Down-Regulation of CRMP-1 in Patients with Epilepsy and a Rat Model

The Collapsin Response Mediator Protein-1 (CRMP-1) is a brain specific protein identified as a signaling molecule of Semaphorin-3A and act as axon repellent guidance factor in nervous system. Recent studies indicated that axon guidance molecules may play a role in synaptic reorganization in the adult brain and thereby promote epileptogenesis. This study aimed to investigate expression pattern of CRMP-1 in epileptogenesis. Using double immunofluorescence labeling, immunohistochemistry and western blot analysis, we looked into the CRMP-1 expression in temporal neocortex from patients with temporal lobe epilepsy (TLE) and histological normal temporal neocortex from the controls. We also studied the expression pattern of CRMP-1 in hippocampus and adjacent cortex of a TLE rat model on 6, 24, 72 h, 1, 2 weeks, 1 month, and 2 months post-seizure, and from control rats. CRMP-1 was mainly expressed in the neuronal cytoplasm in the temporal lobe of intractable TLE patients, which was co-expressed with -2. CRMP-1 expression was downregulated in temporal neocortical of TLE patients. In addition, in pilocarpine-induced animal model of epilepsy, CRMP-1 dynamically decreased in a range of 2 months. Thus, our results indicate that CRMP-1 may be involved in the development of TLE.     

Expression of SHANK3 in the Temporal Neocortex of Patients with Intractable Temporal Epilepsy and Epilepsy Rat Models

SH3 and multiple ankyrin (ANK) repeat domain 3 (SHANK3) is a synaptic scaffolding protein enriched in the postsynaptic density of excitatory synapses. SHANK3 plays an important role in the formation and maturation of excitatory synapses. In the brain, SHANK3 directly or indirectly interacts with various synaptic molecules including N-methyl-D-aspartate receptor, the metabotropic glutamate receptor (mGluR), and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor. Previous studies have shown that Autism spectrum disorder is a result of mutations of the main SHANK3 isoforms, which may be due to deficit in excitatory synaptic transmission and plasticity. Recently, accumulating evidence has demonstrated that overexpression of SHANK3 could induce seizures in vivo. However, little is known about the role of SHANK3 in refractory temporal lobe epilepsy (TLE). Therefore, we investigated the expression pattern of SHANK3 in patients with intractable temporal lobe epilepsy and in pilocarpine-induced models of epilepsy. Immunofluorescence, immunohistochemistry, and western blot analysis were used to locate and determine the expression of SHANK3 in the temporal neocortex of patients with epilepsy, and in the hippocampus and temporal lobe cortex of rats in a pilocarpine-induced epilepsy model. Double-labeled immunofluorescence showed that SHANK3 was mainly expressed in neurons. Western blot analysis confirmed that SHANK3 expression was increased in the neocortex of TLE patients and rats. These results indicate that SHANK3 participates in the pathology of epilepsy.     

Up-regulation of apelin in brain tissue of patients with epilepsy and an epileptic rat model

Prolonged epileptic seizures or SE can cause neuronal cell death. However, the exact role of neuroprotectant against brain injury during epileptic seizure needs to be further elucidated. The aim of this study was to investigate the expression of the apelin, a novel neuroprotective peptide, in brain tissues of the patients with temporal lobe epilepsy (TLE) and experimental rats using immunohistochemistry, immunofluorescence and Western blotting analysis and to discuss the possible role of apelin in TLE. Thirty temporal neocortical tissue samples from the patients with drug-refractory TLE underwent surgical therapy and nine histologically normal temporal lobes tissues as controls were used in our study. Fifty-six Sprague-Dawley rats were randomly divided into seven groups, including one control group and six groups with epilepsy induced by lithium-pilocarpine. Hippocampus and adjacent cortex were taken from the controls and epileptic rats at 1, 3, 7, 14, 30, and 60 days after onset of seizures. Apelin was mainly expressed in the neurons of TLE patients and controls, and was significantly increased in TLE patients compared with the controls. Apelin was also expressed in the neurons of experimental and control rats, it was gradually increased in the experimental rat post-seizure and reached a stable high level in chronic epileptic phase. Our results demonstrated that the increased expression of apelin in the brain may be involved in human TLE.     

伴海马硬化的颞叶癫痫患者海马组织内BDNF 表达变化

目的:研究伴海马硬化的难治性颞叶癫痫(TLE)患者海马组织内脑源性神经营养因子(brain derived neurotrophic factor,BDNF)的表达变化,探讨其在难治性颞叶癫痫发病机制中的作用。方法:采集5例伴海马硬化的难治性TLE患者手术中切除的海马组织,用逆转录-聚合酶链反应(RT-PCR)法检测BDNF mRNA表达,并与3例非海马硬化TLE患者对照。结果:与非海马硬化组比较,伴海马硬化的难治性TLE患者海马组织中的BDNF mRNA表达明显增加(P<0.01)。结论:伴海马硬化的难治性TLE患者海马组织中BDNF mRNA表达表达增高,可能在海马硬化和难治性颞叶癫痫发生、发展中具有重要作用。     

伴海马硬化的颞叶癫痫患者海马组织内BDNF表达变化

目的：研究伴海马硬化的难治性颞叶癫痫（TLE）患者海马组织内脑源性神经营养因子（brain derivedneurotrophic factor,BDNF）的表达变化,探讨其在难治性颞叶癫痫发病机制中的作用。方法：采集5例伴海马硬化的难治性TLE患者手术中切除的海马组织,用逆转录-聚合酶链反应（RT—PCR）法检测BDNFmRNA表达,并与3例非海马硬化TLE患者对照。结果：与非海马硬化组比较,伴海马硬化的难治性TLE患者海马组织中的BDNFmRNA表达明显增加（P〈0．01）。结论：伴海马硬化的难治性TLE患者海马组织中BDNFmRNA表达表达增高,可能在海马硬化和难治性颞叶癫痫发生、发展中具有重要作用。     

颞叶癫痫脑“默认模式”的fMRI研究

功能磁共振成像(functional magnetic resonance imaging,fMRI)被用于检测静息时脑功能神经网络.作者运用静息fMRI检测海马硬化颞叶癫痫(temporal lobe epilepsy,TLE)脑"默认模式",采用感兴趣区域功能连接分析检测16例TLE患者和16名正常对照静息时脑的"默认模式",并进行组内和组间分析.研究发现,与正常对照相比,TLE静息时海马、颞极、额叶、颞叶、壳核及楔前叶等脑区与后扣带回的功能连接增强.研究结果表明TLE患者的固有脑功能组织模式有可能出现紊乱.这一研究将有助于从脑功能的角度了解癫痫患者某些临床症状的发病机理,为今后癫痫诊治的发展提供一定的帮助.     

Decreased Astroglial Monocarboxylate Transporter 4 Expression in Temporal Lobe Epilepsy

Efflux of monocaroxylates like lactate, pyruvate, and ketone bodies from astrocytes through monocarboxylate transporter 4 (MCT4) supplies the local neuron population with metabolic intermediates to meet energy requirements under conditions of increased demand. Disruption of this astroglial-neuron metabolic coupling pathway may contribute to epileptogenesis. We measured MCT4 expression in temporal lobe epileptic foci excised from patients with intractable epilepsy and in rats injected with pilocarpine, an animal model of temporal lobe epilepsy (TLE). Cortical MCT4 expression levels were significantly lower in TLE patients compared with controls, due at least partially to MCT4 promoter methylation. Expression of MCT4 also decreased progressively in pilocarpine-treated rats from 12 h to 14 days post-administration. Underexpression of MCT4 in cultured astrocytes induced by a short hairpin RNA promoted apoptosis. Knockdown of astrocyte MCT4 also suppressed excitatory amino acid transporter 1 (EAAT1) expression. Reduced MCT4 and EAAT1 expression by astrocytes may lead to neuronal hyperexcitability and epileptogenesis in the temporal lobe by reducing the supply of metabolic intermediates and by allowing accumulation of extracellular glutamate.     

Wogonin preventive impact on hippocampal neurodegeneration,inflammation and cognitive defects in temporal lobe epilepsy

Previous studies demonstrated that the pathophysiological changes after temporal lobe epilepsy (TLE) such as oxidative stress, inflammatory reaction contribute to cognitive defect and neuronal damage. The present study was conducted to evaluate the anticonvulsant effect of wogonin ameliorates kainate-induced TLE, and to investigate the mechanism underlying these effects. Rats were divided into control, wogonin, kainate, and wogonin-pretreated kainate groups. The rat model of TLE was induced by unilateral intrahippocampal injection of 0.4 ug/ul of kainate. The results showed that the cognitive function in TLE rats was significantly impaired, and wogonin treatment improved cognitive function in the Morris water maze (MWM). H & E staining and TUNEL staining showed obvious damage in the hippocampus of TLE rats, and wogonin alleviated the damage. To evaluate the oxidative stress, the expression of MDA and GSH in plasma were detected. Nrf-2 and HO-1 mRNA expression in the hippocampus were detected. The levels of MDA in plasma increased in TLE rats, and the levels of GSH in plasma and Nrf-2, HO-1 in the brain decreased. Treatment with wogonin alleviated these changes. We also detected the mRNA expression of inflammatory mediators like IL-1β, TNF-α, and NF kB in the brain. The inflammatory reaction was significantly activated in the brain of TLE rats, and wogonin alleviated neuroinflammation. We detected the mRNA expression of Bcl-2, Bax, caspase-3, in the hippocampus. The levels of Bcl-2 decreased in TLE rats, Bax and caspase-3 increased, while wogonin alleviated these changes. The present study indicated that wogonin exerted a noticeable neuroprotective effect in kainate-induced TLE rats.     

Increased Intrinsic Connectivity of the Default Mode Network in Temporal Lobe Epilepsy: Evidence from Resting-State MEG Recordings

The electrophysiological signature of resting state oscillatory functional connectivity within the default mode network (DMN) during spike-free periods in temporal lobe epilepsy (TLE) remains unclear. Using magnetoencephalographic (MEG) recordings, this study investigated how the connectivity within the DMN was altered in TLE, and we examined the effect of lateralized TLE on functional connectivity. Sixteen medically intractable TLE patients and 22 controls participated in this study. Whole-scalp 306-channel MEG epochs without interictal spikes generated from both MEG and EEG data were analyzed using a minimum norm estimate (MNE) and source-based imaginary coherence analysis. With this processing, we obtained the cortical activation and functional connectivity within the DMN. The functional connectivity was increased between DMN and the right medial temporal (MT) region at the delta band and between DMN and the bilateral anterior cingulate cortex (ACC) regions at the theta band. The functional change was associated with the lateralization of TLE. The right TLE showed enhanced DMN connectivity with the right MT while the left TLE demonstrated increased DMN connectivity with the bilateral MT. There was no lateralization effect of TLE upon the DMN connectivity with ACC. These findings suggest that the resting-state functional connectivity within the DMN is reinforced in temporal lobe epilepsy during spike-free periods. Future studies are needed to examine if the altered functional connectivity can be used as a biomarker for treatment responses, cognitive dysfunction and prognosis in patients with TLE.     

Upregulated P2X3 Receptor Expression in Patients with Intractable Temporal Lobe Epilepsy and in a Rat Model of Epilepsy

Purinergic P2X3 receptors (P2X3Rs) play extensive roles in nerve cells in the central nervous system, particularly in hyperexcitability and calcium (Ca²⁺) influx. However, the role of P2X3Rs in epilepsy has not been previously investigated. To determine the relationship between P2X3Rs and epilepsy, the expression and cellular location of P2X3Rs in patients with intractable temporal lobe epilepsy (TLE) and in a lithium chloride-pilocarpine-induced chronic rat model of epilepsy were assessed. Furthermore, the function of P2X3Rs was assessed in vitro. Real-time quantitative polymerase chain reaction (RT-qPCR) and Western blot analysis were used to evaluate the expression levels of P2X3Rs in brain tissues from TLE patients and an epileptic rat model, whereas immunofluorescence labeling was applied to determine the distribution of target proteins. Whole-cell recording was subsequently performed to identify the influence of P2X3Rs on seizure-like discharges. P2X3Rs were located at the cell bodies and dendrites of neurons with significantly increased expression in the TLE patients and epileptic rat model. In vitro, P2X3R activation accelerated sustained repetitive firing, whereas P2X3R inhibition led to relatively low-frequency discharges. To the best of our knowledge, this is the first study provide evidence that upregulated P2X3R expression exists in both epileptic humans and rats and may aggravate the epileptic state in vitro. Thus, P2X3Rs may represent a novel therapeutic target for antiepileptic drugs.     

Time-Dependent Modulation of Mitogen Activated Protein Kinases and AKT in Rat Hippocampus and Cortex in the Pilocarpine Model of Epilepsy

The epileptogenesis may involve a variety of signaling events that culminate with synaptic reorganization. Mitogen-activated protein kinases (MAPKs) and AKT may be activated by diverse stimulus including neurotransmitter, oxidative stress, growth factors and cytokines and are involved in synaptic plasticity in the hippocampus and cerebral cortex. The pilocarpine model in rodents reproduces the main features of mesial temporal lobe epilepsy related to hippocampus sclerosis (MTLE-HS) in humans. We analyze the phosphorylation profile of MAPKs (ERK1/2, p38(MAPK), JNK1/2/3) and AKT by western blotting in the hippocampus (Hip) and cortex (Ctx) of male adult wistar rats in different periods, after pilocarpine induced status epilepticus (Pilo-SE) and compared with control animals. Biochemical analysis were done in the Hip and Ctx at 1, 3, 12?h (acute period), 5?days (latent period) and 50?days (chronic period) after Pilo-SE onset. Hence, the main findings include increased phosphorylation of ERK1 and p38(MAPK) in the Hip and Ctx 1 and 12?h after the Pilo-SE onset. The JNK2/3 isoform (54?kDa) phosphorylation was decreased at 3?h after the Pilo-SE onset and in the chronic period in the Hip and Ctx. The AKT phosphorylation increased only in the Hip during the latent period. Our study demonstrates, in a systematic manner, the profile of MAPKs and AKT modulation in the hippocampus and cerebral cortex in response to pilocarpine. Based in the role of each signaling enzyme is possible that these changes may be related, at least partially, to modifications in the intrinsic neuronal physiology and epileptogenic synaptic network that appears in the MTLE-HS.     

Increased Expression of Rac1 in Epilepsy Patients and Animal Models

The mechanisms of epilepsy remain incompletely understood. Rac1 (ras-related C3 botulinum toxin substrate 1) belongs to the Rho family of small GTPases. Rac1 play important roles in cytoskeleton rearrangement and neuronal synaptic plasticity, which had also been implicated in epilepsy. However, little is known regarding the expression of Rac1 in the epileptic brain or whether Rac1-targeted interventions affect the progression of epilepsy. The aim of this study was to investigate the expression profile of Rac1 in brain tissues from patients suffering from temporal lobe epilepsy (TLE) and experimental epileptic rats and determine the possible role of Rac1 in epilepsy. We demonstrated that the expression of Rac1 is significantly increased in TLE patients and in lithium-pilocarpine epilepsy model animals compared to the corresponding controls. Rac1 inhibitor NSC23766 reduced the severity of status epilepticus during the acute stage in a lithium-pilocarpine animal model. Consistent with these results, the latent period of a PTZ kindling animal model also increased. Our results demonstrated that the increased expression of Rac1 may contribute to pathophysiology of epilepsy.

     

LncRNA-UCA1 inhibits the astrocyte activation in the temporal lobe epilepsy via regulating the JAK/STAT signaling pathway

This article aimed to reveal the mechanism of long noncoding RNA (lncRNA) urothelial cancer-associated 1 (UCA1) regulated astrocyte activation in temporal lobe epilepsy (TLE) rats via mediating the activation of the JAK/STAT signaling pathway. A model of TLE was established based on rats via kainic acid (KA) injection. All rats were divided into the Sham group (without any treatments), KA group, normal control (NC; injection with empty vector) + KA group, and UCA1 + KA group. The Morris water maze was used to test the learning and memory ability of rats, and the expression of UCA1 in the hippocampus was determined by quantitative real time polymerase chain reaction (qRT-PCR). Surviving neurons were counted by Nissl staining, and expression levels of glial cells glial fibrillary acidic protein (GFAP), p-JAK1, and p-STAT3 and glutamate/aspartate transporter (GLAST) were analyzed by immunofluorescence and Western blot analysis. A rat model of TLE was established by intraperitoneal injection of KA. qRT-PCR and fluorescence analyses showed that UCA1 inhibited astrocyte activation in the hippocampus of epileptic rats. Meanwhile, the Morris water maze analysis indicated that UCA1 improved the learning and memory in epilepsy rats. Moreover, the Nissl staining showed that UCA1 might have a protective effect on neuronal injury induced by KA injection. Furthermore, the immunofluorescence and Western blot analysis revealed that the overexpression of UCA1 inhibited KA-induced abnormal elevation of GLAST, astrocyte activation of the JAK/STAT signaling pathway, as well as hippocampus of epilepsy rats. UCA1 inhibited hippocampal astrocyte activation and JAK/STAT/GLAST expression in TLE rats and improved the adverse reactions caused by epilepsy.     

GPER1 Modulates Synaptic Plasticity During the Development of Temporal Lobe Epilepsy in Rats

G-protein coupled estrogen receptor 1 (GPER1) is a novel type of estrogen receptor. Several studies have shown that it has an anti-inflammatory action,which plays an important role in remyelination and cognitive ability adjustment. However, whether it is involved in the development of temporal lobe epilepsy (TLE) is still unknown. The present study established a TLE model by intraperitoneal injection of lithium chloride (3 mmol/kg) and pilocarpine (50 mg/kg) in rats to study the effect of GPER1 in the synaptic plasticity during the development of temporal lobe epilepsy. A microinjection cannula was implanted into the lateral ventricle region of rats via a stereotaxic instrument. G-1 is the specific GPER1 agonist and G15 is the specific GPER1 antagonist. The G1 or G15 and Dimethyl sulfoxide were injected into the rat brains in the intervention groups and control group, respectively. After G1 intervention, the learning and memory abilities and hippocampal neuron damage in epileptic rats were significantly improved, while G15 weakened the neuroprotective effect of GPER1. Meanwhile, G1 controlled the abnormal formation of hippocampal mossy fiber sprouting caused by seizures, and participated in the regulation of synaptic plasticity by reducing the expression of Synapsin I and increasing the expression of gephyrin. Inhibitory synapse gephyrin may play a significant role in synaptic plasticity.

     

Induction of astrocytic cyclooxygenase-2 in epileptic patients with hippocampal sclerosis

Induction of cyclooxygenase-2 (COX-2) has been described in a wide range of neurological diseases including animal models of epilepsy. The present study was undertaken to assess COX-2 expression in hippocampal biopsies from patients with therapy-refractive temporal lobe epilepsy (TLE). For this purpose, hippocampal CA1 subfield was dissected from epileptic patients with (n=5) or without (n=2) hippocampal sclerosis (HS). COX-2 expression was investigated using immunohistochemistry and semi-quantitative RT-PCR. COX-2 immunoreactivity in TLE patient material in the absence of HS was restricted to a few neurons of the hippocampus. In the presence of HS, on the other hand, a significant induction of astrocytic COX-2 immunoreactivity associated with a concomitant increase in the steady-state level of COX-2 mRNA was observed in the CA1 subfield. These findings suggest that induction of astrocytic COX-2 is implicated in the pathogenesis of HS in TLE and is consistent with the previous findings of increased concentrations of prostaglandins in the cerebrospinal fluid of these patients.     

The levels of renin-angiotensin related components are modified in the hippocampus of rats submitted to pilocarpine model of epilepsy

We previously showed that patients with temporal lobe epilepsy (TLE) present an increased expression of angiotensin II (AngII) AT1 and AT2 receptors in the hippocampus, supporting the idea of an upregulation of renin-angiotensin system (RAS) in this disease. This study aimed to verify the relationship between the RAS and TLE during epileptogenesis. Levels of the peptides angiotensin I (AngI), angiotensin II (AngII) and angiotensin 1-7 (Ang 1-7), were detected by HPLC assay. Angiotensin AT1 and AT2 receptors, Mas mRNA receptors and angiotensin converting enzyme (ACE), tonin and neutral endopeptidase (NEP) mRNA were also quantified at the hippocampus of Wistar rats by real time PCR, during acute (n=10), silent (n=10) and chronic (n=10) phases of pilocarpine-induced epilepsy. We observed an increased peptide level of Ang1-7 into acute and silent phases, decreasing importantly (p≤0.05) in the chronic phase, suggesting that AngI may be converted into Ang 1-7 by NEP, which is present in high levels in these periods. Our results also showed increased peptide level of AngII in the chronic phase of this model. In contraposition, the ACE expression is reduced in all periods. These data suggest that angiotensinogen or AngI may be cleaved to AngII by tonin, which showed increased expression in all phases. We found changes in AT1, AT2 and Mas mRNA receptors levels suggesting that Ang1-7 could act at Mas receptor during the silent period. Herein, we demonstrated for the first time, changes in angiotensin-related peptides, their receptors as well as the releasing enzymes in the hippocampus of rats during pilocarpine-induced epilepsy.     

Comparative proteomics and correlated signaling network of rat hippocampus in the pilocarpine model of temporal lobe epilepsy

In temporal lobe epilepsy (TLE), the seizure origin typically involves the hippocampal formation. The pilocarpine-induced TLE provides a model to investigate the molecular and functional characterization of epileptogenesis by mimicking the human epileptic condition. Here, we employed a 2-D gel-based proteomic technique to profile proteome changes in the rat hippocampus after pilocarpine treatment. Using MALDI MS and MS/MS, 57 differentially expressed proteins were identified, which were found either up-regulated and/or down-regulated at the two time points 12 h (acute period; Ap) and 72 h (silent period; Sp) compared with the control. These proteins can be related to underlying mechanism of pilocarpine-induced TLE, indicating cytoskeleton modification, altered synaptic function, mitochondrial dysfunction, changed ion channel, and chaperone. Five of the identified proteins, synaptosomal-associated protein 25 (SNAP25), synapsin-2 (SYN2), homer protein homolog 2 (HOMER2), alpha-internexin (INA), and voltage-dependent anion channel 2 (VDAC2) were investigated by semiquantitative RT-PCR, and SNAP25 and INA were further validated by Western blot and immunohistochemistry staining. Furthermore, association of these pilocarpine-induced proteins with biological functions using the Ingenuity Pathway Analysis (IPA) tool showed that nucleic acid metabolism, system development, tissue and cell morphology were significantly altered. IPA of the canonical networks indicated that six membrane proteins (e.g., SNAP25, SYN2, and HOMER2) participated in three biological networks as starting proteins. Our results offer a clue to identify biomarkers for the development of pharmacological therapies targeted at epilepsy.