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Focal cortical dysplasia type IIb (FCDIIb) and tuberous sclerosis complex (TSC) are typical causes of developmental delay and refractory epilepsy. G‐protein‐coupled receptor 30 (GPR30) is a specific estrogen receptor that is critical in neurodevelopment, neuroinflammation, and neuronal excitability, suggesting that it plays a potential role in the epilepsy of patients with FCDIIb and TSC. Therefore, we investigated the role of GPR30 in patients with FCDIIb and TSC. We found that the expression of GPR30 and its downstream protein kinase A (PKA) pathway were decreased and negatively correlated with seizure frequency in female patients with FCDIIb and TSC, but not in male patients. GPR30 was widely distributed in neurons, astrocytes, and microglia, and its downregulation was especially notable in microglia. The GPR30 agonist G‐1 increased the expression of PKA and p‐PKA in cultured cortical neurons, and the GPR30 antagonist G‐15 exhibited the opposite effects of G‐1. The NF‐κB signaling pathway was also activated in the specimens of female patients with FCDIIb and TSC, and was regulated by G‐1 and G‐15 in cultured cortical neurons. We also found that GPR30 regulated cortical neuronal excitability by altering the frequency of spontaneous excitatory postsynaptic currents and the expression of NR2A/B. Further, the relationship between GPR30 and glycometabolism was evaluated by analyzing the correlations between GPR30 and 18F‐FDG PET‐CT values (standardized uptake values, SUVs). Positive correlations between GPR30 and SUVs were found in female patients, but not in male patients. Intriguingly, GPR30 expression and SUVs were significantly decreased in the epileptogenic tubers of female TSC patients, and ROC curves indicated that SUVs could predict the localization of epileptogenic tubers. Taken together, our results suggest a potential protective effect of GPR30 in the epileptogenesis of female patients with FCDIIb and TSC.

Epilepsy is a highly prevalent and drug-refractory neurological disorder characterized by spontaneous recurrent seizures. Estrogen is identified to be proconvulsant and lowers the seizure threshold of female epilepsy. Estrogen receptor β (ERβ) has been proposed to mediate neuroprotection in epilepsy, although the underlying mechanism remains unknown. Rationale: In this study, we investigated the role of ERβ in the epileptogenesis of female temporal lobe epilepsy (TLE). Methods: Immunohistochemistry, immunofluorescence, western blots, Golgi staining, 1H MRS and whole-cell patch-clamp were used to evaluate ERβ expression, pathological changes, and synaptic excitation /inhibition (E/I) balance in female TLE patients and ovariectomized (OVX) chronic epileptic mice. Electroencephalogram (EEG) recordings were recorded to evaluate the epileptic susceptibility in OVX WT and ERβ-/- mice. And high-throughput RNA-sequence was performed to identify differential expression genes (DEGs) which can elucidate the potential mechanism of ERβ regulating the seizure susceptibility. Results: ERβ expression was decreased in the brains of female TLE patients and OVX chronic epileptic mice. ERβ deletion enhanced seizure susceptibility and exacerbated the imbalance of synaptic E/I in hippocampal CA1 area of OVX epileptic mice. In line with these observations, RNA-sequence data further identified glutamine ligase (GLUL) as the target of ERβ involved in regulating synaptic E/I in CA1. Furthermore, ERβ agonist WAY-200070 markedly suppressed epileptic phenotypes and normalized GLUL expression in CA1 region of kainic acid (KA) induced OVX chronic epileptic model. Conclusions: Our data provide novel insight into the pathogenesis of female TLE, and indicate ERβ provides a new therapeutic strategy for female TLE patients.

Tuberous sclerosis complex (TSC) is an autosomal dominant multisystem disorder characterized by hamartomas in multiple organ systems. The TSC1 and TSC2 genes have been identified as the genetic basis of TSC. Two gene tests were used for definitive genetic diagnosis. In our study, the case of a Chinese pediatric patient with seizures, hypomelanotic macules, hyperpigmented patches, multiple parenchymal lesions in the ventricle, and developmental retardation is detailed. Whole-genome sequencing (WGS) and multiplex ligation-dependent probe amplification (MLPA) were employed to detect genetic variations and copy number variations of TSC1 and TSC2. A novel heterozygous nonsense mutation in the TSC2 gene (c.3751A>T, p.Lys1251Ter) was identified in a Chinese pediatric patient suffering from TSC, whose unaffected parents did not carry this mutation. The mutation was classified as “pathogenic” according to the American College of Medical Genetics (ACMG) guidelines. WGS was carried out to definitively diagnose and detect variations in the exon and noncoding region of the gene and copy number variations in the whole genome simultaneously. For diseases with complex genetic mechanisms, WGS as the first-line test can be efficient and cost-effective for clinical diagnosis.

Aim Nod-like receptor protein 3 (NLRP3) inflammasome-mediated inflammation has emerged as a contributor to epileptogenesis. Endoplasmic reticulum stress (ERS) plays an important role in epilepsy-induced neurodegeneration. NLRP3 activation and ERS reactions share the same induction factors, suggesting that these processes may be interdependent. However, the correlation between NLRP3 and ERS in TLE has not been confirmed. Methods The expression patterns of NLRP3 inflammasome and ERS-related markers in the temporal neocortices of TLE patients were investigated by western blotting, immunohistochemistry and immunofluorescent labelling. Correlations between the protein levels of NLRP3 and the expression of ERS-related markers were assessed using Spearman's rank correlation test. To observe the relationship between the NLRP3 inflammasome and ERS, inhibitors were used in a status epilepticus (SE) model. Results Our results show that NLRP3 inflammasome components and ERS-related markers were upregulated in the temporal neocortices of TLE patients, and were mainly localized to neurons, astrocytes and microglia. We found a positive correlation between the protein levels of NLRP3 and the expression of ERS-related markers in the temporal neocortices of 20 TLE patients. Furthermore, after blocking the NLRP3 inflammasome with MCC950, the expression of ERS-related markers was markedly decreased in the hippocampi of SE mice. Moreover, TUDCA, a specific ERS inhibitor, also reduced the expression of NLRP3 components in the hippocampus under SE conditions. Conclusion Taken together, our data reveal the interdependence of the NLRP3 inflammasome and ERS in the epileptogenic zone of TLE patients and in the hippocampi of mice in the early post-SE phase.

BackgroundA hallmark of temporal lobe epilepsy (TLE) is brain inflammation accompanied by neuronal demise. Accumulating evidence demonstrates that Rev-Erbα is involved in regulating neuroinflammation and determining the fate of neurons. Therefore, we studied the expression and cellular distribution of Rev-Erbα in the epileptogenic zone of TLE and the effect of treatment with the Rev-Erbα specific agonist SR9009 in the pilocarpine model.MethodsThe expression pattern of Rev-Erbα was investigated by western blotting, immunohistochemistry, and immunofluorescence labeling in patients with TLE. Next, the effects of SR9009 on neuroinflammation, neuronal apoptosis, and neuronal loss in the mouse hippocampus 7 days after status epilepticus (SE) were assessed by western blotting, immunofluorescence labeling staining, and TUNEL staining.ResultsThe western blotting, immunohistochemistry, and immunofluorescence labeling results revealed that Rev-Erbα was downregulated in the epileptogenic zone of TLE patients and mainly localized in neurons, astrocytes, and presumably microglia. Meanwhile, the expression of Rev-Erbα was decreased in the hippocampus and temporal neocortex of mice treated with pilocarpine in the early post-SE and chronic phases. Interestingly, the expression of Rev-Erbα in the normal hippocampus showed a 24-h rhythm; however, the rhythmicity was disturbed in the early phase after SE, and this disturbance was still present in epileptic animals. Our further findings revealed that treatment with SR9009 inhibited NLRP3 inflammasome activation, inflammatory cytokine (IL-1β, IL-18, IL-6, and TNF-α) production, astrocytosis, microgliosis, and neuronal damage in the hippocampus after SE.ConclusionsTaken together, these results suggested that a decrease in Rev-Erbα in the epileptogenic zone may contribute to the process of TLE and that the activation of Rev-Erbα may have anti-inflammatory and neuroprotective effects.

Objective: Central nervous system infections (CNSIs), especially viral encephalitis and meningitis, are well-recognized causes of medically refractory epilepsy. Although surgery is an effective and durable intervention against these infections, the seizure control outcomes described in previous surgical series have been variable. Accordingly, it is not clear which variables are most valuable in predicting seizure control following surgery for CNSI. The aim of this meta-analysis was to identify the predictors of favorable surgical outcomes in CNSI-related epilepsy.Methods: The PubMed, EMBASE, Cochrane Library, WANGFANG, VIP, CBM, and CNKI databases were searched for studies according to the inclusion criteria. Prognostic factors, surgical outcomes, and patient characteristics were extracted. Heterogeneity was detected by the I2 and Q statistics.Results: Seventeen studies were included in our meta-analysis. Eight predictors of favorable outcomes (Engel Class I/II) were determined, including abnormal MRI findings, meningitis, temporal location only, regional ictal pattern, unilateral ictal pattern, older age at epilepsy, longer silent period, and longer time from infection, as follows: OR = 3.34 (95% CI 1.44–7.74), OR = 0.31 (95% CI 0.13–0.70), OR = 0.34 (95% CI 0.16–0.74), OR = 5.65 (95% CI 1.75–18.30), and OR = 9.53 (95% CI 2.36–38.48), respectively, and MD = 2.15 (95% CI 0.20–4.11), MD = 2.40 (95% CI 0.09–4.70), and MD = 8.49 (95% CI 1.50–15.48), respectively. A subgroup analysis found the following associations: regional and unilateral ictal patterns in viral encephalitis, a younger age at infection in parasitic encephalopathy, an older age at surgery, a longer time from onset, and a longer time from infection in unexplained meningitis. A sensitivity analysis restricted to studies that included each variable yielded robust results. Little evidence of publication bias was observed.Conclusions: This meta-analysis suggests that abnormal MRI findings, meningitis, temporal location only, regional and unilateral ictal patterns, older age at epilepsy, longer silent period, and longer time from infection are predictive factors in patients with favorable surgical outcomes in CNSI-related epilepsy. In addition, different infective agents influenced the results in regional and unilateral ictal patterns in ictal electroencephalography, as well as the relationship between age at infection and surgery and the time from epilepsy onset and infection.

Temporal lobe epilepsy (TLE) is defined as the sporadic occurrence of spontaneous recurrent seizures, and its pathogenesis is complex. SHP‐2 (Src homology 2‐containing protein tyrosine phosphatase 2) is a widely expressed cytosolic tyrosine phosphatase protein that participates in the regulation of inflammation, angiogenesis, gliosis, neurogenesis and apoptosis, suggesting a potential role of SHP‐2 in TLE. Therefore, we investigated the expression patterns of SHP‐2 in the epileptogenic brain tissue of intractable TLE patients and the various effects of treatment with the SHP‐2‐specific inhibitor SHP099 on a pilocarpine model. Western blotting and immunohistochemistry results confirmed that SHP‐2 expression was upregulated in the temporal neocortex of patients with TLE. Double‐labeling experiments revealed that SHP‐2 was highly expressed in neurons, astrocytes, microglia and vascular endothelial cells in the epileptic foci of TLE patients. In the pilocarpine‐induced C57BL/6 mouse model, SHP‐2 upregulation in the hippocampus began one day after status epilepticus, reached a peak at 21 days and then maintained a significantly high level until day 60. Similarly, we found a remarkable increase in SHP‐2 expression at 1, 7, 21 and 60 days post‐SE in the temporal neocortex. In addition, we also showed that SHP099 increased reactive gliosis, the release of IL‐1β, neuronal apoptosis and neuronal loss, while reduced neurogenesis and albumin leakage. Taken together, the increased expression of SHP‐2 in the epileptic zone may be involved in the process of TLE.

Background Meningiomas typically manifest on magnetic resonance imaging (MRI) as iso- to hypointense on T1-weighted imaging and iso- to hyperintense on T2-weighted imaging. After contrast administration, they usually homogeneously enhance and exhibit a visible dural tail. Meningiomas with atypical findings may be misdiagnosed. Case presentation We report a 50-year-old female patient with a pathologically diagnosed fibrous meningioma (World Health Organization grade I) that exhibited ring enhancement on MRI. Conclusions Meningiomas may rarely present with ring enhancement on MRI. The natural history and mechanisms of cystic degeneration and enhancement in the various types of meningioma require further study.

Cortical tubers in patients with tuberous sclerosis complex (TSC) are highly associated with intractable epilepsy. Recent evidence suggests a close relationship between FGF13 and seizures. To understand the role of FGF13 in the pathogenesis of cortical tubers, we investigated the expression pattern of FGF13 in cortical tubers of TSC compared with normal control cortices (CTX). We found that both the mRNA and protein levels of FGF13 were significantly higher in the cortical tubers from patients with TSC than in the control cortices. The immunohistochemical results showed strong FGF13 immunoreactivity in abnormal cells, including dysplastic neurons (DNs) and giant cells (GCs). Moreover, double-label immunofluorescence analyses confirmed that FGF13 was mainly localized in neurons and nearly absent in glia-like cells. The protein levels of FGF13 in the TSC samples were positively correlated with the frequency of seizures before surgery. Taken together, these results suggest that the overexpression and distribution pattern of FGF13 may be related to intractable epilepsy caused by TSC.

Focal cortical dysplasias (FCDs) are well recognized as important causes of medically intractable epilepsy in both children and adults. To explore the potential role of fibroblast growth factor 13 (FGF13) in intractable epilepsy caused by FCDs, we examined the expression of FGF13 in cortical lesions from 23 patients with FCD type Ia (FCDIa), 24 patients with FCD type IIa (FCDIIa), and 12 patients with FCD type IIb (FCDIIb), and we compared the results with the FGF13 expression levels in control cortex (CTX) brain tissues from 12 nonepileptic normal subjects. Both the mRNA levels and protein levels of FGF13 were significantly higher in the cortical lesions from patients with FCD than in the control cortices. The immunohistochemical results showed that strong FGF13 immunoreactivity was observed in misshapen cells, including neuronal microcolumns, hypertrophic neurons, dysmorphic neurons, and most balloon cells. Moreover, double-label immunofluorescence analyses confirmed that FGF13 was mainly localized in neurons and nearly absent in glia-like cells. Taken together, our results suggest that the overexpression of FGF13 in FCDs and the cell-specific distribution patterns of FGF13 in misshapen neurons in FCDs could potentially contribute to intractable epilepsy caused by FCDs.

Focal cortical dysplasia (FCD) is one of the main causes of medically intractable epilepsy. Some studies have reported that transient receptor potential canonical channel 3 (TRPC3) may play an important role in the occurrence of seizures. In this study, we investigated the expression patterns of TRPC3 in different types of FCD. Forty-five FCD specimens and 12 control samples from autopsies were used in our study. Western blotting, immunohistochemistry, and immunofluorescence staining were employed to detect protein expression and distribution. The amount of TRPC3 protein was markedly elevated in the FCD group. The immunohistochemistry results revealed that TRPC3 staining was strong in the malformed cells and microcolumns. Most of the TRPC3-positive cells were colabeled with glutamatergic and GABAergic markers. The overexpression and altered cellular distribution of TRPC3 in the FCD samples suggest that TRPC3 may be related to epileptogenesis in FCD.

Background Temporal lobe epilepsy (TLE) is the most common intractable epilepsy in adults, and elucidation of the underlying pathological mechanisms is needed. Voltage-gated chloride channels (ClC) play diverse physiological roles in neurons. However, less is known regarding their functions in the epilepogenesis of TLE. Methods ClC-mediated current and the spontaneous inhibitory synaptic currents (sIPSC) in hippocampal neurons of epileptic lesions were investigated by electrophysiological recording. The EEG data were analyzed by Z-scored wavelet and Fourier transformations. The expression of ClC-3, a member of ClC gene family, was detected by immunostaining and western blot. Findings ClC-mediated current was increased in the hippocampal neurons of chronic TLE mice. Application of chloride channel blockers, NPPB (5-Nitro-2- [3-phenylpropylamino] benzoic acid) and DIDS (4,4’-Diisothiocyanato-2,2’-stilbenedisulfonic acid disodium salt) reduced ClC-mediated current and increased inhibitory synaptic transmission in TLE mice. NPPB and DIDS reduced the seizure frequency and the average absolute power of ictal high-frequency oscillations (HFOs, 80-500 Hz) in TLE mice. In addition, both drugs induced outwardly rectified currents, which might be tonic inhibitory currents in the hippocampal neurons of TLE patients. Furthermore, the expression of ClC-3 was increased in the hippocampus of TLE mice and patients and positively correlated with both the absolute power and number of ictal HFOs per seizure in the sclerotic hippocampus. Interpretation These data suggest that ClC participate in the epilepogenetic process of TLE and the inhibition of ClC may have anti-epileptic effect. Funding This work was supported by National Natural Science Foundation of China (No. 81601143, No. 81771217).

Focal cortical dysplasia type II (FCD II) and tuberous sclerosis complex (TSC) are well-known causes of chronic refractory epilepsy in children. Canonical transient receptor potential channels (TRPCs) are non-selective cation channels that are commonly activated by phospholipase C (PLC) stimulation. Previous studies found that TRPC4 may participate in the process of epileptogenesis. This study aimed to examine the expression and distribution of TRPC4 in FCD II (n = 24) and TSC (n = 11) surgical specimens compared with that in age-matched autopsy control samples (n = 12). We found that the protein levels of TRPC4 and its upstream factor, PLC delta 1 (PLCD1), were elevated in FCD II and TSC samples compared to those of control samples. Immunohistochemistry assays revealed that TRPC4 staining was stronger in malformed cells, such as dysmorphic neurons, balloon cells and giant cells. Moderate-to-strong staining of the upstream factor PLCD1 was also identified in abnormal neurons. Moreover, double immunofluorescence staining revealed that TRPC4 was colocalised with glutamatergic and GABAergic neuron markers. Taken together, our results indicate that overexpression of TRPC4 protein may be involved in the epileptogenesis of FCD II and TSC.

// Song Li 1 , Zhenle Zang 1 , Jiaojiang He 2 , Xin Chen 1 , Sixun Yu 3 , Yuchun Pei 1 , Zhi Hou 1 , Ning An 1 , Hui Yang 1 , Chunqing Zhang 1 , Shiyong Liu 1 1 Epilepsy Research Center of PLA, Department of Neurosurgery, Xinqiao Hospital, Third Military Medical University, Chongqing 400037, China 2 Department of Neurosurgery, General Hospital of the People's Liberation Army Lanzhou Military Region, Lanzhou, Gansu, 730050, China 3 Department of Neurosurgery, General Hospital of the People's Liberation Army Chengdu Military Region, Chengdu, Sichuan, 610083, China Correspondence to: Chunqing Zhang, email: cqzhang@tmmu.edu.cn Shiyong Liu, email: shiyongxinqiao@163.com Keywords: focal cortical dysplasia, epileptogenesis, pathogenesis, pannexin 1, pannexin 2 Received: May 16, 2016 Accepted: December 16, 2016 Published: December 28, 2016 ABSTRACT Focal cortical dysplasia (FCD) is a major cause of intractable epilepsy in children however the mechanisms underlying the pathogenesis of FCD and FCD induced epilepsy remain unclear. Increasing evidence suggests that the large-pore ion channels, pannexin 1 (Panx1) and 2 (Panx2), are involved in epilepsy and brain development. In this study, we investigated the expression of Panx1 and Panx2 in surgical samples from patients with FCD type Ia (FCDIa), type IIa (FCDIIa), and type IIb (FCDIIb) and in age-matched autopsy control samples. We found Panx1 mRNA and protein levels were both increased in all these FCD samples. Immunohistochemical analyses revealed that Panx1 was mainly distributed in microcolumn neurons, dysmorphic neurons (DNs), balloon cells (BCs) and reactive astrocytes. Double-labeled staining showed that the Panx1-positive neurons were mostly glutamatergic DNs and occasionally GABAergic normal-appearing neurons. Importantly, the protein levels of Panx1 positively correlated with the frequency of seizures. Intriguingly, the Panx2 mRNA and protein levels were only upregulated in FCDIIb lesions and characteristically expressed on SOX2-positive multipotential BCs. Immunofluorescent experiments identified that Panx2-positive BCs mainly expressed the neuronal differentiation transcription factor MASH1 but not the immature glial marker vimentin. Taken together, our results established a potential role of the specific expression and cellular distribution patterns of Panx1 and Panx2 in FCD-associated epileptogenesis and pathogenesis.

Introduction: Lennox-Gastaut syndrome (LGS) is a severe subtype of childhood-onset epileptic encephalopathy with drug-resistant and poor surgical prognosis. However, electroencephalogram (EEG) patterns of symptomatic LGS or LG phenotypes with structural brain lesions including focal abnormalities or asymmetric slow-spike-wave (SSW) patterns remain largely unknown. Due to the contradictory lateralization difference between MRI lesions and EEG pattern in symptomatic LGS or LG phenotypes, it is difficult to determine the precise lateralization of epileptic lesions, which is crucial to better surgical prognosis. This study is aim to ascertain the clinical characteristics of the EEG patterns, and its relationship with MRI lesions and to evaluate its prognostic value of surgical treatment in symptomatic LGS or LG phenotypes. Methods: Twenty-four symptomatic LGS cases with asymmetric EEG SSW patterns and contralaterally independent or contralaterally dominant MRI lesions were collected, and their clinical features were analyzed retrospectively. Results: In this cohort, most of lesions were perinatal or acquired during the first 6 months of life. The most common etiology was intracerebral hemorrhage. The LGS patients with both asymmetric SSW and focal sporadic epileptic waves (SEW) patterns showed the best surgical outcome with Engel class I level. Asymmetric SSW patterns with maximal discharges contralateral to MRI lesions were frequently observed in most of symptomatic LGS or LG phenotypes. Predominantly diffuse destructive lesions led to an attenuated voltage of ipsilateral scalp EEG producing an asymmetric SSW pattern in those patients with symptomatic LGS or LG phenotypes. Conclusions: Our study reveals a special SEW EEG pattern in symptomatic LG patients with asymmetric SSW and MRI lesions contralateral to the dominant EEG patterns. Contradictory lateralization difference between MRI and EEG probably arises from the relative voltage attenuation presenting in EEG ipsilateral to huge destructive lesions from early life. Our study suggests that the independent focal SEW activity remaining ipsilateral to the MRI lesion can potentially predict better surgical prognosis in symptomatic LGS or LG phenotypes.

Backgrounds Focal cortical dysplasia type IIb (FCD IIb) and tuberous sclerosis complex (TSC) are very frequently associated with epilepsy in pediatric patients. Human leukocyte immunoglobulin-like receptor B2 (LILRB2) participates in the process of neurite growth, synaptic plasticity, and inflammatory reaction, suggesting a potential role of LILRB2 in epilepsy. However, little is known about the distribution and expression of LILRB2 in cortical lesions of FCD IIb and cortical tubers of TSC. Methods In this study, we have described the distribution and expression of LILRB2 signaling pathway in cortical lesions of pediatric patients with FCD IIb (n = 15) and TSC (n = 12) relative to age-matched autopsy control samples (CTX, n = 10), respectively. The protein levels of LILRB2 pathway molecules were assessed by western blotting and immunohistochemistry. The expression pattern was investigated by immunohistochemistry and double labeling experiment. Spearman correlation analysis to explore the correlation between LILRB2 protein level and seizure frequency. Results The protein levels of LILRB2 and its downstream molecules POSH, SHROOM3, ROCK1, ROCK2 were increased in cortices of patients compared to CTX. Protein levels of LILRB2 negatively correlated with the frequency of seizures in FCD IIb and TSC patients, respectively. Moreover, all LILRB2 pathway molecules were strongly expressed in dysmorphic neurons, balloon cells, and giant cells, LILRB2 co-localized with neuron marker and astrocyte marker. Conclusion Taken together, the special expression patterns of LILRB2 signaling pathway in cortical lesions of FCD IIb and TSC implies that it may be involved in the process of epilepsy.

At least 50% of patients with tuberous sclerosis complex present with intractable epilepsy; for these patients, resective surgery is a treatment option. Here, we report a nationwide multicentre retrospective study and analyse the long-term seizure and neuropsychological outcomes of epilepsy surgery in patients with tuberous sclerosis complex. There were 364 patients who underwent epilepsy surgery in the study. Patients’ clinical data, postoperative seizure outcomes at 1-, 4-, and 10-year follow-ups, preoperative and postoperative intelligence quotients, and quality of life at 1-year follow-up were collected. The patients’ ages at surgery were 10.35 ± 7.70 years (range: 0.5–47). The percentage of postoperative seizure freedom was 71% (258/364) at 1-year, 60% (118/196) at 4-year, and 51% (36/71) at 10-year follow-up. Influence factors of postoperative seizure freedom were the total removal of epileptogenic tubers and the presence of outstanding tuber on MRI at 1- and 4-year follow-ups. Furthermore, monthly seizure (versus daily seizure) was also a positive influence factor for postoperative seizure freedom at 1-year follow-up. The presence of an outstanding tuber on MRI was the only factor influencing seizure freedom at 10-year follow-up. Postoperative quality of life and intelligence quotient improvements were found in 43% (112/262) and 28% (67/242) of patients, respectively. Influence factors of postoperative quality of life and intelligence quotient improvement were postoperative seizure freedom and preoperative low intelligence quotient. The percentage of seizure freedom in the tuberectomy group was significantly lower compared to the tuberectomy plus and lobectomy groups at 1- and 4-year follow-ups. In conclusion, this study, the largest nationwide multi-centre study on resective epilepsy surgery, resulted in improved seizure outcomes and quality of life and intelligence quotient improvements in patients with tuberous sclerosis complex. Seizure freedom was often achieved in patients with an outstanding tuber on MRI, total removal of epileptogenic tubers, and tuberectomy plus. Quality of life and intelligence quotient improvements were frequently observed in patients with postoperative seizure freedom and preoperative low intelligence quotient.

SummaryAim Infantile spasms (IS) are an age-specific epileptic syndrome with specific clinical symptom and electroencephalogram (EEG) features, lacking treatment options, and a poor prognosis. Excessive endogenous corticotropin-releasing hormone (CRH) in infant brain might result in IS. However, the data from human IS are limited. In our study, we investigated the expressions of CRH and its receptor type 1 (CRHR1) in surgical tissues from patients with IS and autopsy controls. Methods Specimens surgically removed from 17 patients with IS, and six autopsy controls were included in the study. Real-time PCR, Western blotting, and immunostaining were used to detect the expressions of mRNA, protein expression, and distribution. The correlation between variates was analyzed by Spearman rank correlation. Results The expressions of CRH and CRHR1 were significantly upregulated in the epileptogenic tissues of IS patients compared with the control group. CRH was distributed mainly in neurons, while CRHR1 was distributed in neurons, astrocytes, and microglia. The expression levels of CRH and CRHR1 were positively correlated with the frequency of epileptic spasms. Moreover, the expression of protein kinase C (PKC), which was an important downstream factor of CRHR1, was significantly upregulated in the epileptogenic tissues of patients with IS and was positively correlated with the CRHR1 expression levels and the frequency of epileptic spasms. Conclusion These results suggest that the CRH signal transduction pathway might participate in the epileptogenesis of IS, supporting the hypothesis that CRH is related to the pathogenesis of IS.

Summary Aim Focal cortical dysplasia (FCD) represents a well-known cause of medically intractable epilepsy. Studies found that transient receptor potential vanilloid receptor 4 (TRPV4) may participate in the occurrence of seizures. This study investigated the expression patterns of TRPV4 in FCD and the cascade that regulate functional state of TRPV4 in cortical neurons. Methods Thirty-nine surgical specimens from FCD patients and 10 age-matched control samples from autopsies were included in this study. Protein expression and distribution were detected by Western blot, immunohistochemistry, and immunofluorescence staining. Calcium imaging was used to detect the TRPV4-mediated Ca2+ influx in cortical neurons. Results (1) The protein levels of TRPV4 and of an upstream factor, protein kinase C (PKC), were markedly elevated in FCD. (2) TRPV4 staining was stronger in the dysplastic cortices of FCD and mainly observed in neuronal microcolumns and malformed cells. (3) The activation of TRPV4 was central for [Ca2+]i elevation in cortical neurons, and this activity of TRPV4 in cortical neurons was regulated by the PKC, but not the PKA, pathway. Conclusion The overexpression and altered cellular distribution of TRPV4 in FCD suggest that TRPV4 may potentially contribute to the epileptogenesis of FCD.

Focal cortical dysplasias (FCDs) are major brain malformations that commonly lead to medically intractable epilepsy. The purinergic ionotropic P2X7 receptor (P2X7R) is an atypical P2X subtype that gates calcium and sodium ions. Previous animal studies have suggested that P2X7R is a contributing factor in epileptogenesis. This study aimed to define the distribution and expression of P2X7R in 35 FCD patient-surgical-resection specimens relative to autopsy control samples (n = 8). Immunohistochemical colocalization assays revealed that P2X7R was primarily expressed in neurons, astrocytes, and microglia. In FCD samples, P2X7R protein levels were increased in abnormal cell types such as dysmorphic neurons and balloon cells, which are characteristic of FCD. By real-time PCR and Western blotting, P2X7R mRNA and protein expression levels were elevated in FCD patient samples vs control samples; P2X7R expression was also higher in FCDII vs FCDIa patient samples. Because interleukin-1β is a downstream factor of the P2X7R signaling pathway, we determined that there was also moderate-to-strong interleukin-1β expression in the dysmorphic neurons, balloon cells, and microglia in FCD patient lesions. These results indicate that increasing P2X7R levels may contribute to the pathogenesis of human FCD and that P2X7R represents a potential anti-epileptogenic target.

The aim of this study was to determine the predictors of seizure recurrence in surgery for focal cortical dysplasia (FCD) by conducting a meta-analysis. Publications that met the pre-stated inclusion criteria were selected from PubMed and CNKI databases. Two authors extracted data independently about prognostic factors, surgical outcome, and clinical characteristics of participants. A fixed-effects model was used to calculate the summary of odds ratio (OR) with 95% confidence interval (CI). Forty-eight studies were included in our meta-analysis. Three predictors of seizure recurrence (Engel class III/IV)—histological FCD type I, incomplete resection, and extratemporal location were determined; combined OR with 95% CI were 1.94 (95%CI 1.53–2.46), 12.06 (95%CI 7.32–19.88), and 1.91 (95%CI 1.06–3.44), respectively. Trial sequential analysis revealed that the outcomes had a sufficient sample size to reach firm conclusions. Furthermore, seizure location was not substantially modified by geographic region, while histological FCD type I and incomplete resection showed a significant association with seizure recurrence in different continents except Asia for incomplete resection. Sensitivity analyses restricted to studies for each variable yielded robust results. Little evidence of publication bias was observed. Meanwhile, the difference in the standard for outcome failed to influence the results for prognosis. Network meta-analysis including 13 trials comparing subtypes of FCD found the FCD IIb had the lowest seizure recurrence rate. This meta-analysis suggests that histological FCD type I, incomplete resection, and extratemporal location are recurrence factors in patients with epilepsy surgery for FCD. In addition, FCD IIb is associated with the highest rates of postoperative seizure control among the subtypes of FCD, type I and type II.

Focal cortical dysplasia (FCD) is the main cause of medically intractable pediatric epilepsy. Previous studies have suggested that alteration of cortical interneurons and abnormal cytoarchitecture have been linked to initiation and development for seizure. However, whether each individual subpopulation of cortical interneurons is linked to distinct FCD subtypes remains largely unknown. Here, we retrospectively analyzed both control samples and epileptic specimens pathologically diagnosed with FCD types Ia, IIa, or IIb. We quantified three major interneuron (IN) subpopulations, including parvalbumin (PV)-, somatostatin (Sst)-, and vasoactive intestinal peptide (Vip)-positive INs across all the subgroups. Additionally, we calculated the ratio of the subpopulations of INs to the major INs (mINs) by defining the total number of the PV-, Sst-, and Vip-INs as mINs. Compared with the control, the density of the PV-INs in FCD type IIb was significantly lower, and the ratio of PV/mINs was lower in the superficial part of the cortex of the FCD type Ia and IIb groups. Interestingly, we found a significant increase in the ratio of Vip/mINs only in FCD type IIb. Overall, these results suggest that in addition to a reduction in PV-INs, the increase in Vip/mINs may be related to the initiation of epilepsy in FCD type IIb. Furthermore, the increase in Vip/mINs in FCD type IIb may, from the IN development perspective, indicate that FCD type IIb forms during earlier stages of pregnancy than FCD type Ia.

Toll-like receptors (TLRs) that mediate inflammatory responses play an important role in epilepsy; however, whether TLR1 is also involved in epileptogenesis remains unclear. Thus, in this study, we investigated the extent and pattern of TLR1 expression in epileptic tissues. One-hundred and thirty-two mice were intra-cerebroventricularly injected with PBS or kainic acid (KA) and were examined at 1, 3, 8 and 24 h. The expression pattern and distribution of TLR1 were examined by reverse-transcriptase polymerase chain reaction (RT-PCR), western blot analysis and immunohistochemistry staining. The mRNA and protein levels of TLR1 were significantly upregulated in the hippocampus and temporal cortex of epileptic mice compared with those of controls. TLR1 expression was increased as early as 1 h following KA treatment and peaked at 8 and 24 h. Immunohistochemistry staining demonstrated that TLR1 was distributed in the CA1-3, dentate gyrus and hilus regions of the hippocampus and different cortical regions. Immunofluorescent staining further revealed that TLR1 was primarily expressed in the neurons, microglia, and astrocytes of epileptogenic tissue. These results demonstrate that cortical and hippocampal sub-regional expression of TLR1 is altered during epileptogenesis in a time- and location-specific manner, suggesting a close association with the process of epilepsy.

Increasing evidence indicates that inflammatory processes play a crucial role in the etiopathology of epilepsy and seizure disorders. The Toll/IL-1R domain-containing adapter-inducing IFN-β (TRIF) activated several transcriptions leading to the production of pro-inflammatory cytokines in the central nervous system, which suggests a potential role for TRIF in the epileptogenesis of epilepsy. In this study, we investigated the roles of TRIF in human and mice epileptogenic tissues. Western blot and immunohistochemistry assays showed that the expression of TRIF was significantly upregulated in neurons and glial cells in both human epileptic tissues and mouse models, and positively correlated with seizure frequency. TRIF expression positively correlated with high-mobility group box 1 (HMGB1) expression. In TRIF-deficient mice, electroencephalograms displayed a significant decrease in seizure frequency and duration time, while KA induced seizures compared with wild-type (WT) mice. The number and duration time of spontaneous seizures were also decreased in the chronic KA-induced TRIF-deficient mouse models. In TLR4-deficient hippocampal neurons and mouse models, TRIF expression was lower compared with WT mice during HMGB1 and KA stimulation. Meanwhile, in KA-induced TRIF-deficient mouse models, microglia activation was significantly suppressed; pro-inflammatory factors including IL-1β, TNF-α, iNOS, HMGB1 and IFN-β were reduced; and the survival of the neurons in the hippocampus increased compared with WT mice. Our findings suggested that TRIF may be involved in the epileptogenesis of temporal lobe epilepsy, which would make it a potential therapeutic target for the treatment of epilepsy.

Focal cortical dysplasia (FCD) is a well-known cause of medically intractable epilepsy. To understand the potential role of the inflammatory cytokine interleukin 2 (IL-2) in the pathogenesis of FCD, we investigated the expression patterns of IL-2 and its receptors (IL-2Rs) in FCD and control samples that included epileptic neocortex from mesial temporal lobe epilepsy patients and nonepileptic normal cortex (CTX). Greater mRNA and protein levels of IL-2 and IL-2Rs were observed in FCD versus CTX samples. Moreover, the expression of IL-2 and IL-2Rs was significantly higher in FCD II than FCD I. In situ hybridization and immunohistochemistry results indicated that IL-2 and IL-2Rs were strongly expressed in hypertrophic neurons and neuronal microcolumns in FCD I and highly expressed in malformed cells in FCD II. In addition, the protein levels of Janus kinase 1, Janus kinase 3, phosphorylated signal transducer and activator of transcription 5, which are important downstream factors in the IL-2 signaling pathway, were increased in FCD lesions. Soluble IL-2R was decreased in FCD compared with that in CTX samples. These results suggest that upregulation of IL-2 and IL-2Rs combined with activation of IL-2-dependent signaling pathways may contribute to the pathogenesis of FCD.

The role of interleukin 17 (IL-17) to epilepsy-associated cortical tubers of tuberous sclerosis complex (TSC) is unknown. We investigated the expression patterns of the IL-17 and IL-17 receptor (IL-17R) in cortical tubers of TSC compared with normal control cortex (CTX). We found that IL-17 and IL-17R were clearly upregulated in cortical tubers at the protein levels. Immunostaining indicated that IL-17 was specifically distributed in the innate immunity cells (DNs, GCs, astrocytes, and microglia) and adaptive immunity cells (T-lymphocytes) as well as the endothelial cells of blood vessels. The overexpression and distribution patterns of IL-17 may be involved in the epileptogenicity of cortical tubers in TSC.

Tuberous sclerosis complex (TSC) and focal cortical dysplasia type IIb (FCDIIb) are recognized as causes of intractable epilepsy. Transient receptor potential vanilloid receptor 1 (TRPV1), a member of the transient receptor potential family, is the capsaicin receptor and is known to be involved in peripheral nociception. Recent evidence suggested that TRPV1 may be a contributing factor in epileptogenicity. Here, we evaluated the expression of TRPV1 in the cortical lesions of TSC and FCDIIb relative to normal control cortex. TRPV1 was studied in epilepsy surgery cases with TSC (cortical tubers; n=12) and FCDIIb (n=12) using immunocytochemistry, confocal analysis, and Western blotting (WB). Immunohistochemical location of the TRPV1 was predominately detected in the abnormal cell types, such as dysmorphic neurons, balloon cells (BCs) and giant cells. Co-localization assays further revealed that cells expressing TRPV1 mainly had a neuronal lineage, apart from some BCs in FCDIIb, which obviously were of astrocytic lineage. The increased TRPV1 expression within the dysplastic cortex of TSC and FCDIIb was confirmed by WB. Interestingly, both immunohistochemical and WB data indicated that TRPV1 might have both cytoplasm and nuclear distribution, suggesting a potential nuclear role of TRPV1. The over-expression of TRPV1 in cortical lesions of TSC and FCDIIb suggested the possible involvement of TRPV1 in the intrinsic and increased epileptogenicity of malformations of cortical development associated epilepsy diseases and may represent a potential antiepileptogenic target. However, the current data are merely descriptive, and further electrophysiological investigation is needed in the future.

Aim To investigate the application of neuronavigation in the implantation of depth electrodes in patients with epilepsy. Material and methods Thirty-six patients with epilepsy who were implanted with depth electrodes using neuronavigation were assessed for accuracy of implantation and associated complications. Results In the imaging navigation group, patients were implanted with 2-14 depth electrodes. The average number of implantations was 4.8 electrodes/case. The average implantation error was 2.03 ± 0.98 mm, exhibiting no significant difference compared to the frame-based stereotactic group. In the imaging group, an average of 19.4 min was required to implant each electrode, which was significantly shorter than the time required in the frame group (34.5 min). The temporal lobe was elucidated as the factor that affects electrode implantation accuracy. One patient in the imaging group exhibited a small amount of bleeding, and one suffered from cerebrospinal fluid leakage; however, the overall complication rate in the imaging group was lower than that in the frame group. Conclusion Imaging navigation provides better means of depth electrode implantation; its implantation accuracy is similar to that of the frame-based stereotactic method and it is less time consuming and causes less complications, and is especially suitable for stereoelectroencephalography, which requires multiple depth electrodes.

Focal cortical dysplasia (FCD) is strongly associated with medically intractable epilepsy. Studies suggest that liver X receptor beta (LXRβ) may participate in the pathogenesis of FCD. The present study investigated the expression pattern of LXRβ in FCD and the distribution of LXRβ in different neural precursor cells. Twenty-five surgical specimens from FCD patients and 11 age-matched control samples from autopsies were included in our study. Protein levels and distribution were detected by western blot, immunohistochemistry, and immunofluorescence staining. We found that (1) the level of LXRβ protein was markedly reduced in FCD. (2) LXRβ staining was weaker in the dysplastic cortices of FCD and was mainly observed in neuronal microcolumns, and malformed cells. (3) LXRβ was co-localized with radial glial cells (RGCs) markers and oligodendrocyte precursor cells (OPCs) markers in malformed cells. (4) RGCs marker and OPCs marker were down-regulated while LXRβ downstream factors were up-regulated in FCD specimens. Taken together, our results indicate that LXRβ may interact with β-catenin to regulate the generation of OPCs and the transformation of RGCs. LXRβ therefore potentially contributes to the pathogenesis of FCD.

Temporal lobe epilepsy (TLE) is a frequent form of focal intractable epilepsy in adults, but the specific mechanism underlying the epileptogenesis of TLE is still unknown. Human leukocyte immunoglobulin-like receptor B2 (LILRB2) (the murine homolog gene called paired immunoglobulin-like receptor B, or PirB), participates in the process of synaptic plasticity and neurite growth in the central nervous system (CNS), suggesting a potential role of LILRB2 in epilepsy. However, the expression pattern of LILRB2 and the downstream molecular signal in intractable TLE remains poorly understood. In the present study, western blotting and immunohistochemistry results showed that LILRB2 expression was upregulated in the temporal neocortex of patients with TLE. Moreover, protein levels of LILRB2 negatively correlated with the frequency of seizures in TLE patients. In the pilocarpine-induced C57BL/6 mouse model, PirB upregulation in the hippocampus began 12h after status epilepticus (SE), reached a peak at 7days and then maintained a significantly high level until day 60. Similarly, we found a remarkable increase in PirB expression at 1day, 7days and30days post-SE in the temporal cortex. Double-labeled immunofluorescence showed that LILRB2/PirB were highly expressed in neurons and astrocytes but not microglia. In addition, protein levels of POSH, SHROOM3, ROCK1 and ROCK2, the important downstream factors of the LILRB2 pathway, were significantly increased in the epileptic foci of TLE patients and located on the NeuN-positive neurons and GFAP-positive astrocytes. Taken together, our results indicate that LILRB2/PirB may be involved in the process of TLE.

Summary The efficacy of surgery for the treatment of epilepsy in patients with West syndrome secondary to tuberous sclerosis is unclear. The charts of 17 patients with tuberous sclerosis and secondary West syndrome who underwent a one-stage surgical resection with a combined palliative operative procedure were reviewed. Engel classification was used to classify the patients with regard to seizure status following surgery. After surgery, 11 patients were in Engel class I, 4 in class II, and 2 in class III. The EEG after surgery was normal in 8 patients, significantly improved in 8, and without significant improvement in 1 patient. Six patients had a recurrence of seizures after surgery, which included 3 patients with continuing spasms and 3 patients where the spasms had resolved but had developed either partial seizures or generalized tonic–clonic seizures. There were significant improvements in the Gesell Developmental Schedules for motor field ( P =0.003), adaptive field ( P =0.003), language field ( P =0.033), and personal–social field ( P =0.007). Thus, a one-stage surgical approach can be used to produce satisfactory outcomes in young children with tuberous sclerosis who have secondary West syndrome and seizures that do not respond to conventional antiepileptic therapy, even in when there are multiple epileptogenic foci.

The malformative cortical lesions in the cerebral cortex that are characteristic of focal cortical dysplasia type IIb (FCDIIb) and tuberous sclerosis complex (TSC) are well-recognized causes of chronic intractable epilepsy in children. Increasing evidence suggests that extracellular matrix molecules play important roles in epileptogenesis. Matrix metalloproteinase 9 (MMP9), a typical extracellular matrix proteolytic protease, has been shown to participate in the occurrence of seizures in experimental models. In the present study, we used immunoblotting to analyze the levels of MMP9 protein in FCDIIb lesions, TSC tubers and control samples, which included epileptic neocortices from temporal lobe epilepsy and non-epileptic normal cortices (CTX). The cellular distribution of MMP9 was further investigated by immunohistochemical methods. Our findings demonstrated the elevated levels of the inactive and active forms of MMP9 protein in FCDIIb and TSC lesions compared with CTX. Furthermore, the immunohistochemical results showed that MMP9 was characteristically expressed in the following misshapen cells: hypertrophic neurons, dysmorphic neurons, balloon cells and giant cells. Additionally, double immunofluorescent staining revealed that the reactive astrocytes, but not the microglia, expressed high levels of MMP9. Taken together, our findings suggest that the overexpression and spatial distribution patterns of MMP9 may be linked with the intractable epilepsy caused by FCDIIb and TSC.

Mesial temporal lobe epilepsy (MTLE) is a frequent form of focal intractable epilepsy in adults. We previously reported overexpression of vascular endothelial growth factor C (VEGF-C) and its receptors, VEGFR-2 and VEGFR-3, in epilepsy-associated tuberous sclerosis complex. To identify whether VEGF-C and its receptors are involved in epileptogenesis of MTLE, we investigated the levels and expression pattern of VEGF-C and its receptors in temporal neocortex and hippocampus (HPC) from 28 patients with MTLE and ten control (CTX) subjects. Real-time quantitative polymerase chain reaction and Western blotting results revealed upregulated mRNA and immunoreactive protein levels of VEGF-C, VEGFR-2, and VEGFR-3 in the MTLE group compared to the control groups. Immunohistochemistry and double-labeled immunofluorescence showed that VEGF-C was highly expressed in neurons and astrocytes, including reactive astrocytes and vascular endothelial cells, VEGFR-2 was expressed at a high level in reactive astrocytes and vascular endothelial cells, but not in neurons, whereas VEGFR-3 was only overexpressed in reactive astrocytes. Taken together, these findings suggest that VEGF-C and its receptors, VEGFR-2 and VEGFR-3, may contribute to the epileptogenesis of MTLE.

Cortical tubers are malformations of cortical development in patients with tuberous sclerosis complex (TSC), and highly associated with pediatric intractable epilepsy. Recent evidence has shown that signaling mediated through vascular endothelial growth factor-C (VEGF-C) and its receptors, VEGFR-2 and VEGFR-3, has direct effects on both neurons and glial cells. To understand the potential role of VEGF-C system in the pathogenesis of cortical tubers, we investigated the expression patterns of VEGF-C signaling in cortical tubers compared with age-matched normal control cortex (CTX). We found that VEGF-C, VEGFR-2 and VEGFR-3 were clearly upregulated in tubers at both the mRNA and protein levels, compared with CTX. The in situ hybridization and immunostaining results demonstrated that VEGF-C, VEGFR-2 and VEGFR-3 were highly expressed in dysplastic neurons (DNs), giant cells (GCs) and reactive astrocytes within tubers. Most DNs/GCs expressing VEGF-C and its receptors co-labeled with neuronal rather than astrocytic markers, suggesting a neuronal lineage. In addition, protein levels of Akt-1, p-Bad and ERK1/2, the important downstream factors of the VEGF-C pathway, were significantly increased in cortical tubers, indicating involvement of VEGF-C-dependent prosurvival signaling in cortical tubers. Taken together, our results suggest a putative role for the VEGF-C signaling pathway in the pathogenesis of cortical tubers.

Tuberous sclerosis complex (TSC) and focal cortical dysplasia type IIb (FCDIIb) are characterized by epilepsy-associated cerebral cortical malformations. To understand the potential role of the inflammatory cytokine interleukin 6 (IL-6) in the pathogenesis of these lesions, we analyzed the IL-6 system in TSC and FCDIIb cortical lesions and in control cortex (CTX). Greater messenger RNA and protein levels of IL-6 and of its receptors (i.e. IL-6 receptor [IL-6R] and glycoprotein 130 [gp130]) were observed in TSC and FCDIIb lesions versus CTX. Immunohistochemical analyses indicated that IL-6 and IL-6R were strongly expressed in misshapen cells, namely, dysmorphic neurons, giant neurons, and balloon cells. Glycoprotein 130 was diffusely expressed in nearly all cell types. Most IL-6/IL-6R+ misshapen cells colabeled with neuronal rather than astrocytic markers, suggesting a neuronal lineage; most IL-6/IL-6R+ balloon cells in FCDIIb expressed glial fibrillary acidic protein. Protein levels of Janus kinase 2 and phosphorylated signal transducer and activator of transcription 3 were greater than in CTX, suggesting involvement of the gp130-Janus kinase 2-signal transducer and activator of transcription 3 pathway in IL-6 signal transduction. Soluble IL-6R, but not soluble gp130, was greater in TSC and FCDIIb lesions than in CTX, indicating activation of this trans-signaling pathway. These results suggest that overexpression in the IL-6 system and activation of IL-6 signal transduction pathways may contribute to the pathogenesis of cortical lesions in TSC and FCDIIb.

The subventricular zone (SVZ) located adjacent to the lateral ventricles is the major site where neural progenitor cells (NPCs) are concentrated in the adult brain. NPCs in the anterior subventricular zone (SVZa) generate neuronal precursors and migrate along a highly localized pathway--the rostral migratory stream (RMS) to the olfactory bulb (OB), where they differentiate into interneurons. To investigate the modulatory effects of basic helix-loop-helix (bHLH) transcription factors on differentiation from SVZa NPCs, we firstly examined the distribution of bHLH family members (Mash1, Id2, and Hes1) in cultured mouse SVZa NPCs and evaluated their regulatory effects on differentiation by transfection with Mash1, Id2, or Hes1 eukaryotic expression plasmid. Furthermore, we assessed the effects of bHLH transcription factors on the expression of downstream molecules of the Wnt/beta-catenin pathway, beta-catenin and (Glycogen synthase kinase-3beta). Our results demonstrated that Mash1, Id2, Hes1 were all widely expressed in in vitro progenies from mouse SVZa NPCs. Analyses of SVZa NPCs transfected with eukaryotic expression plasmids showed that Mash1 promoted neuronal differentiation from SVZa NPCs, while Id2 and Hes1 repressed neuronal differentiation. In addition, we found that Id2 and Hes1 simulated expression of beta-catenin and GSK-3beta, while Mash1 inhibited their expression. Our results suggest that the classic bHLH transcription factors, Mash1, Id2 and Hes1, play important roles in the regulation of differentiation from SVZa NPCs. This modulation is possibly mediated by a coordination of bHLH and Wnt/beta-catenin signaling.

The dentate gyrus (DG) of the hippocampus is one of a few regions in the adult mammalian brain characterized by ongoing neurogenesis. Proliferation of neural precursors in the granule cell layer of the DG has been identified in pentylenetetrazol (PTZ) kindling epilepsy model, however, little is known about the molecular mechanism. We previously reported that the expression pattern of bone morphogenetic proteins-4 (BMP4) mRNA in the hippocampus was developmentally regulated and mainly localized in the DG of the adult. To explore the role of BMP4 in epileptic activity, we detected BMP4 expression in the DG during PTZ kindling process and explore its correlation with cell proliferation combined with bromodeoxyuridine (BrdU) labeling technique. We found that dynamic changes in BMP4 level and BrdU labeled cells dependent on the kindling stage of PTZ induced seizure-prone state. The number of BMP4 mRNA-positive cells and BrdU labeled cells reached the top level 1 day after PTZ kindled, then declined to base level 2 months later. Furthermore, there was a significant correlation between increased BMP4 mRNA expression and increased number of BrdU labeled cells. After effectively blocked expression of BMP4 with antisense oligodeoxynucleotides(ASODN), the BrdU labeled cells in the dentate gyrus subgranular zone(DG-SGZ) and hilus were significantly decreased 16d after the first PTZ injection and 1, 3, 7, 14d after kindled respectively. These findings suggest that increased proliferation in the DG of the hippocampus resulted from kindling epilepsy elicited by PTZ maybe be modulated by BMP4 over-expression.

Focal cortical dysplasia (FCD) is known as a common cause of chronic refractory epilepsy, but the underlying mechanisms of the factors that lead to FCD-related epilepsy are unclear. Previous studies have shown that canonical transient receptor potential channels (TRPCs) might be involved in the process of epileptogenesis. Canonical transient receptor potential channel 1 (TRPC1), which is ubiquitously expressed in the brain, has been shown to be involved in epileptiform bust firing in knockout mice. In this study, we examined the expression of TRPC1 in FCD type Ia (FCDIa), FCD type IIa (FCDIIa), and FCD type IIb (FCDIIb) surgical specimens from patients and age-matched autopsy control samples. Real-time quantitative PCR and western blotting indicated that TRPC1 mRNA and protein levels were increased in FCDIa, FCDIIa, and FCDIIb samples compared to control samples. Immunohistochemistry results revealed that TRPC1 was mainly distributed in microcolumns, dysmorphic neurons, and balloon cells. Further double immunofluorescent staining showed that TRPC1 was co-localized with glutamatergic and GABAergic markers. Taken together, our results demonstrate that the overexpression and specific cellular location of TRPC1 might be related to the epileptogenesis of FCD.

Neural stem cells (NSCs) reside in the anterior portion of the forebrain subventricular zone (SVZa) and generate the progenitors which will differentiate into neurons, and via a tangential migratory pathway, known as the rostral migratory stream (RMS), migrate to the olfactory bulbs (OB). Bone morphogenetic proteins (BMPs) play significant roles in neural development at different stages and locations, but their roles have not been determined in the SVZa. To explore possible roles of BMPs in SVZa NSCs, BMP4 at various concentrations were tested for their capacity to induce SVZa NSCs. The expression of BMP4 was also examined in living cells using a reportor vector, in which the BMP4 promotor was conjugated with red fluorescent protein (RFP). In the meantime, the differentiation of SVZa NSCs was dynamically monitored by using reportor vectors of the Nestin enhancer and the promoters of TH and GFAP. In the OB, high expression of BMP4 was found using both promoter activity analysis and in situ hybridization. However, low BMP4 expression was found in the RMS and only moderate expression of BMP4 was displayed in the SVZa. The results also demonstrated that low concentrations (1-5 ng/ml) of BMP4 promoted the proliferation of SVZa NSCs but high concentrations (10-100 ng/ml) of BMP4 inhibited this proliferation. BMP4 enhanced neuron commitment before 4 days but inhibited it after 4 days. As the antagonist of BMP4, Noggin almost completely blocked all these BMP4 responses. Thus, our findings indicate that BMP4 promotes the exit from the cell cycle and triggers the differentiation of neuron progenitors in the OB. BMP4 also promotes the proliferation of the committed neuron progenitors in the RMS, but in the SVZa, BMP4 may facilitate the commitment of NSCs into astrocytes.

The dentate gyrus of the hippocampus is one of few regions in the adult mammalian brain characterized by ongoing neurogenesis. It has been demonstrated that Noggin antagonizes bone morphogenetic protein-4 (BMP4) to create a niche for subventricular zone neurogenesis. We previously demonstrated that Noggin and BMP4 showed strong expression in the proliferative subgranular layer of the dentate gyrus in adult rats. To examine the action of Noggin on cell proliferation in the dentate gyrus of adult rats, we administered antisense oligodeoxynucleotide (ASODN) to Noggin by continuous infusion into the lateral ventricle of rats. Antisense-infused rats displayed significant reduction in number of bromodeoxyuridine (BrdU) labeled cells in the dentate gyrus. This indicated that endogenous Noggin activity is important for naturally occurring cell proliferation in the dentate gyrus, and perhaps neurogenesis, and is one of the many factors involved in its regulation.

Mutations in RNA-binding Fox 1 (RBFOX1) are known to be associated with neurodevelopmental disorders including epilepsy, mental retardation and autism spectrum disorder. The deletion of the Rbfox1 gene in mice has been shown to result in heightened susceptibility to seizures. However, other studies have revealed mutations or the downregulation of RBFOX1 in specimens obtained from patients with epilepsy or malformations of cortical development (MCD). Generally, the expression of RBFOX1 varies according to tissue type. In this study, we demonstrated the upregulation of RBFOX1 protein in the cortex of patients with MCD and intractable epilepsy. Electrophysiological recordings of cultured rat cortical neurons with increased Rbfox1 expression also revealed a significantly increased amplitude of action potential (AP) and Na+ current density. Some of these neurons (26.32%) even displayed spontaneous, recurrent, epileptiform discharges (SREDs). Additionally, certain Rbfox1 target transcripts associated with epilepsy, including glutamate receptor, ionotropic, N-methyl D-aspartate 1 [Grin1, also known as N-methyl-D-aspartate receptor subunit NR1 (NMDAR1)], synaptosomal-associated protein, 25 kDa (SNAP-25 or Snap25) and sodium channel, voltage gated, type VIII, alpha subunit (Scn8a, also known as Nav1.6) were identified to be upregulated in these cultured cortical neurons with an upregulated Rbfox1 expression. These data suggest that the upregulation of RBFOX1 contributes to neuronal hyperexcitation and seizures. The upregulation of NMDAR1 (Grin1), SNAP-25 (Snap25) and Scn8a may thus be involved in Rbfox1-related neuronal hyperexcitation.

Focal cortical dysplasia (FCD) represents a well-recognized cause of medically intractable epilepsy. Previous studies have indicated that seizures can reduce brain pH and then eliminate seizure discharges. Acid-sensing ion channels (ASICs) are H(+)-gated cation channels that are widely expressed in the central and peripheral nervous systems. To understand the potential roles of ASIC1a in the epileptogenesis of FCD, we investigated the expression and distribution patterns of ASIC1a in surgical specimens from patients with FCD and age-matched normal cortices (CTX). Decreased ASIC1a messenger RNA (mRNA) and protein expression were detected in FCD compared with CTX. Moreover, the expression of ASIC1a was significantly lower in FCD type II than FCD type I. Immunohistochemistry results indicated that the overall immunoreactivity of the ASIC1a staining was diminished in the dysplastic cortices of FCD compared to the CTX samples. In FCD, ASIC1a immunoreactivity was mainly observed in reactive astrocytes and a minority of malformed cells, including hypertrophic neurons, dysmorphic neurons, and balloon cells. Confocal analysis demonstrated that most malformed cells expressing ASIC1a were co-labeled with neuronal rather than astrocytic markers, indicating a neuronal lineage. In conclusion, the downregulation and altered cellular distribution of ASIC1a in FCD suggest that ASIC1a may potentially contribute to the epileptogenesis of FCD.

Microgyria is associated with epilepsy and due to developmental disruption of neuronal migration. However, the role of endogenous subventricular zone-derived neural progenitors (SDNPs) in formation and hyperexcitability has not been fully elucidated. Here, we establish a neonatal cortex freeze-lesion (FL) model, which was considered as a model for focal microgyria, and simultaneously label SDNPs by CM-DiI. Morphological investigation showed that SDNPs migrated into FL and differentiated into neuronal and glia cell types, suggesting the involvement of endogenous SDNPs in the formation of FL-induced microgyria. Patch-clamp recordings in CM-DiI positive (CM-DiI(+)) pyramidal neurons within FL indicated an increase in frequency of spontaneous action potentials, while the resting membrane potential did not differ from the controls. We also found that spontaneous excitatory postsynaptic currents (EPSCs) increased in frequency but not in amplitude compared with controls. The evoked EPSCs showed a significant increase of 10-90% in rise time and decay time in the CM-DiI(+) neurons. Moreover, paired-pulse facilitation was dramatically larger in CM-DiI(+) pyramidal neurons. Western blotting data showed that AMPA and NMDA receptors were increased to some extent in the FL cortex compared with controls, and the NMDA/AMPA ratio of eEPSCs at CM-DiI(+) pyramidal neurons was significantly increased. Taken together, our findings provide novel evidence for the contribution of endogenous SDNPs in the formation and epileptogenicity of FL-induced focal microgyria.

Focal cortical dysplasias (FCDs) are increasingly recognized as important causes of medically intractable epilepsy. To understand the potential role of the interleukin 17 (IL-17) system in the epileptogenesis of FCDs, we studied the expression patterns of the IL-17 system in 15 FCD type Ia (FCDIa), 12 FCD type IIa (FCDIIa), and 12 FCD type IIb (FCDIIb) cortical lesions and compared the results with those in cerebral cortex from 10 control patients. Protein levels of IL-17, IL-17 receptor (IL-17R), and downstream factors of the IL-17 pathway (nuclear factor-κB activator 1 [NFκB; ACT1] and NFκB-p65) were markedly elevated in FCDIa, FCDIIa, and FCDIIb. Moreover, protein levels of IL-17 and IL-17R positively correlated with the frequency of seizures in FCD patients. Immunostaining indicated that IL-17 and IL-17R are highly expressed in neuronal microcolumns, dysmorphic neurons, balloon cells, astrocytes, and vascular endothelial cells. Nuclear factor-κB activator 1 and NFκB-p65 were diffusely expressed in FCDs. In addition, we detected a few IL-17-positive, CD4-positive T lymphocytes in FCDIIa and FCDIIb but not in FCDIa. Taken together, these findings suggest that the overexpression of the IL-17 system and the activation of the IL-17 signal transduction pathway may be involved in the epileptogenicity of cortical lesions in FCDs, thus representing a novel potential target for antiepileptic therapy.

The reticulon protein Nogo-A is an important regulator of neurite growth, axonal plasticity, and cell migration in the central nervous system. Previous studies have shown markedly elevated levels of Nogo-A in human temporal lobe epilepsy. In the present study, we examined the expression pattern of the Nogo-A system in cortical lesions of pediatric patients with tuberous sclerosis complex and focal cortical dysplasia type IIb. These disorders are characterized by malformations of cortical development and are frequently associated with intractable epilepsy. We found that the messenger RNA and protein levels of the Nogo-A receptor (NgR) and the downstream targets of Nogo-A, LINGO-1, TROY, and RhoA but not P75 were upregulated in the cortices of patients compared with autopsy control samples. Immunohistochemical analyses indicated that Nogo-A and NgR were strongly expressed in misshapen cells, particularly dysmorphic neurons, balloon cells, and giant cells. TROY was diffusely expressed in the malformations of cortical development. Most of the Nogo-A/NgR-positive misshapen cells were colabeled with neuronal rather than astrocytic markers. Taken together, our results suggest that the activation of Nogo-A via the NgR/LINGO-1/TROY signal transduction pathways, but not NgR/LINGO-1/P75, may be involved in the development and/or seizure activity of cortical lesions in tuberous sclerosis complex and focal cortical dysplasia type IIb.

International audience; The similarities between brain tumor stem cells and neural stem cells suggest a possible stem cell origin of tumorigenesis. Recently, cells with features of stem cells have been observed in lesions of adult and pediatric cortical dysplasia (CD). Given the evidence for a close relationship between CD and certain brain tumors, together with the finding that CD neural stem cells/progenitors are abnormally developed, we propose that CD is a possible substrate for brain tumors. The neural stem cells/progenitors in CD have accumulating abnormalities, and these abnormal stem/progenitor cells may be the initiating, transformed cells of brain tumors, when subsequently exposed to a carcinogen.

Lennox-Gastaut syndrome (LGS) is a devastating and refractory generalized epilepsy affecting children and adolescents. In this study we report the results of resective surgery in 18 patients with LGS phenotype who underwent single-lobe/lesionectomy or multilobe resection plus multiple subpial transection and/or callosotomy. After surgery, seven patients became completely seizure-free (Engel Class I) and five almost seizure-free (Engel Class II). Additional four had significant seizure control (Engel Class III), and two had no change in seizure frequency (Engel Class IV). Of the 4 patients without any lesion on brain MRI, 2 ended with Engel Class II, 1 with III and the other with IV in Engels’ classification. Mean intelligence quotient (IQ) increased from 56.1 ± 8.1 (mean ± SD) before operation to 67.4 ± 8.2 (mean ± SD) after operation, a significant improvement (P=0.001). Results also indicated that the younger the patient at surgery, or the shorter the interval between onset of seizure and resective operation, the better the intellectual outcome. Our data suggest that resective epilepsy surgery can be successful in patients with LGS phenotype as long as the EEG shows dominance of discharges in one hemisphere and corresponding ipsilateral imaging findings, even with contralateral ictal discharges.