MiR-23b-3p Improves Brain Damage after Status Epilepticus by Reducing the Formation of Pathological High-Frequency Oscillations via Inhibition of cx43 in Rat Hippocampus.

In order to investigate the effectiveness and safety of miR-23b-3p in anti-seizure activity and to elucidate the regulatory relationship between miR-23b-3p and Cx43 in the nervous system, we have established a lithium chloride-pilocarpine (PILO) status epilepticus (SE) model. Rats were randomly divided into the following groups: seizure control (PILO), valproate sodium (VPA+PILO), recombinant miR-23b-3p overexpression (miR+PILO), miR-23b-3p sponges (Sponges+PILO), and scramble sequence negative control (Scramble+PILO) (n = 6/group). After experiments, we got the following results. In the acute phase, the time required for rats to reach stage IV after PILO injection was significantly longer in VPA+PILO and miR+PILO. In the chronic phase after SE, the frequency of spontaneous recurrent seizures (SRSs) in VPA+PILO and miR+PILO was significantly reduced. At 10 min before seizure cessation, the average energy expression of fast ripples (FRs) in VPA+PILO and miR+PILO was significantly lower than in PILO. After 28 days of seizure, Cx43 expression in PILO was significantly increased, and Beclin1expression in all groups was significantly increased. After 28 days of SE,the number of synapses in the CA1 region of the hippocampus was significantly higher in the VPA+PILO and miR+PILO groups compared to that in the PILO group. After 28 days of SE ,hippocampal necrotic cells in the CA3 region were significantly lower in the VPA+PILO and miR+PILO groups compared to those in the PILO group. There were no significant differences in biochemical indicators among the experimental group rats 28 days after SE compared to the seizure control group. Based on the previous facts, we can reach the conclusion that MiR-23b-3p targets and blocks the expression of hippocampal Cx43 which can reduce the formation of pathological FRs, thereby alleviating the severity of seizures, improving seizure-induced brain damage.

Frequency-dependent seizure-suppressing effects of optogenetic activation of septal inhibitory cells in mesial temporal lobe epilepsy.

Mesial temporal lobe epilepsy (MTLE) is characterized by recurring focal seizures that arise from limbic areas and are often refractory to pharmacological interventions. We have reported that optogenetic stimulation of PV-positive cells in the medial septum at 0.5 Hz exerts seizure-suppressive effects. Therefore, we compared here these results with those obtained by optogenetic stimulation of medial septum PV-positive neurons at 8 Hz in male PV-ChR2 mice (P60-P100) undergoing an initial, pilocarpine-induced status epilepticus (SE). Optogenetic stimulation (5 min ON, 10 min OFF) was performed from day 8 to day 12 after SE at a frequency of 8 Hz (n = 6 animals) or 0.5 Hz (n = 8 animals). Surprisingly, in both groups, no effects were observed on the occurrence of interictal spikes and interictal high frequency oscillations (HFOs). However, 0.5 Hz stimulation induced a significant decrease of seizure occurrence (p < 0.05). Such anti-ictogenic effect was not observed in the 8 Hz protocol that instead triggered seizures (p < 0.05); these seizures were significantly longer under optogenetic stimulation compared to when optogenetic stimulation was not implemented (p < 0.05). Analysis of ictal HFOs revealed that in the 0.5 Hz group, but not in the 8 Hz group, seizures occurring under optogenetic stimulation were associated with significantly lower rates of fast ripples compared to when optogenetic stimulation was not performed (p < 0.05). Our results indicate that activation of GABAergic PV-positive neurons in the medial septum exerts seizure-suppressing effects that are frequency-dependent and associated with low rates of fast ripples. Optogenetic activation of medial septum PV-positive neurons at 0.5 Hz is efficient in blocking seizures in the pilocarpine model of MTLE, an effect that did not occur with 8 Hz stimulation.

Dingxian pill alleviates hippocampal neuronal apoptosis in epileptic mice through TNF-α/TNFR1 signaling pathway inhibition.

ETHNOPHARMACOLOGICAL RELEVANCE: Dingxian Pill (DXP), a famous traditional Chinese medicine prescription, and has been widely proven to have positive therapeutic effects on "Xianzheng" (the name of epilepsy in ancient China). However, the anti-epileptic molecular mechanisms of DXP are not yet fully understood and remain to be further investigated. AIM OF THE STUDY: To elucidate the molecular mechanism of DXP's improvement in epileptic neuronal loss, damage and apoptosis by regulating TNF-α/TNFR1 signaling pathway. MATERIALS AND METHODS: Sixty Kunming mice were randomly divided in 6 groups: control group (equal volume of normal saline), model group (180 mg kg-1 pilocarpine hydrochloride - used to establish the epilepsy animal model), carbamazepine group (30 mg kg-1), and low, medium, and high-dose Dingxian Pill groups (4.08, 8.16, and 16.32 g kg-1, respectively - oral administration once daily for 2 weeks). Successful establishment of the epileptic mouse model was monitored with electroencephalography. Pathological changes in hippocampal tissue were analyzed with hematoxylin-eosin staining. Hippocampal neuronal apoptosis was analyzed with TUNEL staining. TNF-α, TNFR1, TRADD, FADD, and caspase-8 mRNA and protein expression levels in hippocampal tissue were analyzed with real-time quantitative polymerase chain reaction, immunohistochemistry, and Western blot, respectively. Cleaved caspase-8 protein levels in hippocampal tissue were measured with immunohistochemistry and Western blot. RESULTS: Compared to control, the model group showed an increase in continuous epileptic discharge waves on EEG, a damaged hippocampal neuron morphological structure, increased hippocampal neuronal apoptosis, and significantly increased TNF-α, TNFR1, TRADD, FADD, and caspase-8 mRNA and protein levels, and increased caspase-8 cleavage (P < 0.05). Compared to the model group, the carbamazepine group as well as the low-, medium-, and high-dose Dingxian Pill groups showed decreased epileptic discharges on EEG, an obvious hippocampal neuron morphological structure restoration, varying degrees of attenuated hippocampal neuronal apoptosis, and significantly decreased TNF-α, TNFR1, TRADD, FADD, and caspase-8 mRNA and protein levels as well as decreased caspase-8 cleavage (P < 0.05). CONCLUSIONS: Dingxian Pill exerts an anti-epileptic effect through inhibition of TNF-α/TNFR1 signaling pathway-mediated apoptosis in hippocampal neurons.

Rat strain differences in seizure frequency and hilar neuron loss after systemic treatment with pilocarpine.

At least 3 months after systemic treatment with pilocarpine to induce status epilepticus, Long-Evans and Sprague-Dawley rats were video-EEG monitored for seizures continuously for 1 month. Rats were then perfused, hippocampi were processed for Nissl staining, and hilar neurons were quantified. Seizure frequency in Long-Evans rats was 1/10th of that in Sprague-Dawley rats, and more variable. Hilar neuron loss was also less severe in Long-Evans rats. However, there was no correlation between hilar neuron loss and seizure frequency in either strain. The low and variable seizure frequency suggests limited usefulness of pilocarpine-treated Long-Evans rats for some epilepsy experiments.

The latency to awake from induced-obstructive sleep apnea is reduced in rats with chronic epilepsy.

OSA is known to increase the risk for SUDEP in persons with epilepsy, but the relationship between these two factors is not clear. Also, there is no study showing the acute responses to obstructive apnea in a chronic epilepsy model. Therefore, this study aimed to characterize cardiorespiratory responses to obstructive apnea and chemoreceptor stimulation in rats. In addition, we analyzed respiratory centers in the brain stem by immunohistochemistry. Epilepsy was induced with pilocarpine. About 30-60 days after the first spontaneous seizure, tracheal and thoracic balloons, and electrodes for recording the electroencephalogram, electromyogram, and electrocardiogram were implanted. Intermittent apneas were made by inflation of the tracheal balloon during wakefulness, NREM sleep, and REM sleep. During apnea, respiratory effort increased, and heart rate fell, especially with apneas made during wakefulness, both in control rats and rats with epilepsy. Latency to awake from apnea was longer with apneas made during REM than NREM, but rats with epilepsy awoke more rapidly than controls with apneas made during REM sleep. Rats with epilepsy also had less REM sleep. Cardiorespiratory responses to stimulation of carotid chemoreceptors with cyanide were similar in rats with epilepsy and controls. Immunohistochemical analysis of Phox2b, tryptophan hydroxylase, and NK1 in brain stem nuclei involved in breathing and sleep (retrotrapezoid nucleus, pre-Bötzinger complex, Bötzinger complex, and caudal raphe nuclei) revealed no differences between control rats and rats with epilepsy. In conclusion, our study showed that rats with epilepsy had a decrease in the latency to awaken from apneas during REM sleep, which may be related to neuroplasticity in some other brain regions related to respiratory control, awakening mechanisms, and autonomic modulation.

Crack cocaine inhalation increases seizure susceptibility by reducing acetylcholinesterase activity.

Crack cocaine is a highly addictive and potent stimulant drug. Animal studies have shown that the cholinergic system plays a role in neurotoxicity induced by cocaine or its active metabolites inhalation. Behavioral alterations associated with crack cocaine use include hyperactivity, depressed mood, and decreased seizure threshold. Here we evaluate the acetylcholinesterase (AChE) and reactive oxygen species (ROS) activity, behavioral profile, and the threshold for epileptic seizures in rats that received intrahippocampal pilocarpine (H-PILO) followed by exposure to crack cocaine (H-PILO + CRACK). Animals exposed to H-PILO + CRACK demonstrated increased severity and frequency of limbic seizures. The AChE activity was reduced in the groups exposed to crack cocaine alone (CRACK) and H-PILO + CRACK, whereas levels of ROS remained unchanged. In addition, crack cocaine exposure increased vertical locomotor activity, without changing water and sucrose intake. Short-term memory consolidation remained unchanged after H-PILO, H-PILO + CRACK, and CRACK administration. Overall, our data suggest that crack cocaine inhalation reduced the threshold for epileptic seizures in rats submitted to low doses of pilocarpine through the inhibition of AChE. Taken together, our findings can be useful in the development of effective strategies for preventing and treating the harmful effects of cocaine and crack cocaine on the central nervous system.

Glycoprotein Non-metastatic Melanoma Protein B (GPNMB) Protects Against Neuroinflammation and Neuronal Loss in Pilocarpine-induced Epilepsy via the Regulation of Microglial Polarization.

Epilepsy is a progressive neurodegenerative disease highlighted by recurrent seizures, neuroinflammation, and the loss of neurons. Microglial dysfunction is commonly found in epileptic foci and contributes to neuroinflammation in the initiation and progression of epilepsy. Glycoprotein non-metastatic melanoma protein B (GPNMB), a transmembrane glycoprotein, has been involved in the microglial activation and neuroinflammation response. The present study investigated the functional significance of GPNMB in epilepsy. A proven model of epilepsy was established by intraperitoneal injection of pilocarpine to male Sprague Dawley rats. Lentivirus vectors carrying GPNMB or GPNMB short hairpin RNA (shGPNMB) were injected into the hippocampus to induce overexpression or knockdown of GPNMB. GPNMB expression was significantly upregulated and overexpression of GPNMB in the hippocampus reduced seizure activity and neuronal loss after status epilepticus (SE). We here focused on the effects of GPNMB deficiency on neuronal injury and microglia polarization 28 days after SE. GPNMB knockdown accelerated neuronal damage in the hippocampus, evidenced by increased neuron loss and neuronal cell apoptosis. Following GPNMB knockdown, M1 polarization (iNOS) and secretion of pro-inflammatory cytokines IL-6, IL-1ß, and TNF-α were increased, and M2 polarization (Arg1) and secretion of anti-inflammatory cytokines IL-4, IL-10, and TGF-ß were decreased. BV2 cells were used to further confirm the regulatory role of GPNMB in modulating phenotypic transformations and inflammatory cytokine expressions in microglia. In conclusion, these results indicated that GPNMB suppressed epilepsy through repression of hippocampal neuroinflammation, suggesting that GPNMB might be considered the potential neurotherapeutic target for epilepsy management and play a protective role against epilepsy by modulating the polarization of microglia.

Compartmental neuronal degeneration in the ventral striatum induced by status epilepticus in young rats' brain in comparison with adults.

According to experimental and clinical studies, status epilepticus (SE) causes neurodegenerative morphological changes not only in the hippocampus and other limbic structures, it also affects the thalamus and the neocortex. In addition, several studies reported atrophy, metabolic changes, and neuronal degeneration in the dorsal striatum. The literature lacks studies investigating potential neuronal damage in the ventral component of the striatopallidal complex (ventral striatum [VS] and ventral pallidum) in SE experimentations. To better understand the development of neuronal damage in the striatopallidal complex associated with SE, the detected neuronal degeneration in the compartments of the VS, namely, the nucleus accumbens (NAc) and the olfactory tubercle (OT), was analyzed. The experiments were performed on Wistar rats at age of 25-day-old pups and 3-month-old adult animals. Lithium-pilocarpine model of SE was used. Lithium chloride (3 mmol/kg, ip) was injected 24 h before administering pilocarpine (40 mg/kg, ip). This presented study demonstrates the variability of post SE neuronal damage in 25-day-old pups in comparison with 3-month-old adult rats. The NAc exhibited small to moderate number of Fluoro-Jade B (FJB)-positive neurons detected 4 and 8 h post SE intervals. The number of degenerated neurons in the shell subdivision of the NAc significantly increased at survival interval of 12 h after the SE. FJB-positive neurons were evidently more prominent occupying the whole anteroposterior and mediolateral extent of the nucleus at longer survival intervals of 24 and 48 h after the SE. This was also the case in the bordering vicinity between the shell and the core compartments but with clusters of degenerating cells. The severity of damage of the shell subdivision of the NAc reached its peak at an interval of 24 h post SE. Isolated FJB-positive neurons were detected in the ventral peripheral part of the core compartment. Degenerated neurons persisted in the shell subdivision of the NAc 1 week after SE. However, the quantity of cell damage had significantly reduced in comparison with the aforementioned shorter intervals. The third layer of the OT exhibited more degenerated neurons than the second layer. The FJB-positive cells in the young animals were higher than in the adult animals. The morphology of those cells was identical in the two age groups except in the OT.

Alterations in HCN1 expression and distribution during epileptogenesis in rats.

BACKGROUND: The hyperpolarization-activated cyclic nucleotide-gated cation channel (HCN1) is predominantly located in key regions associated with epilepsy, such as the neocortex and hippocampus. Under normal physiological conditions, HCN1 plays a crucial role in the excitatory and inhibitory regulation of neuronal networks. In temporal lobe epilepsy, the expression of HCN1 is decreased in the hippocampi of both animal models and patients. However, whether HCN1 expression changes during epileptogenesis preceding spontaneous seizures remains unclear. OBJECTIVE: The aim of this study was to determine whether the expression of HCN1 is altered during the epileptic prodromal phase, thereby providing evidence for its role in epileptogenesis. METHODS: We utilized a cobalt wire-induced rat epilepsy model to observe changes in HCN1 during epileptogenesis and epilepsy. Additionally, we also compared HCN1 alterations in epileptogenic tissues between cobalt wire- and pilocarpine-induced epilepsy rat models. Long-term video EEG recordings were used to confirm seizures development. Transcriptional changes, translation, and distribution of HCN1 were assessed using high-throughput transcriptome sequencing, total protein extraction, membrane and cytoplasmic protein fractionation, western blotting, immunohistochemistry, and immunofluorescence techniques. RESULTS: In the cobalt wire-induced rat epilepsy model during the epileptogenesis phase, total HCN1 mRNA and protein levels were downregulated. Specifically, the membrane expression of HCN1 was decreased, whereas cytoplasmic HCN1 expression showed no significant change. The distribution of HCN1 in the distal dendrites of neurons decreased. During the epilepsy period, similar HCN1 alterations were observed in the neocortex of rats with cobalt wire-induced epilepsy and hippocampus of rats with lithium pilocarpine-induced epilepsy, including downregulation of mRNA levels, decreased total protein expression, decreased membrane expression, and decreased distal dendrite expression. CONCLUSIONS: Alterations in HCN1 expression and distribution are involved in epileptogenesis beyond their association with seizure occurrence. Similarities in HCN1 alterations observed in epileptogenesis-related tissues from different models suggest a shared pathophysiological pathway in epileptogenesis involving HCN1 dysregulation. Therefore, the upregulation of HCN1 expression in neurons, maintenance of the HCN1 membrane, and distal dendrite distribution in neurons may represent promising disease-modifying strategies in epilepsy.

Effect of the closed-loop hippocampal low-frequency stimulation on seizure severity, learning, and memory in pilocarpine epilepsy rat model.

AIMS: In this study, the anticonvulsant action of closed-loop, low-frequency deep brain stimulation (DBS) was investigated. In addition, the changes in brain rhythms and functional connectivity of the hippocampus and prefrontal cortex were evaluated. METHODS: Epilepsy was induced by pilocarpine in male Wistar rats. After the chronic phase, a tripolar electrode was implanted in the right ventral hippocampus and a monopolar electrode in medial prefrontal cortex (mPFC). Subjects' spontaneous seizure behaviors were observed in continuous video recording, while the local field potentials (LFPs) were recorded simultaneously. In addition, spatial memory was evaluated by the Barnes maze test. RESULTS: Applying hippocampal DBS, immediately after seizure detection in epileptic animals, reduced their seizure severity and duration, and improved their performance in Barnes maze test. DBS reduced the increment in power of delta, theta, and gamma waves in pre-ictal, ictal, and post-ictal periods. Meanwhile, DBS increased the post-ictal-to-pre-ictal ratio of theta band. DBS decreased delta and increased theta coherences, and also increased the post-ictal-to-pre-ictal ratio of coherence. In addition, DBS increased the hippocampal-mPFC coupling in pre-ictal period and decreased the coupling in the ictal and post-ictal periods. CONCLUSION: Applying closed-loop, low-frequency DBS at seizure onset reduced seizure severity and improved memory. In addition, the changes in power, coherence, and coupling of the LFP oscillations in the hippocampus and mPFC demonstrate low-frequency DBS efficacy as an antiepileptic treatment, returning LFPs to a seemingly non-seizure state in subjects that received DBS.

Increased Cyclic Adenosine Monophosphate Responsive Element is Closely Associated with the Pathogenesis of Drug-resistant Epilepsy.

BACKGROUND: Drug-resistant epilepsy (DRE) is a refractory neurological disorder. There is ample evidence that suggest that γ-aminobutyric acid-a (GABAA) receptors could be one of the mechanisms responsible for the development of drug resistance in epilepsy. It is also known that the cAMP response element binding protein (CREB) plays a possible key role in the transcriptional regulation of GABAA. OBJECTIVE: This study explores the role of CREB in the development of DRE and the effect of CREB on GABA-related receptors in DRE. METHODS: The CREB expression was increased or decreased in the hippocampus of normal rats by lentiviral transfection, who then underwent the lithium-pilocarpine-induced epilepsy model. Phenobarbital (PB) sodium and carbamazepine (CBZ) were used to select a drug-resistant epileptic model. The expression levels of GABAA receptor α1, ß2, and γ2 subunits and CREB protein were measured in the rat hippocampus by western blot and fluorescent quantitative PCR. RESULTS: The frequency and duration of seizures increased in the overexpression group compared to that in the control group. In addition, the severity, frequency, and duration of seizures decreased in the group with decreased expression. The hippocampus analysis of the expression levels of the CREB protein and CREB mRNA yielded similar findings. Altering the CREB protein expression in the rat hippocampus could negatively regulate the expression and transcript levels of GABAA receptors α1, ß2, and γ2, suggesting that CREB may serve as a potential target for the development of treatment protocols and drugs for epilepsy. CONCLUSION: Our study shows that enhanced CREB expression promotes the development of DRE and negatively regulates GABAA receptor levels and that the inhibition of CREB expression may reduce the incidence of DRE.

Saikosaponin D mitigate pilocarpine-induced astrocyte injury by regulating the NLRP3/caspase-1 signaling pathway.

Studies have shown that saikosaponin D (SSD) has favorable neurotherapeutic effects. Therefore, the objective of this study was to explore the efficacy and possible molecular mechanisms of SSD on pilocarpine (PP)-induced astrocyte injury. Primary astrocytes were isolated from juvenile rats and identified using immunofluorescence. The cells were treated with PP and/or SSD for 6 h and 12 h, respectively, followed by measurement of their viability through 3-(4,5-dimethylthiazol)-2,5-diphenyl-tetrazolium bromide (MTT) assay. Next, quantitative real-time polymerase chain reaction (qRT-PCR) was used to measure the expression levels of Glial fibrillary acidic protein (GFAP), C3, S100 calcium binding protein A10 (S100a10), pentraxin 3 (Ptx3), toll-like receptor 4 (TLR4), and RAG in astrocytes after different treatments. Enzyme-linked immunosorbent assay and biochemical tests were utilized to evaluate the level of inflammatory factors [interleukin (IL)-1ß, IL-6, and tumor necrosis factor alpha (TNF-α)] secreted by cells and the content of oxidative stress-related factors (malondialdehyde [MDA] and glutathione [GSH]) or enzyme activity (catalase [CAT] and glutathione peroxidase [GPX]) in cells. The JC-1 mitochondrial membrane potential (MMP) fluorescence probe was used to measure the MMP in astrocytes. Additionally, western blot was applied to test the expression of proteins related to the nod-like receptor protein 3 (NLRP3)/caspase-1 signaling pathway. PP treatment (1 mM) induced cell injury by significantly reducing the viability of astrocytes and expression of cellular markers. SSD treatment (4 µM) had no toxicity to astrocytes. Besides, SSD (4 µM) treatment could significantly up-regulate the cell viability and marker expression of PP-induced astrocytes. Furthermore, SSD could be employed to inhibit inflammation (reduce IL-1ß, IL-6, and TNF-α levels) and oxidative stress (decrease MDA level, elevate GSH level, the activity of CAT and GPX), and ameliorate mitochondrial dysfunction (upregulate JC-1 ratio) in PP-induced astrocytes. Moreover, further mechanism exploration revealed that SSD treatment significantly reduced the activity of the NLRP3/caspase-1 signaling pathway activated by PP induction. SSD increased cell viability, inhibited inflammation and oxidative stress response, and ameliorated mitochondrial dysfunction in PP-induced astrocyte injury model, thus playing a neuroprotective role. The mechanism of SSD may be related to the inhibition of the NLRP3/caspase-1 inflammasome.

Neuroprotective effects of quinpirole on lithium chloride pilocarpine-induced epilepsy in rats and its underlying mechanisms.

INTRODUCTION: Epilepsy is a common neurological disorder that presents with challenging mechanisms and treatment strategies. This study investigated the neuroprotective effects of quinpirole on lithium chloride pilocarpine-induced epileptic rats and explored its potential mechanisms. METHODS: Lithium chloride pilocarpine was used to induce an epileptic model in rats, and the effects of quinpirole on seizure symptoms and cognitive function were evaluated. The Racine scoring method, electroencephalography, and Morris water maze test were used to assess seizure severity and learning and memory functions in rats in the epileptic group. Additionally, immunohistochemistry and Western blot techniques were used to analyze the protein expression levels and morphological changes in glutamate receptor 2 (GluR2; GRIA2), BAX, and BCL2 in the hippocampi of rats in the epileptic group. RESULTS: First, it was confirmed that the symptoms in rats in the epileptic group were consistent with features of epilepsy. Furthermore, these rats demonstrated decreased learning and memory function in the Morris water maze test. Additionally, gene and protein levels of GluR2 in the hippocampi of rats in the epileptic group were significantly reduced. Quinpirole treatment significantly delayed seizure onset and decreased the mortality rate after the induction of a seizure. Furthermore, electroencephalography showed a significant decrease in the frequency of the spike waves. In the Morris water maze test, rats from the quinpirole treatment group demonstrated a shorter latency period to reach the platform and an increased number of crossings through the target quadrant. Network pharmacology analysis revealed a close association between quinpirole and GluR2 as well as its involvement in the cAMP signaling pathway, cocaine addiction, and dopaminergic synapses. Furthermore, immunohistochemistry and Western blot analysis showed that quinpirole treatment resulted in a denser arrangement and a more regular morphology of the granule cells in the hippocampi of rats in the epileptic group. Additionally, quinpirole treatment decreased the protein expression of BAX and increased the protein expression of BCL2. CONCLUSION: The current study demonstrated that quinpirole exerted neuroprotective effects in the epileptic rat model induced by lithium chloride pilocarpine. Additionally, it was found that the treatment not only alleviated the rats' seizure symptoms, but also improved their learning and memory abilities. This improvement was linked to the modulation of protein expression levels of GLUR2, BAX, and BCL2. These findings provided clues that would be important for further investigation of the therapeutic potential of quinpirole and its underlying mechanisms for epilepsy treatment.

Low-frequency stimulation in the zona incerta attenuates seizure via driving GABAergic neuronal activity.

BACKGROUND: Managing refractory epilepsy presents a significant a substantial clinical challenge. Deep brain stimulation (DBS) has emerged as a promising avenue for addressing refractory epilepsy. However, the optimal stimulation targets and effective parameters of DBS to reduce seizures remian unidentified. OBJECTIVES: This study endeavors to scrutinize the therapeutic potential of DBS within the zona incerta (ZI) across diverse seizure models and elucidate the associated underlying mechanisms. METHODS: We evaluated the therapeutic potential of DBS with different frequencies in the ZI on kainic acid (KA)-induced TLE model or M1-cortical seizures model, pilocarpine-induced M1-cortical seizure models, and KA-induced epilepsy model. Further, employing calcium fiber photometry combined with cell-specific ablation, we sought to clarified the causal role of ZI GABAergic neurons in mediating the therapeutic effects of DBS. RESULTS: Our findings reveal that DBS in the ZI alleviated the severity of seizure activities in the KA-induced TLE model. Meanwhile, DBS attenuated seizure activities in KA- or pilocarpine-induced M1-cortical seizure model. In addition, DBS exerts a mitigating influence on KA induced epilepsy model. DBS in the ZI showed anti-seizure effects at low frequency spectrum, with 5 Hz exhibiting optimal efficacy. The low-frequency DBS significantly increased the calcium activities of ZI GABAergic neurons. Furthermore, selective ablation of ZI GABAergic neurons with taCasp3 blocked the anti-seizure effect of low-frequency DBS, indicating the anti-seizure effect of DBS is mediated by the activation of ZI GABAergic neurons. CONCLUSION: Our results demonstrate that low-frequency DBS in the ZI attenuates seizure via driving GABAergic neuronal activity. This suggests that the ZI represents a potential DBS target for treating both hippocampal and cortical seizure through the activation of GABAergic neurons, thereby holding therapeutic significance for seizure treatment.

H2S inhibits LiCl/pilocarpine-induced seizures and promotes neuroprotection by regulating TRPV2 expression via the AC3/cAMP/PKA pathway.

It is widely acknowledged that epilepsy is a neurological disorder characterized by recurrent and atypical neuronal discharges, resulting in transient dysfunction within the brain. The protective role of hydrogen sulfide (H2S) in epilepsy has been elucidated by recent studies, but the underlying mechanisms remain poorly understood. To investigate this, the concentration of H2S was measured by spectrophotometry and a fluorescent probe in LiCl/Pilocarpine (LiCl/Pilo)-induced seizures in rats. The localization of proteins was examined using immunofluorescence. Electroencephalogram and behavioral tests were employed to evaluate the occurrence of seizures. Neuropathological changes in the hippocampus were examined by hematoxylin-eosin staining, Nissl staining, and transmission electron microscopy. Through proteomics and bioinformatics analysis, we identified the differential proteins in the hippocampus of rats following H2S intervention. Protein changes were detected through western blotting. The results showed that H2S treatment significantly alleviated seizures and minimized post-seizures neurological damage in rats. Proteomics analysis revealed adenylate cyclase 3 (AC3) as a protein potentially targeted by H2S. Moreover, the AC3 activator forskolin reversed the downregulation effect of H2S on the AC3/cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA)/transient receptor potential vanilloid 2 (TRPV2) signaling pathway. In conclusion, H2S targets and downregulates the expression of AC3, thereby modulating the AC3/cAMP/PKA signaling pathway to regulate the expression of TRPV2 in LiCl/Pilo-induced seizures, ultimately leading to seizure inhibition and neuroprotection.

Seizure-induced LIN28A disrupts pattern separation via aberrant hippocampal neurogenesis.

Prolonged seizures can disrupt stem cell behavior in the adult hippocampus, an important brain structure for spatial memory. Here, using a mouse model of pilocarpine-induced status epilepticus (SE), we characterized spatiotemporal expression of Lin28a mRNA and proteins after SE. Unlike Lin28a transcripts, induction of LIN28A protein after SE was detected mainly in the subgranular zone, where immunoreactivity was found in progenitors, neuroblasts, and immature and mature granule neurons. To investigate roles of LIN28A in epilepsy, we generated Nestin-Cre:Lin28aloxP/loxP (conditional KO [cKO]) and Nestin-Cre:Lin28a+/+ (WT) mice to block LIN28A upregulation in all neuronal lineages after acute seizure. Adult-generated neuron- and hippocampus-associated cognitive impairments were absent in epileptic LIN28A-cKO mice, as evaluated by pattern separation and contextual fear conditioning tests, respectively, while sham-manipulated WT and cKO animals showed comparable memory function. Moreover, numbers of hilar PROX1-expressing ectopic granule cells (EGCs), together with PROX1+/NEUN+ mature EGCs, were significantly reduced in epileptic cKO mice. Transcriptomics analysis and IHC validation at 3 days after pilocarpine administration provided potential LIN28A downstream targets such as serotonin receptor 4. Collectively, our findings indicate that LIN28A is a potentially novel target for regulation of newborn neuron-associated memory dysfunction in epilepsy by modulating seizure-induced aberrant neurogenesis.

ATF5-regulated Mitochondrial Unfolded Protein Response Attenuates Neuronal Damage in Epileptic Rat by Reducing Endoplasmic Reticulum Stress Through Mitochondrial ROS.

Endoplasmic reticulum (ER) dysfunction caused by excessive ER stress is a crucial mechanism underlying seizures-induced neuronal injury. Studies have shown that mitochondrial reactive oxygen species (ROS) are closely related to ER stress, and our previous study showed that activating transcription factor 5 (ATF5)-regulated mitochondrial unfolded protein response (mtUPR) modulated mitochondrial ROS generation in a hippocampal neuronal culture model of seizures. However, the effects of ATF5-regulated mtUPR on ER stress and the underlying mechanisms remain uncertain in epilepsy. In this study, ATF5 upregulation by lentivirus infection attenuated seizures-induced neuronal damage and apoptosis in a rat model of pilocarpine-induced epilepsy, whereas ATF5 downregulation by lentivirus infection had the opposite effects. ATF5 upregulation potentiated mtUPR by increasing the expression of mitochondrial chaperone heat shock protein 60 (HSP60) and caseinolytic protease proteolytic subunit (ClpP) and reducing mitochondrial ROS generation in pilocarpine-induced seizures in rats. Additionally, upregulation of ATF5 reduced the expression of glucose-regulated protein 78 (GRP78), protein kinase RNA-like endoplasmic reticulum kinase (PERK), activating transcription factor 4 (ATF4), and C/EBP homologous protein (CHOP), suggesting suppression of ER stress; Moreover, ATF5 upregulation attenuated apoptosis-related proteins such as B-cell lymphoma-2 (BCL2) downregulation, BCL2-associated X (BAX) and cleaved-caspase-3 upregulation. However, ATF5 downregulation exerted the opposite effects. Furthermore, pretreatment with the mitochondria-targeted antioxidant mito-TEMPO attenuated the harmful effects of ATF5 downregulation on ER stress and neuronal apoptosis by reducing mitochondrial ROS generation. Overall, our study suggested that ATF5-regulated mtUPR exerted neuroprotective effects against pilocarpine-induced seizures in rats and the underlying mechanisms might involve mitochondrial ROS-mediated ER stress.

Highly dynamic inflammatory and excitability transcriptional profiles in hippocampal CA1 following status epilepticus.

Transient brain insults including status epilepticus (SE) can initiate a process termed 'epileptogenesis' that results in chronic temporal lobe epilepsy. As a consequence, the entire tri-synaptic circuit of the hippocampus is fundamentally impaired. A key role in epileptogenesis has been attributed to the CA1 region as the last relay station in the hippocampal circuit and as site of aberrant plasticity, e.g. mediated by acquired channelopathies. The transcriptional profiles of the distinct hippocampal neurons are highly dynamic during epileptogenesis. Here, we aimed to elucidate the early SE-elicited mRNA signature changes and the respective upstream regulatory cascades in CA1. RNA sequencing of CA1 was performed in the mouse pilocarpine-induced SE model at multiple time points ranging from 6 to 72 h after the initial insult. Bioinformatics was used to decipher altered gene expression, signalling cascades and their corresponding cell type profiles. Robust transcriptomic changes were detected at 6 h after SE and at subsequent time points during early epileptogenesis. Major differentially expressed mRNAs encoded primarily immediate early and excitability-related gene products, as well as genes encoding immune signalling factors. Binding sites for the transcription factors Nfkb1, Spi1, Irf8, and two Runx family members, were enriched within promoters of differentially expressed genes related to major inflammatory processes, whereas the transcriptional repressors Suz12, Nfe2l2 and Rest were associated with hyperexcitability and GABA / glutamate receptor activity. CA1 quickly responds to SE by inducing transcription of genes linked to inflammation and excitation stress. Transcription factors mediating this transcriptomic switch represent targets for new highly selected, cell type and time window-specific anti-epileptogenic strategies.

Differential Regulation of Wnt/ß-catenin Signaling in Acute and Chronic Epilepsy in Repeated Low Dose Lithium-Pilocarpine Rat Model of Status Epilepticus.

Epilepsy is a chronic neurological complication characterized by unprovoked seizure episodes due to the imbalance between excitatory and inhibitory neurons. The epileptogenesis process has been reported to be involved in chronic epilepsy however, the mechanism underlying epileptogenesis remains unclear. Recent studies have shown the possible involvement of Wnt/ß-catenin signaling in the neurogenesis and neuronal reorganization in epileptogenesis. In this study, we used repeated low dose lithium-pilocarpine model of status epilepsy (SE) to study the involvement of Wnt/ß-catenin signaling at acute and chronic stages post SE induction. The acute study ranged from day 0 to day 28 post SE induction and the chronic study ranged from day 0 to day 56 post SE induction. Several neurobehavioral parameters and seizure score and seizure frequency was analysed until the end of the study. The proteins involved in the regulation of Wnt/ß-catenin signaling and downstream cascading were analysed using western blot and quantitative real-time PCR analysis. The Wnt/ß-catenin pathway was found inactive in acute SE, while the same was found activated at the chronic stage. Our findings suggest that the activated Wnt/ß-catenin signaling in chronic epilepsy might be the possible mechanism underlying epileptogenesis as indicated by increased neuronal count, increased synaptic density, astrogliosis and apoptosis in chronic epilepsy. These findings can help target the Wnt/ß-catenin pathway differentially depending upon the type of epilepsy. The acute stage characterized by SE can be improved by targeting GSK-3ß levels and the chronic stage characterized by temporal lobe epilepsy can be improved by targeting ß-catenin and disheveled proteins.

Differential patterns of very high-frequency oscillations in two seizure types of the pilocarpine-induced TLE model.

AIMS: Very high-frequency oscillations (VHFOs, >500 Hz) are considered a highly sensitive biomarker of seizures. We hypothesized that VHFOs may exhibit specificity towards hypersynchronous (HYP) seizures and low-voltage fast (LVF) seizures in temporal lobe epilepsy (TLE). METHODS: Local field potentials were recorded from the hippocampal network in TLE mice induced by pilocarpine. Subsequently, we analyzed the VHFO features, including their temporal-frequency characteristics and VHFO/theta coupling, during three states: baseline, preictal, and postictal for both HYP- and LVF-seizure groups. RESULTS: Significant changes in most of the VHFO features were observed during the preictal state in both seizure groups. In the postictal state, VHFO features in the HYP-seizure group exhibited inverse alterations and appeared to align with those observed during baseline conditions. However, such phenomena were not observed after TLE seizures in the LVF-seizure group. CONCLUSION: Our findings highlight distinct patterns of VHFO feature changes across different states of HYP seizures and LVF seizures. These results suggest that VHFOs could serve as indicative biomarkers for seizure alterations specifically associated with HYP-seizure states.