KA-induced Status Epilepticus Research Articles

Parvalbumin Regulates GAD Expression through Calcium Ion Concentration to Affect the Balance of Glu-GABA and Improve KA-Induced Status Epilepticus in PV-Cre Transgenic Mice.

Aims: the study aimed to (i) use adeno-associated virus technology to modulate parvalbumin (PV) gene expression, both through overexpression and silencing, within the hippocampus of male mice and (ii) assess the impact of PV on the metabolic pathway of glutamate and γ-aminobutyric acid (GABA). Methods: a status epilepticus (SE) mouse model was established by injecting kainic acid into the hippocampus of transgenic mice. When the seizures of mice reached SE, the mice were killed at that time point and 30 min after the onset of SE. Hippocampal tissues were extracted and the mRNA and protein levels of PV and the 65 kDa (GAD65) and 67 kDa (GAD67) isoforms of glutamate decarboxylase were assessed using real-time quantitative polymerase chain reaction and Western blot, respectively. The concentrations of glutamate and GABA were detected with high-performance liquid chromatography (HPLC), and the intracellular calcium concentration was detected using flow cytometry. Results: we demonstrate that the expression of PV is associated with GAD65 and GAD67 and that PV regulates the levels of GAD65 and GAD67. PV was correlated with calcium concentration and GAD expression. Interestingly, PV overexpression resulted in a reduction in calcium ion concentration, upregulation of GAD65 and GAD67, elevation of GABA concentration, reduction in glutamate concentration, and an extension of seizure latency. Conversely, PV silencing induced the opposite effects. Conclusion: parvalbumin may affect the expression of GAD65 and GAD67 by regulating calcium ion concentration, thereby affecting the metabolic pathways associated with glutamate and GABA. In turn, this contributes to the regulation of seizure activity.

Small-molecule caspase-1 inhibitor CZL80 terminates refractory status epilepticus via inhibition of glutamatergic transmission.

Status epilepticus (SE), a serious and often life-threatening medical emergency, is characterized by abnormally prolonged seizures. It is not effectively managed by present first-line anti-seizure medications and could readily develop into drug resistance without timely treatment. In this study, we highlight the therapeutic potential of CZL80, a small molecule that inhibits caspase-1, in SE termination and its related mechanisms. We found that delayed treatment of diazepam (0.5 h) easily induces resistance in kainic acid (KA)-induced SE. CZL80 dose-dependently terminated diazepam-resistant SE, extending the therapeutic time window to 3 h following SE, and also protected against neuronal damage. Interestingly, the effect of CZL80 on SE termination was model-dependent, as evidenced by ineffectiveness in the pilocarpine-induced SE. Further, we found that CZL80 did not terminate KA-induced SE in Caspase-1-/- mice but partially terminated SE in IL1R1-/- mice, suggesting the SE termination effect of CZL80 was dependent on the caspase-1, but not entirely through the downstream IL-1β pathway. Furthermore, in vivo calcium fiber photometry revealed that CZL80 completely reversed the neuroinflammation-augmented glutamatergic transmission in SE. Together, our results demonstrate that caspase-1 inhibitor CZL80 terminates diazepam-resistant SE by blocking glutamatergic transmission. This may be of great therapeutic significance for the clinical treatment of refractory SE.

MiR129-5p-loaded exosomes suppress seizure-associated neurodegeneration in status epilepticus model mice by inhibiting HMGB1/TLR4-mediated neuroinflammation

BackgroundNeuroinflammation contributes to both epileptogenesis and the associated neurodegeneration, so regulation of inflammatory signaling is a potential strategy for suppressing epilepsy development and pathological progression. Exosomes are enriched in microRNAs (miRNAs), considered as vital communication tools between cells, which have been proven as potential therapeutic method for neurological disease. Here, we investigated the role of miR129-5p-loaded mesenchymal stem cell (MSC)-derived exosomes in status epilepticus (SE) mice model.MethodsMice were divided into four groups: untreated control (CON group), kainic acid (KA)-induced SE groups (KA group), control exosome injection (KA + Exo-con group), miR129-5p-loaded exosome injection (KA + Exo-miR129-5p group). Hippocampal expression levels of miR129-5p, HMGB1, and TLR4 were compared among groups. Nissl and Fluoro-jade B staining were conducted to evaluate neuronal damage. In addition, immunofluorescence staining for IBA-1 and GFAP was performed to assess glial cell activation, and inflammatory factor content was determined by ELISA. Hippocampal neurogenesis was assessed by BrdU staining.ResultsThe expression of HMGB1 was increased after KA-induced SE and peaking at 48 h, while hippocampal miR129-5p expression decreased in SE mice. Exo-miR129-5p injection reversed KA-induced upregulation of hippocampal HMGB1 and TLR4, alleviated neuronal damage in the hippocampal CA3, reduced IBA-1 + and GFAP + staining intensity, suppressed SE-associated increases in inflammatory factors, and decreased BrdU + cell number in dentate gyrus.ConclusionsExosomes loaded with miR129-5p can protect neurons against SE-mediated degeneration by inhibiting the pro-inflammatory HMGB1/TLR4 signaling axis.Graphical

The effects of Src tyrosine kinase inhibitor, saracatinib, on the markers of epileptogenesis in a mixed-sex cohort of adult rats in the kainic acid model of epilepsy.

Neurodegeneration and neuroinflammation are key processes of epileptogenesis in temporal lobe epilepsy (TLE). A considerable number (∼30%) of patients with epilepsy are resistant to currently available antiseizure drugs and thus there is a need to develop adjunct therapies to modify disease progression. A vast majority of interventional strategies to treat TLE have utilized males which limits the translational nature of the studies. In this study, we investigated the effects of repeated low-dose kainic acid (KA) injection on the initial status epilepticus (SE) and the effects of Src kinase inhibitor, saracatinib (SAR/AZD0530; 20 mg/kg, oral, daily for 7 days), in a mixed-sex cohort of adult Sprague Dawley rats during early epileptogenesis. There were no sex differences in response to KA-induced SE, and neither did the stage of estrus influence SE severity. KA-induced SE caused significant astrogliosis and microgliosis across the hippocampus, piriform cortex, and amygdala. SAR treatment resulted in a significant reduction of microgliosis across brain regions. Microglial morphometrics such as branch length and the endpoints strongly correlated with CD68 expression in the vehicle-treated group but not in the SAR-treated group, indicating mitigation by SAR. KA-induced SE caused significant neuronal loss, including parvalbumin-positive inhibitory neurons, in both vehicle (VEH) and SAR-treated groups. SAR treatment significantly mitigated FJB-positive neuronal counts as compared to the VEH group. There was an increase in C3-positive reactive astrocytes in the VEH-treated group, and SAR treatment significantly reduced the increase in the piriform cortex. C3-positive astrogliosis significantly correlated with CD68 expression in the amygdala (AMY) of VEH-treated rats, and SAR treatment mitigated this relationship. There was a significant increase of pSrc(Y419)-positive microglia in both KA-treated groups with a statistically insignificant reduction by SAR. KA-induced SE caused the development of classical glial scars in the piriform cortex (PIR) in both KA-treated groups, while SAR treatment led to a 42.17% reduction in the size of glial scars. We did not observe sex differences in any of the parameters in this study. SAR, at the dose tested in the rat kainate model for a week in this study mitigated some of the markers of epileptogenesis in both sexes.

Comparative Analysis of Anticonvulsant Activity of Trans and Cis 5,5'-Diphenylhydantoin Schiff Bases.

Recently, the four 5,5'-diphenylhydantoin Schiff bases, possessing different aromatic species (SB1-Ph, SB2-Ph, SB3-Ph and SB4-Ph) were synthesized, characterized, and evaluated for anticonvulsant activity in combination with phenytoin. In the present study, the SB1-Ph and SB4-Ph compounds were selected, based on their anticonvulsant potency, and compared with their cis isomers, prepared after a one-hour exposure to the UV source, for their anticonvulsant potency in the maximal electroshock (MES) test and the kainate (KA)-induced status epilepticus (SE) test in mice. In the MES test, the cisSB1-Ph compound exhibited superior to phenytoin and trans isomer activity in the three tested doses, while the cisSB4-Ph compound entirely suppressed the electroshock-induced seizure spread at the highest dose of 40 mg/kg. Pretreatment with the cisSB1-Ph compound and the cisSB4-Ph at the doses of 40 mg/kg, respectively, for seven days, significantly attenuated the severity of KA SE compared to the matched control group pretreated with a vehicle, while phenytoin was ineffective in this test. The cisSB4-Ph but not the cisSB1-Ph demonstrated an antioxidant effect against the KA-induced SE in the hippocampus. Our results suggest that trans-cis conversion of 5,5'-diphenylhydantoin Schiff bases has potential against seizure spread in the MES test and mitigated the KA-induced SE. The antioxidant potency of cisSB4-Ph might be associated with its efficacy in mitigating the SE.

CA3 Principal Cell Activation Triggers Hypersynchronous-Onset Seizures in a Mouse Model of Mesial Temporal Lobe Epilepsy.

Mesial temporal lobe epilepsy (MTLE) is the most common form of focal epilepsy and it is characterized by seizures that are often refractory to medications. Seizures in MTLE have two main patterns of onset that have been termed hypersynchronous (HYP) and low-voltage fast (LVF), and are believed to mainly depend on the activity of excitatory principal cells and inhibitory interneurons, respectively. In this study, we investigated whether unilateral open loop optogenetic activation of CaMKII-positive principal cells in the hippocampus CA3 region favors the generation of spontaneous HYP seizures in kainic acid-treated (KA) CaMKII-ChR2 mice. Optogenetic activation of CA3 principal cells (1 Hz, 180 s ON, 220 s OFF) was implemented for 15 days after KA-induced status epilepticus. We found that both LVF and HYP seizures occurred in non-stimulated CaMKII-ChR2 (n= 6) and stimulated CaMKII-Cre (n= 5) mice. In contrast, optogenetic activation of principal cells in CaMKII-ChR2 mice (n= 5), triggered only HYP seizures that were characterized by high fast ripple (250-500 Hz) rates during the pre-ictal and ictal period. These results provide firm evidence that in MTLE spontaneous seizures in MTLE with different onset patterns depend on distinct neuronal network mechanisms of generation. They also demonstrate that HYP seizures occurring in vivo along with their associated fast ripples depend on the activity of principal cells in the CA3 region.

The Apelin/APJ system modulates seizure activity and endocytosis of the NMDA receptor GluN2B subunit

In the central nervous system (CNS), the apelin/APJ system is broadly expressed. According to some studies, activation of this system protects against excitotoxicity mediated by N-methyl-D-aspartate (NMDA) receptors and exerts neuroprotective effects. However, the role of this system in epilepsy remains unclear. In the present study, immunofluorescence staining and western blotting were used to assess APJ localization and expression in the brains of mice with recurrent spontaneous seizures induced by kainic acid (KA). Behavior and local field potentials (LFPs) were assessed in mice with KA-induced seizures. Susceptibility to seizures was assessed in a pentylenetetrazole (PTZ)-induced seizure model. Whole-cell patch-clamp recordings were used to evaluate the role of the apelin/APJ system in regulating synaptic transmission in brain slices from mice in which Mg2+-free medium was used to induce seizures. NMDA receptor GluN2B subunit expression and phosphorylation of GluN2B at Ser1480 were measured in the mouse hippocampus. APJ was primarily localized in neurons, and its expression was upregulated in the epileptic brain. APJ activation after KA-induced status epilepticus (SE) reduced epileptic activity, whereas APJ inhibition aggravated epileptic activity. In the PTZ model, APJ activation reduced and APJ inhibition increased susceptibility to seizures. The apelin/APJ system affected NMDA receptor-mediated postsynaptic currents in patch-clamp recordings. Moreover, APJ regulated the levels of GluN2B phosphorylated at Ser1480 and the abundance of cell-surface GluN2B in neurons. Furthermore, endocytosis of the NMDA receptor GluN2B subunit was regulated by the apelin/APJ system. Together, our findings indicate that the apelin/APJ system modulates seizure activity and may be a novel therapeutic target for epilepsy.

Riluzole and novel naphthalenyl substituted aminothiazole derivatives prevent acute neural excitotoxic injury in a rat model of temporal lobe epilepsy

Epileptogenic seizures, or status epilepticus (SE), leads to excitotoxic injury in hippocampal and limbic neurons in the kainic acid (KA) animal model of temporal lobe epilepsy (TLE). Here, we have further characterized neural activity regulated methylaminoisobutryic acid (MeAIB)/glutamine transport activity in mature rat hippocampal neurons in vitro that is inhibited by riluzole (IC50 = 1 μM), an anti-convulsant benzothiazole agent. We screened a library of riluzole derivatives and identified SKA-41 followed by a second screen and synthesized several novel chlorinated aminothiazoles (SKA-377, SKA-378, SKA-379) that are also potent MeAIB transport inhibitors in vitro, and brain penetrant following systemic administration. When administered before KA, SKA-378 did not prevent seizures but still protected the hippocampus and several other limbic areas against SE-induced neurodegeneration at 3d. When SKA-377 - 379, (30 mg/kg) were administered after KA-induced SE, acute neural injury in the CA3, CA1 and CA4/hilus was also largely attenuated. Riluzole (10 mg/kg) blocks acute neural injury. Kinetic analysis of SKA-378 and riluzoles’ blockade of Ca2+-regulated MeAIB transport in neurons in vitro indicates that inhibition occurs via a non-competitive, indirect mechanism. Sodium channel NaV1.6 antagonism blocks neural activity regulated MeAIB/Gln transport in vitro (IC50 = 60 nM) and SKA-378 is the most potent inhibitor of NaV1.6 (IC50 = 28 μM) compared to NaV1.2 (IC50 = 118 μM) in heterologous cells. However, pharmacokinetic analysis suggests that sodium channel blockade may not be the predominant mechanism of neuroprotection here. Riluzole and our novel aminothiazoles are agents that attenuate acute neural hippocampal injury following KA-induced SE and may help to understand mechanisms involved in the progression of epileptic disease.

Silver nanoparticles synthesized using Tualang honey ameliorate seizures, locomotor activity, and memory function in KA-induced status epilepticus in male rats

Introduction: In recent years, green synthesis of silver nanoparticles using natural products has been increasingly utilized in the biomedical field as a therapeutic approach for managing neurodegenerative diseases. This study aimed to determine the effects of silver nanoparticles synthesized using Tualang honey (THSN) on seizure activity and locomotor and memory functions in rats after kainic acid (KA) induction. Methods: Male Sprague Dawley rats were randomly divided into six groups (n = 6/group), and each group was pre-treated orally with either distilled water or THSN (10 mg/kg or 50 mg/kg), according to their respective groups. Each rat was injected subcutaneously with KA (15 mg/kg) or saline after the last pre-treatment, and the onset of the first generalized seizure was recorded. After 24 hours and five days of KA induction, an open field test (OFT) and a novel object recognition test (NORT) were performed before they were sacrificed. Results: THSN pre-treatment of KA-induced status epilepticus groups demonstrated an increment in latencies to the onset of the first generalized seizure and the number of line crossings in OFT, with a higher recognition index of NORT compared to the untreated KA-induced status epilepticus group. Conclusion: THSN could have neuroprotective effects in ameliorating seizures, locomotor activity, and memory function after KA-induced status epilepticus in male rats.

Reactive microglia are the major source of tumor necrosis factor alpha and contribute to astrocyte dysfunction and acute seizures in experimental temporal lobe epilepsy.

Extensive microglia reactivity has been well described in human and experimental temporal lobe epilepsy (TLE). To date, however, it is not clear whether and based on which molecular mechanisms microglia contribute to the development and progression of focal epilepsy. Astroglial gap junction coupled networks play an important role in regulating neuronal activity and loss of interastrocytic coupling causally contributes to TLE. Here, we show in the unilateral intracortical kainate (KA) mouse model of TLE that reactive microglia are primary producers of tumor necrosis factor (TNF)α and contribute to astrocyte dysfunction and severity of status epilepticus (SE). Immunohistochemical analyses revealed pronounced and persistent microglia reactivity, which already started 4 h after KA-induced SE. Partial depletion of microglia using a colony stimulating factor 1 receptor inhibitor prevented early astrocyte uncoupling and attenuated the severity of SE, but increased the mortality of epileptic mice following surgery. Using microglia-specific inducible TNFα knockout mice we identified microglia as the major source of TNFα during early epileptogenesis. Importantly, microglia-specific TNFα knockout prevented SE-induced gap junction uncoupling in astrocytes. Continuous telemetric EEG recordings revealed that during the first 4 weeks after SE induction, microglial TNFα did not significantly contribute to spontaneous generalized seizure activity. Moreover, the absence of microglial TNFα did not affect the development of hippocampal sclerosis but attenuated gliosis. Taken together, these data implicate reactive microglia in astrocyte dysfunction and network hyperexcitability after an epileptogenic insult.

Modulating the gut microbiota ameliorates spontaneous seizures and cognitive deficits in rats with kainic acid-induced status epilepticus by inhibiting inflammation and oxidative stress.

IntroductionEpilepsy is a highly prevalent neurological disease whose treatment has always been challenging. Hence, it is crucial to explore the molecular mechanisms underlying epilepsy inhibition. Inflammation and oxidative stress are important pathophysiological changes in epilepsy that contribute to the development of spontaneous seizures and cognitive deficits. In recent years, altered gut microbiota composition was found to be involved in epilepsy, but the underlying mechanism remains unclear. Modulation of the gut microbiota showed a positive impact on the brain by regulating oxidative stress and inflammation. Hence, this study evaluated the effect of modulating gut dysbiosis by treating epileptic rats with prebiotics, probiotics, and synbiotics and investigated the underlying molecular mechanism.Materials and methodsEpileptic rat models were established by injecting 1 μl of kainic acid (KA, 0.4 μg/μl) into the right amygdalae. The rats were divided into Sham, KA, KA+prebiotic [inulin:1 g/kg body weight (bw)/day], KA+probiotics (10 × 109cfu of each bacteria/kg, bw/day), and KA+synbiotic groups (1:1 mixture of prebiotics and probiotics). Seizures were monitored, and cognitive function was assessed in all rats. Biochemical indicators, namely, oxidative stress, DNA damage, glutamate levels, and inflammation markers, were also determined.ResultsThe KA-induced status epilepticus (SE) rats exhibited spontaneous seizures and cognitive deficits. This was accompanied by the activation of glial cells, the inflammatory response (IL-1 β, IL-6, and TNF-α), lipid peroxidation (MDA), DNA damage (8-OHdG), the release of glutamate, and a decline in total antioxidant ability (GSH). These changes were alleviated by partial treatment with prebiotics, probiotics, and synbiotics.ConclusionModulating gut dysbiosis ameliorates spontaneous seizures and cognitive deficits in rats with KA-induced status epilepticus. The underlying mechanism may potentially involve the inhibition of inflammation and oxidative stress.

The complement C3-C3aR pathway mediates microglia-astrocyte interaction following status epilepticus.

Gliosis is a histopathological characteristic of epilepsy that comprises activated microglia and astrocytes. It is unclear whether or how crosstalk occurs between microglia and astrocytes in the evolution of epilepsy. Here, we report in a mouse model of status epilepticus, induced by intracerebroventricular injection of kainic acid (KA), sequential activation of microglia and astrocytes and their close spatial interaction in the hippocampal CA3 region. Microglial ablation reduced astrocyte activation and their upregulation of complement C3. When compared to wild-type mice, both C3-/- and C3aR-/- mice had significantly less microglia-astrocyte interaction in response to KA-induced status epilepticus. Additionally, KA-injected C3-/- mice had significantly less histochemical evidence of neurodegeneration. The results suggest that the C3-C3aR pathway contributes to KA-induced neurodegeneration by mediating microglia-astrocyte communication. The C3-C3aR pathway may prove to be a potential therapeutic target for epilepsy treatment.

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.

Transcranial direct current stimulation alleviates seizure severity in kainic acid-induced status epilepticus rats

Status epilepticus (SE) is a state of prolonged and repeated seizures that can lead to permanent brain damage or life-threatening conditions. Transcranial direct current stimulation (tDCS) non-invasively provides a polarity-specific electric current to modulate brain excitability. Little is known about the therapeutic potential of tDCS in SE. Here, we aim to determine the tDCS effects on seizure severity, EEG and post-SE consequences in rats with kainic acid (KA)-induced SE. Rats were subjected to cathodal tDCS or sham stimulation over the dorsal hippocampus for 5 days. KA was intraperitoneally injected to induce SE. We used continuous video-EEG recording to monitor seizure activity, immunostaining and Timm staining to evaluate neuron counts and mossy fiber sprouting, and ELISA for Brain-derived neurotrophic factor (BDNF) protein measurement. Two featured EEG patterns, gamma ranged high-frequency polyspikes and low-frequency spike-and-wave complexes, were identified in the hippocampal CA1 of KA-induced SE rats. tDCS elicited a significant decrease in severe seizures of Racine stages 4–5 in KA-induced SE rats. tDCS-treated rats manifested diminished high-frequency oscillation during SE, decreased chronic spontaneous spike activities and mossy fiber sproutings compared to sham. tDCS-treated rats also exhibited significantly lower hippocampal BDNF protein levels than sham immediately and 4 weeks after SE. A positive correlation between the hippocampal BDNF level and the seizure severity of SE was found. Altogether, our results show that repeated cathodal tDCS can mitigate seizure severity, alter ictal EEG pattern and reduce the chronic adverse consequences in KA-induced SE rats, supporting the therapeutic potential of tDCS in severe prolonged epileptic seizures.

The effects of ammonia stimulation on kainate-induced status epilepticus and anterior piriform cortex electrophysiology

ObjectiveStrong olfactory stimulation (OS) with such substances as toluene or ammonia has been reported to suppress seizures. We aimed to investigate the role of ammonia stimulation on acute kainic acid (KA)-induced seizures. We also investigated any possible effects of ammonia stimulation on the electrophysiology of the anterior piriform cortex (APC). MethodsAdult male Sprague–Dawley rats were implanted with bilateral hippocampal electrodes and an electrode in the left APC. Animals were exposed to either distilled water (control) or ammonia stimulation for 20 s every 5 min during KA induction of status epilepticus (SE). The electroencephalogram (EEG) was analyzed for seizure frequency, duration, severity, and total KA doses given prior to reaching SE. Seizure-free EEG epochs that coincided with OS were chosen and analyzed via wavelet analysis for any spectral changes. ResultsWe found no significant differences in seizure frequency, duration, severity, or administered KA doses before SE between the groups. In the experimental group, a wavelet analysis of variance (WANOVA) revealed a significant stimulation-induced increase of power in the delta and alpha bands prior to the first KA injection and higher power in the delta and theta bands after KA injection. ConclusionsWhereas the spectral analysis of the APC revealed specific OS-induced changes, our findings suggest that OS with ammonia does not result in altering the threshold of attaining KA-induced SE. This does not rule out a potential role for OS in reducing recurrent seizures in the KA or other epilepsy models.

Succinate accumulation induces mitochondrial reactive oxygen species generation and promotes status epilepticus in the kainic acid rat model

Though succinate accumulation is associated with reactive oxygen species (ROS) production and neuronal injury, which play critical roles in epilepsy, it is unclear whether succinate accumulation contributes to the onset of epilepsy or seizures. We sought to investigate changes in succinate, oxidative stress, and mito-SOX levels, as well as mitophagy and neuronal change, in different status epilepticus (SE) rat models. Our results demonstrate that KA-induced SE was accompanied by increased levels of succinate, oxidative stress, and mito-SOX, as well as mitophagy and neuronal degeneration. The similarly increased levels of succinate, oxidative stress, and mito-SOX were also found in pilocarpine-induced SE. Moreover, the reduction of succinate accumulation by the inhibition of succinate dehydrogenase (SDH), malate/aspartate shuttle (MAS), or purine nucleotide cycle (PNC) served to reduce succinate, oxidative stress, and mito-SOX levels, thereby preventing oxidative stress-related neuronal damage and lessening seizure severity. Interestingly, simulating succinate accumulation with succinic acid dimethyl ester may induce succinate accumulation and increased oxidative stress and mito-SOX levels, as well as behavior and seizures in electroencephalograms similar to those observed in rats exposed to KA. Our results indicate that succinate accumulation may contribute to the increased oxidative stress/mitochondrial ROS levels, neuronal degeneration, and SE induced by KA administration. Furthermore, we found that succinate accumulation was mainly due to the inverse catalysis of SDH from fumarate, which was supplemented by the MAS and PNC pathways. These results reveal new insights into the mechanisms underlying SE and that reducing succinate accumulation may be a clinically useful therapeutic target in SE.

Role of Elevated Thrombospondin-1 in Kainic Acid-Induced Status Epilepticus.

Previous studies have suggested that thrombospondin-1 (TSP-1) regulates the transforming growth factor beta 1 (TGF-β1)/phosphorylated Smad2/3 (pSmad2/3) pathway. Moreover, TSP-1 is closely associated with epilepsy. However, the role of the TSP-1-regulated TGF-β1/pSmad2/3 pathway in seizures remains unclear. In this study, changes in this pathway were assessed following kainic acid (KA)-induced status epilepticus (SE) in rats. The results showed that increases in the TSP-1/TGF-β1/pSmad2/3 levels spatially and temporally matched the increases in glial fibrillary acidic protein (GFAP)/chondroitin sulfate (CS56) levels following KA administration. Inhibition of TSP-1 expression by small interfering RNA or inhibition of TGF-β1 activation with a Leu-Ser-Lys-Leu peptide significantly reduced the severity of KA-induced acute seizures. These anti-seizure effects were accompanied by decreased GFAP/CS56 expression and Smad2/3 phosphorylation. Moreover, inhibiting Smad2/3 phosphorylation with ponatinib or SIS3 also significantly reduced seizure severity, alongside reducing GFAP/CS56 immunoreactivity. These results suggest that the TSP-1-regulated TGF-β1/pSmad2/3 pathway plays a key role in KA-induced SE and astrogliosis, and that inhibiting this pathway may be a potential anti-seizure strategy.

Xenon exerts anti-seizure and neuroprotective effects in kainic acid-induced status epilepticus and neonatal hypoxia-induced seizure

Xenon is an inhalation anesthetic with a favorable safety profile, and previous studies have demonstrated its neuroprotective efficacy. However, whether xenon plays a role in the treatment of epilepsy or seizure remains unclear. This study aimed to investigate the role of xenon inhalation and explore the role of different xenon ratio gradients and different delayed treatment times in seizure models. Kainic acid (KA)-induced status epilepticus and neonatal hypoxia-induced seizure models were used in our study. Animals were subject to inhalation of xenon mixture for 60 min after the stimulation used to induce seizures. The control group was treated with 70% nitrogen/30% oxygen, as in previous reports. Behavioral changes, electroencephalography, neuronal injury, and learning and memory function were investigated in each group. The results indicate that xenon mixture significantly reduced the severity of seizures and neurodegeneration in both KA-induced status epilepticus and in neonatal mice with hypoxia-induced seizure. Moreover, treatment with different percentages of xenon (35%, 50%, or 70%), as well as at different intervention time points (immediately, delayed for 15 min, delayed for 30 min) after hypoxia induction significantly attenuated the severity of seizure and neuronal injury. Additionally, 50% or 70% xenon treatment, as well as immediate xenon treatment or with a delay of 15 min attenuated the learning and memory impairments induced by hypoxia. This study confirmed that xenon mixture exerts strong inhibitive effects in seizure and seizure-induced neuronal injury and defects of cognitive function. Moreover, the results suggest that intervention time window and percentage of xenon influence the efficacy of the xenon treatment. Our study supports that xenon inhalation represents a safe means to inhibit seizures and neuronal injury.

Mode-Dependent Effect of Xenon Inhalation on Kainic Acid-Induced Status Epilepticus in Rats

Previous studies have reported the possible neuroprotective effects of xenon treatment. The purpose of this study was to define the range of effective xenon ratio, most effective xenon ratio, and time-window for intervention in the kainic acid (KA) – induced status epilepticus (SE) rat model. Different ratios of xenon (35% xenon, 21% oxygen, 44% nitrogen, 50% xenon, 21% oxygen, 29% nitrogen, 70% xenon, 21% oxygen, and 9% nitrogen) were used to treat the KA-induced SE. Our results confirmed the anti-seizure role of 50 and 70% xenon mixture, with a stronger effect from the latter. Further, 70% xenon mixture was dispensed at three time points (0 min, 15 min delayed, and 30 min delayed) after KA administration, and the results indicated the anti-seizure effect at all treated time points. The results also established that the neuronal injury in the hippocampus and entorhinal cortex (EC), assessed using Fluoro-Jade B (FJB) staining, were reversed by the xenon inhalation, and within 30 min after KA administration. Our study, therefore, indicates the appropriate effective xenon ratio and time-window for intervention that can depress seizures. The prevention of neuronal injury and further reversal of the loss of effective control of depress network in the hippocampus and EC may be the mechanisms underlying the anti-seizure effect of xenon.

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.