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.

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.

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.

Persistent Kv7.2/7.3 downregulation in the rat pilocarpine model of mesial temporal lobe epilepsy.

Mutations within the Kv7.2 and Kv7.3 genes are well described causes for genetic childhood epilepsies. Knowledge on these channels in acquired focal epilepsy, especially in mesial temporal lobe epilepsy (mTLE), however, is scarce. Here, we used the rat pilocarpine model of drug-resistant mTLE to elucidate both expression and function by quantitative polymerase-chain reaction, immunohistochemistry, and electrophysiology, respectively. We found transcriptional downregulation of Kv7.2 and Kv7.3 as well as reduced Kv7.2 expression in epileptic CA1. Consequences were altered synaptic transmission, hyperexcitability which consisted of epileptiform afterpotentials, and increased susceptibility to acute GABAergic disinhibition. Importantly, blocking Kv7 channels with XE991 increased hyperexcitability in control tissue, but not in chronically epileptic tissue suggesting that the Kv7 deficit had precluded XE991 effects in this tissue. Conversely, XE991 resulted in comparable reduction of the paired-pulse ratio in both experimental groups implying preserved presynaptic Kv7.2 function of Schaffer collateral terminals. Consistent with Kv7.2/7.3 downregulation, the Kv7.3 channel opener ß-hydroxybutyrate failed to mitigate hyperexcitability. Our findings demonstrate that compromised Kv7 function is not only relevant in genetic epilepsy, but also in acquired focal epilepsy. Moreover, they help explain reduced anti-seizure efficacy of Kv7 channel openers in drug-resistant epilepsy.

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 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.

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.

Antiepileptogenic and neuroprotective effect of mefloquine after experimental status epilepticus.

Acquired temporal lobe epilepsy (TLE) characterized by spontaneous recurrent seizures (SRS) and hippocampal inhibitory neuron dysfunction is often refractory to current therapies. Gap junctional or electrical coupling between inhibitory neurons has been proposed to facilitate network synchrony and intercellular molecular exchange suggesting a role in both seizures and neurodegeneration. While gap junction blockers can limit acute seizures, whether blocking neuronal gap junctions can modify development of chronic epilepsy has not been examined. This study examined whether mefloquine, a selective blocker of Connexin 36 gap junctions which are well characterized in inhibitory neurons, can limit epileptogenesis and related cellular and behavioral pathology in a model of acquired TLE. A single, systemic dose of mefloquine administered early after pilocarpine-induced status epilepticus (SE) in rat reduced both development of SRS and behavioral co-morbidities. Immunostaining for interneuron subtypes identified that mefloquine treatment likely reduced delayed inhibitory neuronal loss after SE. Uniquely, parvalbumin expressing neurons in the hippocampal dentate gyrus appeared relatively resistant to early cell loss after SE. Functionally, whole cell patch clamp recordings revealed that mefloquine treatment preserved inhibitory synaptic drive to projection neurons one week and one month after SE. These results demonstrate that mefloquine, a drug already approved for malaria prophylaxis, is potentially antiepileptogenic and can protect against progressive interneuron loss and behavioral co-morbidities of epilepsy.

Effect of U50488, a selective kappa opioid receptor agonist and levetiracetam against lithium-pilocarpine-induced status epilepticus, spontaneous convulsive seizures and related cognitive impairment.

PURPOSE: Kappa opioid receptor (KOR) agonists have anticonvulsant effect but their antiepileptogenic effect is unknown. U50488, a selective KOR agonist is used to determine its effect on status epilepticus (SE), spontaneous convulsive seizures (SS) and cognitive impairment in rat lithium-pilocarpine SE model. Effect of an antiepileptic drug levetiracetam is also studied. METHOD: Male Wistar rats with SE were divided into three groups namely, LiP, LiP + U50488 (10 mg/kg, i.p.) and LiP + levetiracetam (400 mg/kg, i.p.) group. SE was terminated after 90 min of its onset with diazepam (15 mg/kg, i.p.) and phenobarbitone (25 mg/kg, i.p.). Drug treatment was started after 15 min of onset of SE and repeated once after 4 h. Rats were video monitored 12 h daily (9 AM to 9 PM) to determine severity of SE using modified Racine scale and onset and frequency of SS from day 0 to day 21. Morris water maze (MWM) test was done at baseline i.e. day -1 (before lithium administration) and day 22, to assess cognitive impairment. RESULTS: As compared to LiP, U50488 decreased the severity of SE (1.98 ± 0.13 vs 2.95 ± 0.12; p-value < 0.0001) but not levetiracetam (2.62 ± 0.09; p-value = 0.3112). Survival increased with both U50488 (90%, n = 10) and levetiracetam (81.8%, n = 11) as compared to NS (56.2%, n = 16). No effect on onset and frequency of SS was found in U50488/levetiracetam group. U50488 improved seizures-induced cognitive impairment. Levetiracetam group showed thigmotactic (wall hugging) behaviour in MWM in 8 out of 9 rats. CONCLUSION: Acute treatment with U50488, a kappa opioid receptor agonist has a beneficial effect on SE, SE-related mortality and memory impairment. The dual protective effect of U50488 on seizures and related cognitive impairment is advantageous over currently used antiseizure drugs which are known to cause cognitive impairment.

Dendritic reorganization in the hippocampus, anterior temporal lobe, and frontal neocortex of lithium-pilocarpine induced Status Epilepticus (SE).

Status Epilepticus (SE) is a distributed network disorder, which involves the hippocampus and extra-hippocampal structures. Epileptogenesis in SE is tightly associated with neurogenesis, plastic changes and neural network reorganization facilitating hyper-excitability. On the other hand, dendritic spines are known to be the excitatory synapse in the brain. Therefore, dendritic spine dynamics could play an intricate role in these network alterations. However, the exact reason behind these structural changes in SE are elusive. In the present study, we have investigated the aforementioned hypothesis in the lithium-pilocarpine treated rat model of SE. We have examined cytoarchitectural and morphological changes using hematoxylin-eosin and Golgi-Cox staining in three different brain regions viz. CA1 pyramidal layer of the dorsal hippocampus, layer V pyramidal neurons of anterior temporal lobe (ATL), and frontal neocortex of the same animals. We observed macrostructural and layer-wise alteration of the pyramidal layer mainly in the hippocampus and ATL of SE rats, which is associated with sclerosis in the hippocampus. Sholl analysis exhibited partial dendritic plasticity in apical and basal dendrites of pyramidal cells as compared to the saline-treated weight-/age-matched control group. These findings indicate that region-specific alterations in dendritogenesis may contribute to the development of independent epileptogenic networks in the hippocampus, ATL, and frontal neocortex of SE rats.

Refinement of the Barnes and Morris water maze protocols improves characterization of spatial cognitive deficits in the lithium-pilocarpine rat model of epilepsy.

Temporal lobe epilepsy (TLE) often causes cognitive impairment, especially a decline in spatial memory. Reductions in spatial memory and learning are also common in rodent models of TLE. The Morris water maze and the Barnes maze are the standard methods for evaluating spatial learning and memory in rodents. However, animals with TLE may exhibit agitation, distress, and fail to follow the paradigmatic context of these tests, making the interpretation of experimental data difficult. This study optimized the procedure of the Morris water maze and the Barnes maze to evaluate spatial learning and memory in rats with the lithium-pilocarpine TLE model (LPM rats). It was demonstrated that LPM rats required a mandatory and prolonged habituation stage for both tests. Therefore, the experimental rats performed relatively well on these tests. Nevertheless, LPM rats exhibited a slower learning process compared to the control rats. LPM rats also showed a reduction in spatial memory formation. This was more pronounced in the Barnes maze. Also, LPM rats utilized a sequential strategy for searching in the Barnes maze and were incapable of developing a more efficient spatial search strategy that is common in control animals. The Barnes maze may be a better choice for assessing search strategies, learning deficits, and spatial memory in rats with TLE when choosing between the two tests. This is because of the risk of unexpected seizure occurrence during the Morris water maze tests, and the potential risks for animal welfare.

The neuroprotective effect of Dl-3-n-butylphthalide in epileptic rats via inhibiting endoplasmic reticulum stress.

INTRODUCTION: The purpose of this study is to investigate whether Dl-3-n-Butylphthalide (NBP) has a neuroprotective effect on pilocarpine-induced epileptic (EP) rats through endoplasmic reticulum stress (ERS)-mediated apoptosis. MATERIAL AND METHODS: The Sprague-Dawley rats were divided into four groups: control (CON), EP, EP + NBP 60 (NBP 60 mg/kg) and EP + NBP 120 (NBP 120 mg/kg) groups. After the successful establishment of the temporal lobe EP model using the lithium-pilocarpine, the rats were given NBP for 28 consecutive days in EP + NBP 60 and EP + NBP 120 groups. Then, the spontaneous recurrent seizure (SRS) latency, SRS frequency and seizure duration were observed in each group. In order to observe the abnormal discharge of rats, the intracranial electrodes were implanted to monitor the electroencephalogram. Nissl staining was used to observe the damage to the hippocampal CA1 neurons, TUNEL staining was employed to observe hippocampal neuronal apoptosis. Western blot was used to detect the expression of ERS and ERS-mediated apoptotic proteins. RESULTS: NBP 60 and NBP 120 decreased SRS frequency (all p < 0.05), shortened seizure duration (all p < 0.05), and reduced the abnormal discharge of the brain. Nissl staining and TUNEL staining results show that NBP protected the hippocampal neurons from damage (all p < 0.05) and inhibited hippocampal neuronal apoptosis in EP rats (all p < 0.05). NBP 60 and NBP 120 could reduce ERS and ERS-mediated apoptotic protein expression in EP rats (all p < 0.05). In addition, the therapeutic effect of NBP on epilepsy in rats is dose-dependent. The SRS frequency of the EP + NBP 120 group was lower, and the seizure duration was shorter than in the EP + NBP 60 group (all p < 0.05), and there were more neurons in the EP + NBP 120 group than in the EP + NBP 60 group ( p < 0.05). CONCLUSIONS: NBP had a significant neuroprotective effect in EP rats. Large doses of NBP are more effective than low doses. The mechanism may be associated with the inhibition of ERS and ERS-mediated apoptosis.

Reduced Cholecystokinin-Expressing Interneuron Input Contributes to Disinhibition of the Hippocampal CA2 Region in a Mouse Model of Temporal Lobe Epilepsy.

A significant proportion of temporal lobe epilepsy (TLE) patients experience drug-resistant seizures associated with mesial temporal sclerosis, in which there is extensive cell loss in the hippocampal CA1 and CA3 subfields, with a relative sparing of dentate gyrus granule cells and CA2 pyramidal neurons (PNs). A role for CA2 in seizure generation was suggested based on findings of a reduction in CA2 synaptic inhibition (Williamson and Spencer, 1994) and the presence of interictal-like spike activity in CA2 in resected hippocampal tissue from TLE patients (Wittner et al., 2009). We recently found that in the pilocarpine-induced status epilepticus (PILO-SE) mouse model of TLE there was an increase in CA2 intrinsic excitability associated with a loss of CA2 synaptic inhibition. Furthermore, chemogenetic silencing of CA2 significantly reduced seizure frequency, consistent with a role of CA2 in promoting seizure generation and/or propagation (Whitebirch et al., 2022). In the present study, we explored the cellular basis of this inhibitory deficit using immunohistochemical and electrophysiological approaches in PILO-SE male and female mice. We report a widespread decrease in the density of pro-cholecystokinin-immunopositive (CCK+) interneurons and a functional impairment of CCK+ interneuron-mediated inhibition of CA2 PNs. We also found a disruption in the perisomatic perineuronal net in the CA2 stratum pyramidale. Such pathologic alterations may contribute to an enhanced excitation of CA2 PNs and CA2-dependent seizure activity in the PILO-SE mouse model.SIGNIFICANCE STATEMENT Impaired synaptic inhibition in hippocampal circuits has been identified as a key feature that contributes to the emergence and propagation of seizure activity in human patients and animal models of temporal lobe epilepsy (TLE). Among the hippocampal subfields, the CA2 region is particularly resilient to seizure-associated neurodegeneration and has been suggested to play a key role in seizure activity in TLE. Here we report that perisomatic inhibition of CA2 pyramidal neurons mediated by cholecystokinin-expressing interneurons is selectively reduced in acute hippocampal slices from epileptic mice. Parvalbumin-expressing interneurons, in contrast, appear relatively conserved in epileptic mice. These findings advance our understanding of the cellular mechanisms underlying inhibitory disruption in hippocampal circuits in a mouse model of spontaneous recurring seizures.

Dynamic effects of miR-20a-5p on hippocampal ripple energy after status epilepticus in rats.

To determine the dynamic effects of miR-20a-5p on hippocampal ripple energy in rats after status epilepticus (SE). A lithium pilocarpine (LiCl-PILO)-induced rat model of status epilepticus (SE) was established, and the rats were divided into the normal control (Control, CTL), epileptic control (PILO), valproic acid (VPA + PILO), miR-20a-5p overexpression lentivirus vector (miR + PILO), sponges blocking lentivirus vector (Sponges + PILO), and scramble sequence negative control (Scramble + PILO) groups (n = 6). Electroencephalograms (EEGs) were used to analyze changes in hippocampal ripple energy before and after SE. Quantitative polymerase chain reaction (q-PCR) analysis showed that miR-20a-5p levels gradually increased after miR-20a-5p overexpression lentivirus vector injection into the lateral ventricle, and the miR-20a-5p levels were significantly higher than that in CTL group on days 7 and 36 (P < 0.001). The miR-20a-5p levels decreased significantly on days 7 and 36 after blocking by sponges lentivirus vector injected into the lateral ventricle (P < 0.001). After injection of PILO, the average ripple energy expression in each group gradually increased, and reached the peak before chloral hydrate injection (compared with 1 day before SE, P < 0.05). The ripple energy in the VPA + PILO and Sponges + PILO groups was significantly lower than that in the PILO group at 60 min and 70 min after PILO injection and before chloral hydrate injection (P < 0.05), and maintained lower until 2 h after chloral hydrate injection in VPA + PILO (P < 0.05). Compared with the VPA + PILO group, the mean ripple energy of the Sponges + PILO group had no difference at all time points (P ≥ 0.05). After SE, ripple distribution of space and energy is closely related to the occurrence of epilepsy. Inhibition of miR20a-5p expression can downregulate ripple oscillation energy during seizure.

The applications of the pilocarpine animal model of status epilepticus: 40 years of progress (1983-2023).

Status epilepticus is a neurological disorder that can result in various neuropathological conditions and presentations. Various studies involving animal models have been accomplished to understand and replicating its prominent manifestations including characteristics of related clinical cases. Up to these days, there are variety of methods and techniques to be utilized in inducing this disorder that can be chemically or electrically applied which depending on the experimental designs and targets of the studies. In particular, the chemically induced pilocarpine animal model of status epilepticus is a reliable choice which has evolved for 40 years from its initial discovery back in 1983. Although the development of the model can be considered as a remarkable breakthrough in understanding status epilepticus, several aspects of the model have been improved, throughout the years. Among the major issues in developing this model are the morbidity and mortality rates during induction process. Several modifications have been introduced in the process by different studies to tackle the related problems including application of dose fractionation, adaptation of pilocarpine to lithium-pilocarpine model and utilization of various drugs. Despite all challenges and drawbacks, this model has proven its pertinent and relevance with improvements that have been adapted since it was introduced 40 years ago. In this review, we emphasize on the evolution of this animal model from the beginning until now (1983 - 2023) and the related issues that have made this model still a popular choice in status epilepticus studies.

(+)-Borneol exerts neuroprotective effects via suppressing the NF-κB pathway in the pilocarpine-induced epileptogenesis rat model.

Neuroinflammation plays a crucial role in the development of epilepsy, and suppressing neuroinflammation can delay epileptogenesis. Recent reports have demonstrated that (+)-borneol has neuroprotective effects in several brain disorders by reducing neuroinflammation. However, its effects on epilepsy have not been reported. In this research, we first studied the effect of different doses of (+)-borneol (3, 6, and 12 mg/kg) on neuroinflammation in a pilocarpine model of epileptogenesis by detecting IL-1ß, TNF-α, and COX-2 expression. We demonstrated that different doses of (+)-borneol decreased IL-1ß, TNF-α, and COX-2 levels, with 12 mg/kg having the most substantial effect. Furthermore, we examined the effects of 12 mg/kg (+)-borneol on neuronal damage, glial cell activation, and apoptosis in the hippocampus at different time points (1, 3, and 7 days) after SE. We found that (+)-borneol significantly ameliorated neuronal injury, decreased glial cell activation, and attenuated apoptosis. We also found that (+)-borneol inhibited the NF-κB pathway activation induced by SE. In conclusion, our results indicated that (+)-borneol reduces neuroinflammation by inhibiting the NF-κB pathway activation, exerts neuroprotective effects, and may have an inhibitory effect in epileptogenesis.