Epilepsy In Mice Research Articles

GLS2 reduces the occurrence of epilepsy by affecting mitophagy function in mouse hippocampal neurons.

Altered mitophagy has been observed in various neurological disorders, such as epilepsy. The role of mitophagy in causing neuronal damage during epileptic episodes is significant, and recent research has indicated that GLS2 plays a crucial role in regulating autophagy. However, exactly how GLS2 affects epilepsy is still unclear. To investigate the expression and distribution characteristics of GLS2 in epilepsy, and then observed the changes in behavior and electrophysiology caused by overexpression of GLS2 in epileptic mice, and determined whether GLS2 regulated seizure-like changes in the mouse model through the protective mechanism of mitophagy. The expression of GLS2 in a kainic acid (KA)-induced epileptic mouse model and aglutamate-inducedneuronal excitatory damage in HT22 cells model was downregulation. In brief, overexpression of GLS2 can alleviate epileptic activity. Subsequently, we demonstrated that GLS2 interacts with mitophagy-related proteins in a KA-induced epilepsy mouse model. Mechanistically, overexpression of GLS2 inhibited mitophagy in epileptic mice, downregulating the expression of LC3 and reducing ROS production. This study proves the GLS2 expression pattern is abnormal in epileptic mice. The function of mitophagy in hippocampal neurons is affected by GLS2, and overexpression of GLS2 can reduce the occurrence of seizure-like events (SLEs) by altering mitophagy function. Thus, GLS2 might control seizures, and our findings provide a fresh avenue for antiepileptic treatment and offer novel insights into treating and preventing epilepsy.

Preoptic area controls sleep-related seizure onset in a genetic epilepsy mouse model.

In genetic and refractory epileptic patients, seizure activity exhibits sleep-related modulation/regulation and sleep and seizure are intermingled. In this study, by using one het Gabrg2 Q390X KI mice as a genetic epilepsy model and optogenetic method in vivo , we found that subcortical POA neurons were active within epileptic network from the het Gabrg2 Q390X KI mice and the POA activity preceded epileptic (poly)spike-wave discharges(SWD/PSDs) in the het Gabrg2 Q390X KI mice. Meanwhile, as expected, the manipulating of the POA activity relatively altered NREM sleep and wake periods in both wt and the het Gabrg2 Q390X KI mice. Most importantly, the short activation of epileptic cortical neurons alone did not effectively trigger seizure activity in the het Gabrg2 Q390X KI mice. In contrast, compared to the wt mice, combined the POA nucleus activation and short activation of the epileptic cortical neurons effectively triggered or suppressed epileptic activity in the het Gabrg2 Q390X KI mice, indicating that the POA activity can control the brain state to trigger seizure incidence in the het Gabrg2 Q390X KI mice in vivo. In addition, the suppression of POA nucleus activity decreased myoclonic jerks in the Gabrg2 Q390X KI mice. Overall, this study discloses an operational mechanism for sleep-dependent seizure incidence in the genetic epilepsy model with the implications for refractory epilepsy. This operational mechanism also underlies myoclonic jerk generation, further with translational implications in seizure treatment for genetic/refractory epileptic patients and with contribution to memory/cognitive deficits in epileptic patients.

SRF Facilitates Transcriptional Inhibition of Gem Expression by m6A Methyltransferase METTL3 to Suppress Neuronal Damage in Epilepsy.

A bioinformatics analysis was conducted to screen for relevant expression datasets of the transcription factor SRF knockout mice. The aim was to investigate the relationship between SRF and m6A-related genes, predict how SRF regulates the m6A modification of GEM genes mediated by METTL3, and explore potential molecular mechanisms associated with neurotrauma. Disease gene databases such as GeneCards, DisGeNET, and Phenolyzer, and transcription factor databases TFDB and TRRUST, were used to obtain epilepsy-related genes and transcription factors. The intersection was then selected. Expression data of SRF knockout epilepsy mice were obtained from the GEO database and used to filter differentially expressed genes. Important module genes related to the disease were selected through WGCNA co-expression analysis. The intersection between these genes and the differentially expressed genes was performed, followed by PPI network analysis and GO/KEGG enrichment analysis. Furthermore, the core genes were selected using the cytoHubba plugin of the Cytoscape software. Differential expression analysis was performed on m6A-related factors in the GEO dataset, and the relationship between SRF and m6A-related factors and core genes was analyzed. The m6A binding sites of SRF with the METTL3 promoter and target gene Gem were predicted using the AnimalTFDB and SRAMP websites, respectively. We found that the transcription factor SRF may be a key gene in epilepsy during neuronal development. Further WGCNA analysis showed that 129 module genes were associated with SRF knockout epilepsy, and these differentially expressed genes were mainly enriched in the neuroactive ligand-receptor interaction pathway. The final results indicate that knocking out SRF may inhibit the transcription of METTL3, thereby inhibiting the m6A modification of Gem and leading to upregulation of Gem expression, thereby playing an important role in neuronal damage. Knocking out the SRF gene may inhibit the transcription of m6A methyltransferase METTL3, thereby inhibiting the m6A modification of GEM genes mediated by METTL3, promoting GEM gene expression, and leading to the occurrence of epilepsy-related neuron injury. Further investigation revealed that SRF overexpression can potentially enhance the transcription of METTL3, thus promoting m6A modification of GEM, resulting in downregulation of GEM expression. This process regulates oxidative stress in epileptic mouse neurons, suppresses inflammatory responses, and mitigates associated damage. Additionally, an in vitro neuronal epileptic model was established, and experimental techniques such as qRT-PCR and WB were employed to assess the expression of SRF, METTL3, and GEM in hippocampal tissues and neurons. The experimental results were consistent with our predictions, demonstrating that overexpression of SRF can inhibit the development of epilepsy-related neuronal damage. This study reveals that knockout of the SRF gene may suppress the transcription of m6A methyltransferase METTL3, thereby inhibiting m6A modification of the GEM gene mediated by METTL3 and subsequently promoting the expression of the GEM gene, leading to the occurrence of epilepsy-related neuronal damage.

Profile of miRNA expression in the hippocampus of epileptic mice and the prediction of potential therapeutic targets.

Epilepsy is a common neurological disease. Increasing evidence has highlighted the role of miRNAs in the molecular mechanisms underlying the development of neurological diseases such as epilepsy. In this study, we established a lithium chloride-pilocarpine epilepsy mouse model, performed miRNA sequencing of hippocampal tissue samples, and compared the obtained miRNA expression profile with that of normal control mice to determine differences in expression levels. We found that 55 miRNAs were differentially expressed in status epilepticus mice compared with normal control mice, with 38 upregulated and 17 downregulated miRNAs. Through subsequent analysis of the five downregulated miRNAs (mmu-let-7a-1-3p, mmu-let-7a-2-3p, mmu-let-7c-5p, mmu-let-7d-5p, and mmu-let-7e-5p) with the most significant differences in expression, the key pathways involved included the MAPK signaling pathway and focal adhesion, among others. Therefore, we believe that let-7 family miRNAs may be potential therapeutic targets for epilepsy.

Cannabinoid receptor 2 agonist AM1241 alleviates epileptic seizures and epilepsy-associated depression via inhibiting neuroinflammation in a pilocarpine-induced chronic epilepsy mouse model

Increasing evidence suggests that cannabinoid receptor 2 (CB2R) serves as a promising anti-inflammatory target. While inflammation is known to play crucial roles in the pathogenesis of epilepsy, the involvement of CB2R in epilepsy remains unclear. This study aimed to investigate the effects of a CB2R agonist, AM1241, on epileptic seizures and depressive-like behaviors in a mouse model of chronic epilepsy induced by pilocarpine. A chronic epilepsy mouse model was established by intraperitoneal administration of pilocarpine. The endogenous cannabinoid system (eCBs) in the hippocampus was examined after status epilepticus (SE). Animals were then treated with AM1241 and compared with a vehicle-treated control group. Additionally, the role of the AMPK/NLRP3 signaling pathway was explored using the selective AMPK inhibitor dorsomorphin. Following SE, CB2R expression increased significantly in hippocampal microglia. Administration of AM1241 significantly reduced seizure frequency, immobility time in the tail suspension test, and neuronal loss in the hippocampus. In addition, AM1241 treatment attenuated microglial activation, inhibited pro-inflammatory polarization of microglia, and suppressed NLRP3 inflammasome activation in the hippocampus after SE. Further, the therapeutic effects of AM1241 were abolished by the AMPK inhibitor dorsomorphin. Our findings suggest that CB2R agonist AM1241 may alleviate epileptic seizures and its associated depression by inhibiting neuroinflammation through the AMPK/NLRP3 signaling pathway. These results provide insight into a novel therapeutic approach for epilepsy.

Withaferin A protects against epilepsy by promoting LCN2-mediated astrocyte polarization to stopping neuronal ferroptosis

BackgroundEpilepsy is among the most frequent severe brain diseases, with few treatment options available. Neuronal ferroptosis is an important pathogenic mechanism in epilepsy. As a result, addressing ferroptosis appears to be a promising treatment approach for epilepsy. Withaferin A (WFA) is a C28 steroidal lactone that has a broad range of neuroprotective properties. Nonetheless, the antiepileptic action of WFA and the intrinsic mechanism by which it inhibits ferroptosis following epilepsy remain unknown. PurposeThis study aimed at investigating to the antiepileptic potential of WFA in epilepsy, as well as to propose a potential therapeutic approach for epilepsy therapy. MethodsWe conducted extensive research to examine the impacts of WFA on epilepsy and ferroptosis, using the kainic acid (KA)-treated primary astrocyte as an in vitro model and KA-induced temporal lobe epilepsy mice as an in vivo model. To analyze the neuroprotective effects of WFA on epileptic mice, electroencephalogram (EEG) recording, Nissl staining, and neurological function assessments such as the Morris water maze (MWM) test, Y-maze test, Elevated-plus maze (O-maze) test, and Open field test were used. Furthermore, the mechanism behind the neuroprotective effect of WFA in epilepsy was investigated using the transcriptomics analysis and verified on epileptic patient and epileptic mouse samples using Western blotting (WB) and immunofluorescence (IF) staining. In addition, WB, IF staining and specific antagonists/agonists were used to investigate astrocyte polarization and the regulatory signaling pathways involved. More critically, ferroptosis was assessed utilizing lipocalin-2 (LCN2) overexpression cell lines, siRNA knockdown, JC-1 staining, WB, IF staining, flow cytometry, electron microscopy (TEM), and ferroptosis-related GSH and MDA indicators. ResultsIn this study, we observed that WFA treatment reduced the number of recurrent seizures and time in seizure, and the loss of neurons in the hippocampal area in in epileptic mice, and even improved cognitive and anxiety impairment after epilepsy in a dose depend. Furthermore, WFA treatment was proven to enhance to the transformation of post-epileptic astrocytes from neurotoxic-type A1 to A2 astrocytes in both in vivo and in vitro experiments by inhibiting the phosphoinositide 3-kinase /AKT signaling pathway. At last, transcriptomics analysis in combination with functional experimental validation, it was discovered that WFA promoted astrocyte polarity transformation and then LCN2 in astrocytes, which inhibited neuronal ferroptosis to exert neuroprotective effects after epilepsy. In addition, we discovered significant astrocytic LCN2 expression in human TLE patient hippocampal samples. ConclusionsTaken together, for the first, our findings suggest that WFA has neuroprotective benefits in epilepsy by modulating astrocyte polarization, and that LCN2 may be a novel potential target for the prevention and treatment of ferroptosis after epilepsy.

Screening for biomarkers of tuberous sclerosis complex-associated epilepsy: a bioinformatics analysis.

The optimal biomarkers for early diagnosis, treatment, and prognosis of tuberous sclerosis complex (TSC)-associated epilepsy are not yet clear. This study identifies the crucial genes involved in the pathophysiology of TSC-associated epilepsy via a bioinformatics analysis. These genes may serve as novel therapeutic targets. Gene chip data sets (GSE62019 and GSE16969) comprising the data of patients with TSC-associated epilepsy and healthy control participants were obtained from the Gene Expression Omnibus (GEO) database. Differentially expressed genes (DEGs) in the GEO database were identified using the GEO2R gene expression analysis tool. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway, Gene Ontology function, and protein-protein interaction (PPI) network analyses were then conducted. The results were analyzed using R language, and are presented in volcano plots, Venn diagrams, heatmaps, and enrichment pathway bubble charts. A gene set enrichment analysis (GSEA), was conducted to examine the KEGG pathways and crucial genes linked to TSC-associated epilepsy. The potential genes were compared with the genes listed in the Online Mendelian Inheritance in Man (OMIM) database and analyzed against the literature to determine their clinical significance. Finally, the expression of the key genes in the TSC-associated epilepsy mice cerebral cortex was examined through immunohistochemical staining. The intersection of the GSE62019 and GSE16969 data sets revealed 151 commonly upregulated DEGs. The KEGG enrichment analysis indicated that these DEGs affected the occurrence and development of TSC-associated epilepsy by modulating complement and coagulation cascades, glycosaminoglycans in cancer, and extracellular matrix-receptor interactions. Four high-scoring clusters emerged, and podoplanin (PDPN) was identified as a key gene through the construction of a PPI network of the common DEGs using the Cytoscape software. A GSEA of the DEGs revealed that the common DEG PDPN was enriched in both data sets in pathways related to platelet activation, aggregation, and the glycoprotein VI (GPVI)-mediated activation cascade. Immunohistochemical staining revealed a significant elevation in PDPN expression in the cerebral cortex of mice with TSC-associated epilepsy. Conversely, the control group mice did not display any significantly positive neurons. The discovery of these crucial genes and signaling pathways extends understanding of the molecular processes underlying the development of TSC-associated epilepsy. Additionally, our findings may provide a theoretical basis for research into targeted clinical treatments.

CRISPR/Cas9-mediated neuronal deletion of 5-lipoxygenase alleviates deficits in mouse models of epilepsy

IntroductionOur previous work reveals a critical role of activation of neuronal Alox5 in exacerbating brain injury post seizures. However, whether neuronal Alox5 impacts the pathological process of epilepsy remains unknown. ObjectivesTo prove the feasibility of neuron-specific deletion of Alox5 via CRISPR-Cas9 in the blockade of seizure onset and epileptic progression. MethodsHere, we employed a Clustered regularly interspaced short-palindromic repeat-associated proteins 9 system (CRISPR/Cas9) system delivered by adeno-associated virus (AAV) to specifically delete neuronal Alox5 gene in the hippocampus to explore its therapeutic potential in various epilepsy mouse models and possible mechanisms. ResultsNeuronal depletion of Alox5 was successfully achieved in the brain. AAV delivery of single guide RNA of Alox5 in hippocampus resulted in reducing seizure severity, delaying epileptic progression and improving epilepsy-associated neuropsychiatric comorbidities especially anxiety, cognitive deficit and autistic-like behaviors in pilocarpine- and kainic acid-induced temporal lobe epilepsy (TLE) models. In addition, neuronal Alox5 deletion also reversed neuron loss, neurodegeneration, astrogliosis and mossy fiber sprouting in TLE model. Moreover, a battery of tests including analysis of routine blood test, hepatic function, renal function, routine urine test and inflammatory factors demonstrated no noticeable toxic effect, suggesting that Alox5 deletion possesses the satisfactory biosafety. Mechanistically, the anti-epileptic effect of Alox5 deletion might be associated with reduction of glutamate level to restore excitatory/inhibitory balance by reducing CAMKII-mediated phosphorylation of Syn ISer603. ConclusionOur findings showed the translational potential of AAV-mediated delivery of CRISPR-Cas9 system including neuronal Alox5 gene for an alternative promising therapeutic approach to treat epilepsy.

IL-17A Aggravated Blood-Brain Barrier Disruption via Activating Src Signaling in Epilepsy Mice.

Inflammation is an important pathogenic driving force in the genesis and development of epilepsy. The latest researches demonstrated that IL-17A mediated blood-brain barrier (BBB) dysfunction through disruption of tight junction protein expression. To investigate whether IL-17A is involved in BBB disruption after acute seizure attack, the pilocarpine model was established with C57BL/6J (wild type, WT) and IL-17R-deficient mice in vivo and with primary cultured rat brain microvascular endothelial cells in vitro. The mortality rate and brain water content were evaluated at 24h after status epilepticus, and IL-17A concentration, endothelial tight junction, adherens junction proteins, and albumin leakage were assessed at 0h, 4h, 12h, and 24h after status epilepticus (SE). IL-17R-deficient mice showed lessen severity of epilepsy than WT mice, accompanied by less albumin leakage, reduced brain water content, decreased IL-17A, and upregulated expression of target proteins (ZO-1, Occludin and VE-cadherin). IL-17R knockout abrogated abnormal upregulation of Src kinase and phosphorylated Src kinase in the setting of SE, and Src kinase inhibitor PP1 abrogated IL-17A-induced SE related endothelial injury in vitro. In conclusion, IL-17A inhibition might be a promising therapeutic option to attenuate endothelial cell injury and further BBB disruption by reducing Src kinase activation.

METTL14/YTHDC1-Mediated m6A Modification in Hippocampus Improves Pentylenetetrazol-Induced Acute Seizures.

Epilepsy is a common neurological disorder which can cause significant morbidity and mortality. N6-methyladenosine (m6A), the most common chemical epigenetic modification among mRNA post-transcriptional modifications, implicated in various physiological and pathological processes, but its role in epilepsy is still unknown. Here, we provide strong evidences in support of an association of m6A and its regulatory proteins with epilepsy. Our results indicated that the level of m6A was declined significantly in the dentate gyrus (DG) of hippocampus of pentylenetetrazol (PTZ)-induced seizure mice. Both the seizure-like behaviors and the excessive activation of DG area neuron were significantly mitigated after the administration of m6A agonist betaine. Mechanically, we found that both the m6A methyltransferase METTL14 and recognition protein YTHDC1 were decreased by PTZ stimulation, which might contribute to the reduced m6A level. Additionally, DG-specific over-expression of METTL14 or YTHDC1 by lentivirus injection could significantly ameliorate seizure-like behaviors and prevent the excessive activation of neuron in epilepsy mice induced by PTZ injection, which might be due to the normalized m6A level. Together, this study identified that METTL14/YTHDC1-mediated m6A modification could participate in seizure-like behaviors, which might provide m6A regulation as a potential and novel therapeutic strategy for epilepsy.

Gut microbiota modification by diosgenin mediates antiepileptic effects in a mouse model of epilepsy.

Diosgenin, a natural steroid saponin, holds promise as a multitarget therapeutic for various diseases, including neurodegenerative conditions. Its efficacy in slowing Alzheimer's disease, Parkinson's disease, multiple sclerosis, and stroke progression has been demonstrated. However, the role of diosgenin in anti-epilepsy and its potential connection to the modulation of the intestinal microbiota remain poorly understood. In this study, exogenous diosgenin significantly mitigated pentylenetetrazole (PTZ)-induced seizures, learning and memory deficits, and hippocampal neuronal injury. 16S ribosomal RNA (16S rRNA) sequencing revealed a reversal in the decrease of Bacteroides and Parabacteroides genera in the PTZ-induced mouse epileptic model following diosgenin treatment. Fecal microbiota transplantation (FMT) experiments illustrated the involvement of diosgenin in modulating gut microbiota and providing neuroprotection against epilepsy. Our results further indicated the repression of enteric glial cells (EGCs) activation and the TLR4-MyD88 pathway, coupled with reduced production of inflammatory cytokines in the colonic lumen, and improved intestinal barrier function in epilepsy mice treated with diosgenin or FMT. This study suggests that diosgenin plays a role in modifying gut microbiota, contributing to the alleviation of intestinal inflammation and neuroinflammation, ultimately inhibiting epilepsy progression in a PTZ-induced mouse model. Diosgenin emerges as a potential therapeutic option for managing epilepsy and its associated comorbidities.

Fecal transfers from children on the ketogenic diet mimic the anti-seizure effect in mice

The ketogenic diet (KD) mediates its anti-seizure effect through the gut microbiota in epilepsy mouse models.1 Lum etal.2 demonstrated that fecal microbiota from children with epilepsy treated with the KD decreases seizure susceptibility in mice after transfer.

MCC950 alleviates seizure severity and angiogenesis by inhibiting NLRP3/ IL-1β signaling pathway-mediated pyroptosis in mouse model of epilepsy

Epilepsy is one of the most common serious chronic brain disorders, affecting up to 70 million people worldwide. Vascular disruption, including blood–brain barrier impairment and pathological angiogenesis, exacerbates its occurrence. However, its underlying mechanisms remain elusive. MCC950 is a specific small-molecule inhibitor that selectively blocks NLRP3 inflammatory vesicle activation across the blood–brain barrier, limits downstream IL-1β maturation and release, and exerts therapeutic effects across multiple diseases. In the present study, an epilepsy model was established by intraperitoneal administration of Kainic acid to adult male C57BL/6J wild-type mice. The results revealed that the epilepsy susceptibility of MCC950-treated mice was decreased, and neural damage following seizure episodes was reduced. In addition, immunofluorescence staining, RT-qPCR, and Western blot demonstrated that MCC950 inhibited the expression of the NLRP3 inflammasome and its related proteins in microglia, whereas microangiogenesis was found to be increased in the cerebral cortex and hippocampus of epileptic mice, and these effects could be reversed by MCC950. Furthermore, neurobehavioral impairment was observed in the epileptic mouse model, and MCC950 similarly alleviated the aforementioned pathological process. To the best of our knowledge, this is the first study to establish that pathological microangiogenesis is associated with NLRP3/IL-1β signaling pathway activation in a Kainic acid-induced epilepsy mouse model and that MCC950 administration attenuates the above-mentioned pathological changes and exerts neuroprotective effects. Therefore, MCC950 is a promising therapeutic agent for the treatment of epilepsy.

Closed‐Loop Optogenetic Modulation of Neural Activities in Freely Moving Mice with Multimodal Fibers

AbstractThe precise manipulation of neural activity is of great significance to reveal the function of complex neural circuits in brain, especially the mechanism of neurological disorders such as epilepsy, Parkinson's, and Alzheimer's disease. In the past decades, optogenetics has become a powerful tool to investigate the function of neural circuits due to its unique advantages of cell specificity and high spatiotemporal resolution. However, to date, most optogenetic manipulation has been conducted in an open‐loop fashion. The closed‐loop neural modulation is still an important task. Here, a closed‐loop optogenetic modulation system is developed for ultra‐fast, ‐accurate, and ‐stable manipulation of neural activities in behaving mice. Specifically, this system is mediated by a functional fiber probe with excellent low optical loss (0.052 dB cm−1 at 589 nm) and electrical impedance (5.40 MΩ µm−2 at 1 kHz). In vivo electrophysiology and behavioral experiments demonstrated that the system can be successfully applied for the identification and intervention of seizures in epilepsy mice with high efficiency. It is envision that the closed‐loop manipulation system has promising applications in brain science, neuroscience, and neuromodulation therapies.

Black Phosphorus Flake-Enabled Wireless Neuromodulation for Epilepsy Treatment.

Epilepsy is a prevalent and severe neurological disorder and generally requires prolonged electrode implantation and tether brain stimulation in refractory cases. However, implants may cause potential chronic immune inflammation and permanent tissue damage due to material property mismatches with soft brain tissue. Here, we demonstrated a nanomaterial-enabled near-infrared (NIR) neuromodulation approach to provide nongenetic and nonimplantable therapeutic benefits in epilepsy mouse models. Our study showed that crystal-exfoliated photothermal black phosphorus (BP) flakes could enhance neural activity by altering the membrane capacitive currents in hippocampus neurons through NIR photothermal neuromodulation. Optical stimulation facilitated by BP flakes in hippocampal slices evoked action potentials with a high spatiotemporal resolution. Furthermore, BP flake-enabled NIR neuromodulation of hippocampus neural circuits can suppress epileptic signals in epilepsy model mice with minimal invasiveness and high biocompatibility. Consequently, nanomaterial-enabled NIR neuromodulation may open up opportunities for nonimplantable optical therapy of epilepsy in nontransgenic organisms.

Decrease of Cellular Communication Network Factor 1 (CCN1) Attenuates PTZ-Kindled Epilepsy in Mice.

To investigate the molecular mechanism of communication network factor 1 (CCN1) regulating pentylenetetrazol (PTZ)-induced epileptogenesis, deepen the understanding of epilepsy seizure pathogenesis, and provide new drug action targets for its clinical prevention and treatment. Differentially expressed genes (DEGs) on microarrays GSE47516 and GSE88992 were analyzed online using GEO2R. Pathway enrichment and protein-protein interaction network (PPI) analysis of DEGs were carried out using Metascape. Brain tissue samples of severe traumatic brain injury patients (named Healthy group) and refractory epilepsy patients (named Epilepsy group) were obtained and analyzed by qRT-PCR and immunohistochemistry (IHC) staining. A PTZ-induced epilepsy mouse model was established and verified. Morphological changes of neurons in mouse brain tissue were detected using hematoxylin and eosin (HE) staining. qRT-PCR was conducted to detect the mRNA expressions of apoptosis-associated proteins Bax, Caspase-3 and bcl2. TUNEL staining was performed to detect brain neuron apoptosis. The levels of myocardial enzymology, GSH, MDA and ROS in blood of mouse were detected by biochemical assay. CCN1 expression was increased in epilepsy brain tissue samples. CCN1 decreasing effectively prolongs seizure incubation period and decreases seizure duration. Silencing of CCN1 also reduces neuronal damage and apoptosis, decreases mRNA and protein expression of proapoptotic proteins Bax and Caspase-3, increases mRNA expression of antiapoptotic protein Bcl2. Moreover, decrease of CCN1 decreases myocardial enzymatic indexes CK and CK-MB levels, reduces myocardial tissue hemorrhage, and relieves oxidative stress response in hippocampal and myocardial tissue. CCN1 expression is increased in epileptic samples. CCN1 decreasing protects brain tissue by attenuating oxidative stress and inhibiting neuronal apoptosis triggered by PTZ injection, which probably by regulating Nrf2/HO-1 pathway.

Anti-inflammatory effects of icariin in the acute and chronic phases of the mouse pilocarpine model of epilepsy

Neuroinflammation mediated by microglia made a significant contribution in the pathophysiology of epilepsy. Icariin (ICA), a bioactive ingredient isolated from Epimedium, has been shown to present both antioxidant and anti-inflammatory properties. This study was to explore the potential therapeutic effects of icariin on mouse pilocarpine model of epilepsy and its underlying mechanisms in vivo and in vitro. To this end, we firstly measured the serum concentrations of the proinflammatory cytokines IL-1β and IL-6 from patients with temporal lobe epilepsy and found that patients with a higher seizure frequency showed correspondingly higher inflammatory reaction. Mouse pharmacokinetic study, transmembrane transportation assay, and cell viability assay collectively demonstrated that ICA was able to cross the blood-brain barrier and has good biocompatibility. The acute and chronic epilepsy models were next established in a pilocarpine mouse model of acquired epilepsy. Icariin has been identified that it could cross the blood-brain barrier and enter the hippocampus to exhibit therapeutic effects. ICA treatment dramatically promoted microglial polarization to the M2 phenotype in epilepsy mice both in the acute and chronic phases. Reduced release of M1-associated proinflammatory factors, such as IL-1β and IL-6, corroborates the altered glial cell polarization. Furthermore, ICA alleviated seizure intensity and mortality in acute phase epileptic mice. Models in the chronic group also showed improved general condition, cognition ability, and memory function after ICA treatment. Taken together, our research strongly suggested that icariin has the potential to treat epilepsy via inhibiting neuroinflammation by promoting microglial polarization to the M2 phenotype.

In Vivo Evaluation of Self-assembled nano-Saikosaponin-a for Epilepsy Treatment.

Saikosaponin-a (SSa) exhibits antiepileptic effects. However, its poor water solubility and inability to pass through the blood-brain barrier greatly limit its clinical development and application. In this study, SSa-loaded Methoxy poly (ethylene glycol)-poly(ε-caprolactone) (MePEG-SSa-PCL) NPs were successfully prepared and characterized. Our objective was to further investigate the effect of this composite on acute seizure in mice. First, we confirmed the particle size and surface potential of the composite (51.00 ± 0.25nm and - 33.77 ± 2.04 mV, respectively). Further, we compared the effects of various MePEG-SSa-PCL doses (low, medium, and high) with those of free SSa, valproic acid (VPA - positive control), and saline only (model group) on acute seizure using three different acute epilepsy mouse models. We observed that compared with the model group, the three MePEG-SSa-PCL treatments showed significantly lowered seizure frequency in mice belonging to the maximum electroconvulsive model group. In the pentylenetetrazol and kainic acid (KA) acute epilepsy models, MePEG-SSa-PCL increased both clonic and convulsion latency periods and shortened convulsion duration more effectively than equivalent SSa-only doses. Furthermore, hematoxylin-eosin and Nissl staining revealed considerably less neuronal damage in the hippocampal CA3 area of KA mice in the SSa, VPA, and three MePEG-SSa-PCL groups relative to mice in the model group. Hippocampal gamma-aminobutyric acid-A (GABA-A) receptor and cleaved caspase-3 expression levels in KA mice were significantly higher and lower, respectively, in the three MePEG-SSa-PCL treatment groups than in the model group. Thus, MePEG-SSa-PCL exhibited a more potent antiepileptic effect than SSa in acute mouse epilepsy models and could alleviate neuronal damage in the hippocampus following epileptic seizures, possibly via GABA-A receptor expression upregulation.

Mechanism of Robo1 in the pentylenetetrazol-kindled epilepsy mouse model.

The neural network hypothesis is one of the important pathogenesis of drug-resistant epilepsy. Axons guide molecules through synaptic remodeling and brain tissue remodeling, which may result in the formation of abnormal neural networks. Therefore, axon guidance plays a crucial role in disease progression. However, although Robo1 is one of the important components of axon guidance, the role of Robo1 in epilepsy remains unclear. In this study, we aimed to explore the mechanism of Robo1 in epilepsy. Male adult C57BL/6 mice were intraperitoneally injected with pentylenetetrazol to establish an epilepsy model. Lentivirus (LV) was given via intracranial injection 2 weeks before pentylenetetrazol injection. Different expressions of Robo1 between the control group, LV-mediated Robo1 short hairpin RNA group, empty vector control LV group, and normal saline group were analyzed using Western blot, immunofluorescence staining, Golgi staining, and video monitoring. Robo1 was increased in the hippocampus in the pentylenetetrazol-induced epilepsy mouse model; lentiviral Robo1 knockdown prolonged the latency of seizure and reduced the seizure grade in mice and resulted in a decrease in dendritic spine density, while the number of mature dendritic spines was maintained. We speculate that Robo1 has been implicated in the development and progression of epilepsy through its effects on dendritic spine morphology and density. Epileptic mice with Robo1 knockdown virus intervention had lower seizure grade and longer latency. Follow-up findings suggest that Robo1 may modulate seizures by affecting dendritic spine density and morphology. Downregulation of Robo1 may negatively regulate epileptogenesis by decreasing the density of dendritic spines and maintaining a greater number of mature dendritic spines.

Inhibition of ANXA2 activity attenuates epileptic susceptibility and GluA1 phosphorylation.

Annexin A2 (ANXA2) participates in the pathology of a variety of diseases. Nevertheless, the impact of ANXA2 on epilepsy remains to be clarified. Hence, the study aimed at investigating the underlying role of ANXA2 in epilepsy through behavioral, electrophysiological, and pathological analyses. It was found that ANXA2 was markedly upregulated in the cortical tissues of temporal lobe epilepsy patients (TLE), kainic acid (KA)-induced epilepsy mice, and in a seizure-like model in vitro. ANXA2 silencing in mice suppressed first seizure latency, number of seizures, and seizure duration in behavioral analysis. In addition, abnormal brain discharges were less frequent and shorter in the hippocampal local field potential (LFP) record. Furthermore, the results showed that the frequency of miniature excitatory postsynaptic currents was decreased in ANXA2 knockdown mice, indicating that the excitatory synaptic transmission is reduced. Co-immunoprecipitation (COIP) experiments demonstrated that ANXA2 interacted with the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) subunit GluA1. Moreover, ANXA2 knockdown decreased GluA1 expression on the cell surface and its phosphorylation onserine 831 and serine 845, related to the decreased phosphorylation levels mediated by protein kinases A and C (PKA and PKC). This study covers a previously unknown and key function of ANXA2 in epilepsy. These findings indicate that ANXA2 can regulate excitatory synaptic activity mediated by AMPAR subunit GluA1 to improve seizure activity, which can provide novel insights for the treatment and prevention of epilepsy.