca2+ influx into TRPV1 expressing HEK293 cells but not in contsrol HEK293 cells. Pretreatment with either capsaicin (1 M) or capsazepine (10 M; TRPV1 antagonist) prevents the effect of KA. Pharmacological inhibition of phospholipase C (PLC) by U73122 or overexpression of phosphatidylinositol 5 phosphatase (Synaptojanin 1; Synj-1) counters the effect of KA. Further, KA treatment causes actin reorganization in HEKTRPV1 cells and PLC inhibition by U73122 prevents this. Molecular modeling data revealed that KA binds to TRPV1 and prebinding with capsaicin prevents the binding of KA to TRPV1. Consistently, the lack of effect of KA in activating chicken TRPV1, which is insensitive to capsaicin, suggests that there is a significant overlap between the sites of KA and capsaicin activation of TRPV1. However, PLC inhibition did not suppress TRPV1 activation by capsaicin. Collectively, our data suggest that KA binds to and activates TRPV1 and causes actin reorganization via PLC-dependent mechanism in vitro. We propose that KA mediates Ca2+ induced toxicity possibly by activating TRPV1. Therefore, inhibiting TRPV1 will be a beneficial strategy in abating Ca2+-induced neurotoxicity.

Drug-Responsive Inhomogeneous Cortical Modulation by Direct Current Stimulation.

Cathodal direct current stimulation (cDCS) induces long-term depression (LTD)-like reduction of cortical excitability (DCS-LTD), which has been tested in the treatment of epilepsy with modest effects. In part, this may be due to variable cortical neuron orientation relative to the electric field. We tested, in vivo and in vitro, whether DCS-LTD occurs throughout the cortical thickness, and if not, then whether drug-DCS pairing can enhance the uniformity of the cortical response and the cDCS antiepileptic effect. cDCS-mediated changes in cortical excitability were measured in vitro in mouse motor cortex (M1) and in human postoperative neocortex, in vivo in mouse somatosensory cortex (S1), and in a mouse kainic acid (KA)-seizure model. Contributions of N-methyl-D-aspartate-type glutamate receptors (NMDARs) to cDCS-mediated plasticity were tested with application of NMDAR blockers (memantine/D-AP5). cDCS reliably induced DCS-LTD in superficial cortical layers, and a long-term potentiation (LTP)-like enhancement (DCS-LTP) was recorded in deep cortical layers. Immunostaining confirmed layer-specific increase of phospho-S6 ribosomal protein in mouse M1. Similar nonuniform cDCS aftereffects on cortical excitability were also found in human neocortex in vitro and in S1 of alert mice in vivo. Application of memantine/D-AP5 either produced a more uniform DCS-LTD throughout the cortical thickness or at least abolished DCS-LTP. Moreover, a combination of memantine and cDCS suppressed KA-induced seizures. cDCS aftereffects are not uniform throughout cortical layers, which may explain the incomplete cDCS clinical efficacy. NMDAR antagonists may augment cDCS efficacy in epilepsy and other disorders where regional depression of cortical excitability is desirable. ANN NEUROL 2020;88:489-502.

Microglial depletion aggravates the severity of acute and chronic seizures in mice

Microglia are the resident immune cells of the center nervous system and participate in various neurological diseases. Here we determined the function of microglia in epileptogenesis using microglial ablation approaches. Three different microglia-specific genetic tools were used, CX3CR1CreER/+:R26iDTA/+, CX3CR1CreER/+:R26iDTR/+, and CX3CR1CreER/+:Csf1rFlox/Flox mice. We found that microglial depletion led to worse kainic acid (KA)-induced status epilepticus, higher mortality rate, and increased neuronal degeneration in the hippocampus. In KA-induced chronic spontaneous recurrent seizures, microglial depletion increased seizure frequency, interictal spiking, and seizure duration. Therefore, microglial depletion aggravates the severity of KA-induced acute and chronic seizures. Interestingly, microglial repopulation reversed the effects of depletion upon KA-induced status epilepticus. Our results demonstrate a beneficial role of microglia in suppressing both acute and chronic seizures, suggesting that microglia are a potential therapeutic target for the management of epilepsy.

PLPP/CIN-mediated Mdm2 dephosphorylation increases seizure susceptibility via abrogating PSD95 ubiquitination

In the brain, murine double minute-2 (Mdm2), an E3-ubiquitin ligase, modulates neuronal excitability by regulating glutamate receptor and postsynaptic density 95 (PSD95) levels through ubiquitination. Thus, Mdm2 is relevant to epileptic seizures in human patients. Although phosphorylation at serine (S) 166 site by AKT increases Mdm2 activity, phosphatases of Mdm2 have been still elusive. Here, we demonstrate the novel function of pyridoxal-5′-phosphate phosphatase/chronophin (PLPP/CIN) in Mdm2 dephosphorylation that may negatively regulate PSD95 ubiquitination. As compared to wild-type mice, PLPP/CIN knockout (PLPP/CIN−/−) mice showed the brief seizure activity and the higher Mdm2-S166 phosphorylation following kainic acid (KA) injection, independent of AKT activity. In addition, PLPP/CIN−/− mice demonstrated the increases in Mdm2-PSD95 binding and PSD95 ubiquitination, accompanied by the decreases in Mdm2 ubiquitination and PSD95-NR2A (a subunit of N-methyl-d-aspartate receptor) bindings. Human PLPP/CIN over-expressing transgenic (PLPP/CINTg) mice reversed these phenomena. In addition, Mdm2 knockdown abolished PSD95 ubiquitination and increased KA-induced seizure activity in PLPP/CIN−/− mice without affecting AKT activity and protein levels of p53 and neural precursor cell expressed developmentally down-regulated gene 4-like (NEDD4–2). Therefore, we suggest that PLPP/CIN may be a potential therapeutic target for epilepsy and Mdm2-associated neurological diseases.

Simultaneous calcium recordings of hippocampal CA1 and primary motor cortex M1 and their relations to behavioral activities in freely moving epileptic mice.

Epilepsy is a common neurological disorder characterized by recurrent epileptic seizures. The cause of most cases of epilepsy is unknown. Although changes of calcium events in a single brain region during seizures have been reported before, there have been few studies on relations between calcium events of two different brain regions and epileptic behaviors in freely moving mice. To analyze calcium events simultaneously recorded in hippocampal CA1 (CA1) and primary motor cortex M1 (M1), and to explore their relations to various epileptic behaviors in freely moving epileptic models. Epileptic models were induced by Kainic acid (KA), a direct agonist of glutamatergic receptor, on adult male C57/BL6J mice. Calcium events of neurons and glia in CA1 and M1 labeled by a calcium indicator dye were recorded simultaneously with a multi-channel fiber photometry system. Three typical types of calcium events associated with KA-induced seizures were observed, including calcium baseline-rising, cortical spreading depression (CSD) and calcium flashing with a steady rate. Our results showed that the calcium baseline-rising occurred in CA1 was synchronized with that in M1, but the CSD waves were not. However, synchronization of calcium flashing in the two areas was uncertain, because it was only detected in CA1. We also observed that different calcium events happened with different epileptic behaviors. Baseline-rising events were accompanied by clonus of forelimbs or trembling, CSD waves were closely related to head movements (15 out of 18, 6 mice). Calcium flashing occurred definitely with drastic convulsive motor seizures (CMS, 6 mice). The results prove that the synchronization of calcium event exists in CA1 and M1, and different calcium events are related with different seizure behaviors. Our results suggest that calcium events involve in the synchronization of neuralnetwork and behaviors in epilepsy.

Ameliorating effect of quercetin on epilepsy by inhibition of inflammation in glial cells.

Epilepsy is a prevalent neurological disorder and it is a significant health risk, affecting >50 million people worldwide. The development of novel and appropriate strategies is required for ameliorating the progression and/or limiting the detrimental consequences of epilepsy. In the current study, kainic acid (KA), a neurotoxin, was used to induce seizures in mice. The flavonoid quercetin has recently been reported to have neuroprotective effects. Therefore, the effects of quercetin on KA-induced epilepsy and the potential underlying molecular mechanisms were examined. It was noted that quercetin attenuated the KA-induced seizure score and proinflammatory cytokine production, including tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β), and activation of nuclear factor κB (NF-κB) in mice. Quercetin attenuated KA-induced proinflammatory cytokine (TNF-α and IL-1β) release from microglia cells, as well as activation of NF-κB and ionized calcium binding adapter molecule 1 in microglia cells. Therefore, quercetin inhibited KA-induced epilepsy by microglia cell inactivation and the production of NF-κB, TNF-α and IL-1β.

MicroRNA-451 Aggravates Kainic Acid-induced Seizure and Neuronal Apoptosis by Targeting GDNF

Epilepsy is a common and serious neurological disease that causes recurrent episodes, but its molecular mechanism remains unclear. Abnormal miRNA expression is associated with epilepsy, including miR-451. This research investigated the role of miR-451 in seizure and its detailed mechanism. The seizure mice model was induced by kainic acid (KA) injection to the right lateral cerebral ventricle. Behavioral changes in mice were observed and evaluated by the Racine Scale. The miR-451 knockout mice were established by adenovirus infection. The in vitro model was performed by miR-451 mimics transfected HEK-293 cells. The amount of neuronal death and morphological changes were evaluated by Nissl staining and H&E staining. The results showed that miR-451 is up regulated in KA-induced seizure models and miR- 451 knockout decreased the behavior score and improved the pathological changes of the hippocampus. Besides, MiR-451 knockout inhibited the apoptosis of hippocampal neurons. Bioinformatics studies have shown that glial cell line-derived neurotrophic factor (GDNF) is a target gene of miR-451. MiR-451 could negatively regulate the expression of GDNF. GDNF overexpression could reverse the effect of miR-451 on KA induced brain injury and neuronal apoptosis. This research demonstrates that miR-451 can affect the behavior of KA-induced epilepsy mice and hippocampal neuronal damage by regulating GDNF expression. The results would provide an experimental foundation for further research about the potential contribution of mi- RNAs to epilepsy pathophysiology.

Dexamethasone after early-life seizures attenuates increased susceptibility to seizures, seizure-induced microglia activation and neuronal injury later in life

Chronic epilepsy can begin with isolated early-life prolonged seizures followed by remission and the re-emergence of seizures later in life. Seizures are known to trigger a neuroinflammatory response to promote neuronal damage and increase the risk of epilepsy. We examined whether post-seizure anti-inflammatory treatment with dexamethasone after early-life seizures could decrease future seizure susceptibility and ameliorate heightened microglia activation and cell injury in response to later-life seizures. Using a “two-hit” model, early-life seizures (SZ) were induced in rats on postnatal day (P) 25 by systemic kainic acid (KA) injection followed by later-life KA at P39. P25 animals were administered anti-inflammatory drugs for 2 or 7 days after first KA exposure to inhibit seizure-induced inflammation. Hippocampal microglial activation was measured after first or second KA treatments to assay neuroinflammation, and the latency and severity of seizures to the second KA treatment were measured to determine seizure susceptibility. In situ end labeling for DNA fragmentation was used to compare KA-induced neuronal injury between treatment groups after the second KA administration. KA-SZ at P25 caused marked microglia activation within 48 hours. At P39, KA-SZ in rats without prior seizures caused a modest (2-fold) increase in microglia assayed 72 hours after KA. In contrast, microglia were markedly activated (5-fold) in response to a second KA-SZ at P39. Short-course (2 days) dexamethasone significantly decreased seizure-induced microglia activation at P25, and ameliorated the exaggerated microglia activation, cell injury, and heightened susceptibility to second-hit seizures. Although short-course dexamethasone was effective, longer term (7 days) administration of dexamethasone resulted in decreased weight gain and increased mortality in animals with or without KA-induced seizures. These data indicated that acute short-term steroid therapy after SZ could inhibit seizure-induced microglia activation and decrease the long-term damaging effects of early-life SZ. These results further implicate seizure-induced inflammation and activation of innate immunity mediated by microglia in the pathogenesis of childhood epilepsy.