Purpose: In this study, we evaluated the presence of tau deposition and protein expression of Reelin/GSK-3(3/p-Tau signaling pathway in the hippocampus of patients with temporal lobe epilepsy (TLE). Methods: A total of 37 cases of TLE with and without hippocampal sclerosis (HS) were selected histopathologically for our study with 5 autopsy cases as the control group. Immunohistochemistry and the histelide assay, a novel technique quantifying antigens in paraffin section, were used to confirm the distribution of protein within Reelin/GSK-3(3/p-Tau signaling pathway and validate the expression of GSK-3(3 and AT8 (hyperphosphorylated tau) in this study. Results: Immunohistochemical staining for AT8 revealed punctate and filamentous positive expression in the CA1, CA2 and CA3 regions of the hippocampus with TLE under the ependyma, distributed in a band-like pattern. By contrast, the control group did not exhibit any immunopositivity. GSK-3(3 was strongly positive in the neuronal bodies, apical dendrites and axons in both groups of TLE, while all controls were negative. In addition, there was no significant difference in the immunohistochemical labelling of Reelin among all cases. The histelide assay indicated that the amounts of AT8 and GSK-3(3 were significantly increased in the two TLE groups (P < 0.05). Notably, there was a positive correlation between AT8 and GSK-3(3 in TLE without HS (P < 0.05). Conclusion: The present data indicates that phosphorylated tau protein and GSK-3(3 are activated in the hippocampus of patients with TLE, and this is the first study to examine relevant proteins with the histelide assay in paraffin samples of human tissue. We consider that the regulatory network of tau protein between the two groups may be similar but not identical. Significance: This study emphasized the Reelin/GSK-3(3/p-Tau signaling pathway in TLE with a quantitative data of human tissues innovatively, revealing inspiration for mechanism exploration.

Focal cortical dysplasia (FCD) type IIb (FCD IIb) is an epileptogenic malformation of the neocortex that is characterized by cortical dyslamination, dysmorphic neurons (DNs) and balloon cells (BCs). Approximately 30–60

Meningiomas are the most common tumours that primarily arise in the central nervous system, but their intratumoural heterogeneity has not yet been thoroughly studied. We aimed to investigate the transcriptome characteristics and biological properties of ECM-remodeling meningioma cells. Single-cell RNA sequencing (ScRNA-seq) data from meningioma samples were acquired and used for analyses. We conducted comprehensive bioinformatics analyses, including screening for differentially expressed genes (DEGs), Kyoto Encyclopedia of Genes and Genomes (KEGG) signaling pathway and Gene Ontology (GO) term enrichment analyses, Gene Set Enrichment Analysis (GSEA), protein–protein interaction (PPI) analysis, and copy number variation (CNV) analysis on single-cell sequencing data from meningiomas. Eighteen cell types, including six meningioma subtypes, were identified in the data. ECM-remodeling meningioma cells (MGCs) were mainly distributed in brain-tumour interface tissues. KEGG and GO enrichment analyses revealed that 908 DEGs were mainly related to cell adhesion, extracellular matrix organization, and ECM-receptor interaction. GSEA analysis demonstrated that homophilic cell adhesion via plasma membrane adhesion molecules was significantly enriched (NES = 2.375, P < 0.001). CNV analysis suggested that ECM-remodeling MGCs showed considerably lower average CNV scores. ECM-remodeling MGCs predominantly localized at the brain-tumour interface area and adhere stably to the basement membrane with a lower degree of malignancy. This study provides novel insights into the malignancy of meningiomas.

Focal Cortical Dysplasia (FCD) is a frequent cause of drug-resistant focal epilepsy in children and young adults. The international FCD classifications of 2011 and 2022 have identified several clinico-pathological subtypes, either occurring isolated, i.e., FCD ILAE Type 1 or 2, or in association with a principal cortical lesion, i.e., FCD Type 3. Here, we addressed the DNA methylation signature of a previously described new subtype of FCD 3D occurring in the occipital lobe of very young children and microscopically defined by neuronal cell loss in cortical layer 4. We studied the DNA methylation profile using 850 K BeadChip arrays in a retrospective cohort of 104 patients with FCD 1 A, 2 A, 2B, 3D, TLE without FCD, and 16 postmortem specimens without neurological disorders as controls, operated in China or Germany. DNA was extracted from formalin-fixed paraffin-embedded tissue blocks with microscopically confirmed lesions, and DNA methylation profiles were bioinformatically analyzed with a recently developed deep learning algorithm. Our results revealed a distinct position of FCD 3D in the DNA methylation map of common FCD subtypes, also different from non-FCD epilepsy surgery controls or non-epileptic postmortem controls. Within the FCD 3D cohort, the DNA methylation signature separated three histopathology subtypes, i.e., glial scarring around porencephalic cysts, loss of layer 4, and Rasmussen encephalitis. Differential methylation in FCD 3D with loss of layer 4 mapped explicitly to biological pathways related to neurodegeneration, biogenesis of the extracellular matrix (ECM) components, axon guidance, and regulation of the actin cytoskeleton. Our data suggest that DNA methylation signatures in cortical malformations are not only of diagnostic value but also phenotypically relevant, providing the molecular underpinnings of structural and histopathological features associated with epilepsy. Further studies will be necessary to confirm these results and clarify their functional relevance and epileptogenic potential in these difficult-to-treat children.

Focal cortical dysplasia (FCD) is a major cause of drug‐resistant paediatric epilepsy and is amenable to successful neurosurgical resection. FCD ILAE Type IIb is the most common FCD subtype, and brain somatic mutations affecting the mTOR pathway play a major pathogenic role. The aim of this study was to comprehensively describe the genotype–phenotype association of 20 patients with histopathologically confirmed FCDIIb using next generation sequencing (NGS) of paired blood–brain samples.

In the United States (US), the Surveillance, Epidemiology, and End Results (SEER) program is the only comprehensive source of population-based information that includes stage of cancer at the time of diagnosis and patient survival data. This program aims to provide a database about cancer incidence and survival for studies of surveillance and the development of analytical and methodological tools in the cancer field. Currently, the SEER program covers approximately half of the total cancer patients in the US. A growing number of clinical studies have applied the SEER database in various aspects. However, the intrinsic features of the SEER database, such as the huge data volume and complexity of data types, have hindered its application. In this review, we provided a systematic overview of the commonly used methodologies and study designs for retrospective epidemiological research in order to illustrate the application of the SEER database. Therefore, the goal of this review is to assist researchers in the selection of appropriate methods and study designs for enhancing the robustness and reliability of clinical studies by mining the SEER database.

Background The correlations of epidemiological characteristics and clinical outcomes with different tumor sites in patients with intracranial typical site germinomas (ICTSGs) have not yet been well established. We analyzed ICTSGs using a multicenter database, focusing on its demographic, management patterns, and long-term survival outcomes. Methods Patients diagnosed with ICTSGs were selected from the Surveillance, Epidemiology, and End-Results (SEER) database. Demographic information and management patterns of ICTSGs were extracted for data analysis stratified by different tumor sites. Kaplan–Meier curves were used to evaluate the survival outcome stratified by treatment, tumor site and tumor size. Results Among the 327 patients enrolled in the study, 16.21% had tumors located in the suprasellar region and 83.79% in the pineal region. The proportion of males was significantly higher among pineal germinomas (94.16 vs 66.04%; P < .001). Smaller tumors (<24 mm) were more common in the suprasellar region (37.74 vs 18.87%; P < .001). A higher percentage of patients with suprasellar germinomas underwent surgery. Radiotherapy (RT) and chemotherapy (CT) was, respectively, administered to 82.97 and 60.61% of patients during the treatment period, with no significant difference between suprasellar and pineal germinomas. CT plus RT was the most common treatment modality for both pituitary (30.19%) and pineal (33.94%) germinomas. Both RT and CT were associated with improved long-term survival. No survival difference was observed between suprasellar and pineal germinomas. Conclusions Despite significant differences in epidemiology and management, pineal and suprasellar germinomas had a similar long-term clinical outcome.

AbstractBackgroundPopulation‐based estimates of the impact of gender throughout the whole course of brain metastases (BMs) at the time of diagnosis of systemic malignancies are insufficient. We aimed to discover the influence of gender on the presence of BMs in newly diagnosed malignancies and the survival of those patients on a population‐based level.MethodsMidlife patients (40 years ≤ age ≤60 years) with newly diagnosed malignancies and BMs at the time of diagnosis were abstracted from the Surveillance, Epidemiology, and End Results (SEER) database of the National Cancer Institute. Clinical variables adjusted patient data. The LASSO regression was performed to exclude the possibility of collinearity. Univariable and multivariable logistic regression analyses were applied to find independent predictors for the presence of BMs, while univariable and multivariable Cox proportional hazard regression analyses were used to determine prognosticators of survival. K‐M curves were used to perform the survival analysis.Result276,327 population‐based samples met inclusion criteria between 2014 and 2016, and 5747 (2.08%) patients were diagnosed with BMs at the time of diagnosis of systematic malignancies. Among all midlife patients with cancer, 44.02% (121,634) were male, while 51.68% (2970) were male among patients with BMs at the time of diagnosis. The most frequent tumor type was breast cancer (23.11%), and lung cancer had the highest incidence proportion of BMs among the entire cohort (19.34%). The multivariable logistic regression model suggested that female (vs. male, odds ratio [OR] 1.07, 95% CI: 1.01–1.14, p < 0.001) was associated with a higher risk of the presence of BMs at the time of diagnosis. Moreover, in the multivariable Cox model for all‐cause mortality in individuals with BMs at diagnosis, female (vs. male, hazard ratio [HR], 0.86, 95% CI, 0.80–0.92, p < 0.001) was shown to have a lower risk of decreased all‐cause mortality.ConclusionThe middle‐aged females were at increased risk of developing BMs, while the middle‐aged males with BMs were at higher risk of having poorer survival.

The BRAF p.V600E mutation is the most common genetic alteration in ganglioglioma (GG). Herein, we collected a consecutive series of 30 GG specimens from Xuanwu Hospital in order to corroborate the genetic landscape and genotype–phenotype correlation of this enigmatic and often difficult‐to‐classify epilepsy‐associated brain tumor entity. All specimens with histopathologically confirmed lesions were submitted to targeted next‐generation sequencing using a panel of 131 genes. Genetic alterations in three cases with histologically distinct tumor components, that is, GG plus pleomorphic xanthoastrocytoma (PXA), dysembryoplastic neuroepithelial tumor (DNT), or an oligodendroglioma (ODG)‐like tumor component, were separately studied. A mean post‐surgical follow‐up time‐period of 23 months was available in 24 patients. Seventy seven percent of GG in our series can be explained by genetic alterations, with BRAF p.V600E mutations being most prevalent (n = 20). Three additional cases showed KRAS p.Q22R and KRAS p.G13R , IRS2 copy number gain (CNG) and a KIAA1549‐BRAF fusion. When genetically studying different histopathology patterns from the same tumor we identified composite features with BRAF p.V600E plus CDKN2A/B homozygous deletion in a GG with PXA features, IRS2 CNG in a GG with DNT features, and a BRAF p.V600E plus CNG of chromosome 7 in a GG with ODG‐like features. Follow‐up revealed no malignant tumor progression but nine patients had seizure recurrence. Eight of these nine GG were immunoreactive for CD34, six patients were male, five were BRAF wildtype, and atypical histopathology features were encountered in four patients, that is, ki‐67 proliferation index above 5% or with PXA component. Our results strongly point to activation of the MAP kinase pathway in the vast majority of GG and their molecular‐genetic differentiation from the cohort of low‐grade pediatric type diffuse glioma remains, however, to be further clarified. In addition, histopathologically distinct tumor components accumulated different genetic alterations suggesting collision or composite glio‐neuronal GG variants.

BackgroundThe extent of the relationship between age and the presence of breast cancer synchronous brain metastases (BCSBMs) and mortality has not yet been well-identified or sufficiently quantified. We aimed to examine the association of age with the presence of BCSBMs and all-cause and cancer-specific mortality outcomes using the SEER database.MethodsAge-associated risk of the presence and survival of BCSBMs were evaluated on a continuous scale (restricted cubic spline, RCS) with logistic or Cox regression models. The main endpoints were the presence of BCSBMs and all-cause mortality or cancer-specific mortality. Cox proportional hazards regression and competing risk models were used in survival analysis.ResultsAmong 374,132 adult breast cancer patients, 1,441 (0.38%) had BMs. The presence of BCSBMs displayed a U-shaped relationship with age, with the highest point of the curve occurring at the age of 62. In both the younger (age ≤ 61) and older (age ≥ 62) groups, the observed curve showed a nearly linear relationship between age and the presence of BCSBMs. The relationship between age and all-cause mortality (ASM) and cancer-specific mortality (CSM) was linear. Older age at diagnosis was associated with a higher risk of ASM (HR 1.019, 95% CI: 1.013–1.024, p < 0.001) and CSM (HR 1.016, 95% CI: 1.010–1.023, p < 0.001) in multivariable Cox models. Age (sHR 1.007, 95% CI 1–1.013, p = 0.049) was substantially related to a significantly increased risk of CSM in competing risk models.ConclusionAge had a non-linear U-shaped relationship with the presence of BCSBMs and a linear relationship with BCSBMs mortality.

Objective:To study the expression of ubiquitin-specific protease 25 (USP25) in the temporal cortex of the kainic acid (KA) induced epilepsy rat model.Methods:Fifty-two male SD rats were randomly divided into the epilepsy model group ( n=39) and sham-operated control group ( n=13) with the random number table. The epilepsy model group was established by injecting KA into the amygdala, then the epileptic rats were randomly divided into 3 groups according to the modeling success time: 1 day for acute period, 7 days for latent period and 30 days for chronic period (13 rats in each group). Those rats were sampled at the end of observation. Rats in the control group were injected with normal saline into the amygdala and sampled together with those in the experimental group. Immunohistochemistry and immunofluorescence double labeling was used to test USP25 expression and its co-expression with neurons (NeuN) and astrocytes (GFAP). Quantitative real-time PCR and Western blot were used to assess the change of USP25 in the temporal cortex of rats. Results:In the ipsilateral temporal cortex, the positive cells of co-expression of USP25 and NeuN were increased in the later stage of epilepsy in the epilepsy model group, and the expression levels of USP25 mRNA and protein in different stages of epilepsy varied significantly ( F= 25.48 and 7.68 respectively, both P<0.05). Compared with the control group (mRNA level: 1.00±0.36, protein level: 1.00±0.46), the expression of USP25 in the latent group (mRNA level: 10.80±4.82, protein level: 1.88±0.32) and the chronic group (mRNA level: 12.97±4.48, protein level: 1.92±0.26) were increased significantly (all P<0.05). In the contralateral temporal cortex, the expression levels of USP25 mRNA and protein in different stages of epilepsy also varied significantly ( F=86.86 and 6.65 respectively, both P<0.05). Conclusions:The increased expression of USP25 in the temporal cortex after the latent period has suggested that the deubiquitination pathway is involved in the chronic pathological process of temporal lobe epilepsy.

Brain edema is a common and serious complication of ischemic stroke with limited effective treatment. We previously reported that methylene blue (MB) attenuated ischemic brain edema in rats, but the underlying mechanisms remained unknown. Aquaporin 4 (AQP4) in astrocytes plays a key role in brain edema. We also found that extracellular signal-regulated kinase 1/2 (ERK1/2) activation was involved in the regulation of AQP4 expression in astrocytes. In the present study, we investigated whether AQP4 and ERK1/2 were involved in the protective effect of MB against cerebral edema. Rats were subjected to transient middle cerebral artery occlusion (tMCAO), MB (3 mg/kg, for 30 min) was infused intravenously through the tail vein started immediately after reperfusion and again at 3 h after ischemia (1.5 mg/kg, for 15 min). Brain edema was determined by MRI at 0.5, 2.5, and 48 h after tMCAO. The decreases of apparent diffusion coefficient (ADC) values on diffusion-weighted MRI indicated cytotoxic brain edema, whereas the increase of T2 MRI values reflected vasogenic brain edema. We found that MB infusion significantly ameliorated cytotoxic brain edema at 2.5 and 48 h after tMCAO and decreased vasogenic brain edema at 48 h after tMCAO. In addition, MB infusion blocked the AQP4 increases and ERK1/2 activation in the cerebral cortex in ischemic penumbra at 48 h after tMCAO. In a cell swelling model established in cultured rat astrocyte exposed to glutamate (1 mM), we consistently found that MB (10 μM) attenuated cell swelling, AQP4 increases and ERK1/2 activation. Moreover, the ERK1/2 inhibitor U0126 (10 μM) had the similar effects as MB. These results demonstrate that MB improves brain edema and astrocyte swelling, which may be mediated by the inhibition of AQP4 expression via ERK1/2 pathway, suggesting that MB may be a potential choice for the treatment of brain edema.

Objective To compare the behavioral manifestations, electroencephalogram ( EEG ) monitoring and hippocampal pathological changes in two rat epilepsy models induced by kainic acid ( KA) injected into hippocampus and amygdala respectively. Methods Male adult Sprague-Dawley rats(n=24) were randomly divided into hippocampus model group ( n=9 ) , amygdala model group ( n=9 ) and control group (n=6). Two epilepsy models were established by kainic acid (0. 6 μg,1. 0 μg/μl) stereotactically injected into hippocampus CA3 region (hippocampus model) or amygdala (amygdala model). The status epilepticus ( SE ) of rats was observed by behavioral manifestations and EEG monitoring. Following successful modeling, those rats were randomly divided into 3 groups, including 1-day ( acute phase) , 7-day ( latent phase) and 30-day ( chronic phase) post SE groups ( n=3 in each group) . Rats in the control group were injected with an equal volume of saline in the hippocampal CA3 or amygdala. Immunohistochemistry&nbsp;was used for observing the pathological changes of neuron (NeuN), astrocyte (GFAP) and microglia (Iba1) in the rat hippocampus. Results Behavioral and EEG monitoring showed that rats from both models had typical seizure behaviors and EEG characteristics in the acute and chronic phases. However, there were different seize types and onset time between the 2 models in the acute phase. The hippocampus model group had partial seizures at 63. 33 ± 4. 41 min post KA injection with intermittent generalized tonic-clonic seizures. Multi-phase spikes were recorded in acute phase. Amygdala model group had severe generalized tonic-clonic seizures at 28. 67 ± 3. 48 min post KA injection, and the main cortical EEG form was sharp wave rhythm. Immunohistochemical staining revealed gradual hippocampal neuron death from acute to chronic phases in both models, which resulted in serious neuronal loss. Meanwhile, the aggravation of astrocyte proliferation and accumulation of microglia occurred in hippocampus in both models. Remarkably, compared with hippocampus model, the amygdala model in chronic phase had more neuronal loss in CA1 region ( 10. 83 ± 1. 52 vs. 22. 43 ± 5. 16, P<0. 01) and CA4 region (12. 87 ± 2. 13 vs. 25. 81 ± 4. 60, P <0. 05), more astrocyte proliferation in CA1 region (61. 20 ± 7. 33 vs. 14. 65 ± 0. 12, P<0. 01) and CA4 region (76. 73 ± 5. 40 vs. 43. 01 ± 1. 35, P<0. 01) as well as more widespread accumulation of microglia in CA1 region (13. 70 ± 3. 88 vs. 1. 08 ± 0. 01, P<0. 01). Conclusions Our results have indicated that both rat epilepsy models simulated human temporal lobe epilepsy. The differences in the behavior, EEG performances and particularly pathological changes of hippocampus between two models should be taken into consideration in the future study.

Atmospheric pressure plasma jet (APPJ) has shown excellent potential prospects in biomedical applications, based on the production of reactive oxygen species and reactive nitrogen species (RNS) from APPJ emissions. The current research focused on the protective effect of APPJ on oxygen and glucose deprivation (OGD)-induced cell death in both the H9C2 cardiac myoblast cell line, a frequently used cardiac cell line in cardioprotective studies, and primary neonatal rat cardiomyocytes (NRCMs). Cells were treated with APPJ for different durations, cultured for 6h and then subjected to OGD for 18h before their use in assays. We found that APPJ treatment could maintain H9C2 cell viability and reduce cell apoptosis in a dose-dependent manner in cells subjected to the OGD conditions. To confirm the cardioprotective effect of APPJ on primary NRCM, we first identified the 'safe dose' of APPJ treatment by evaluating the cytotoxicity of APPJ on primary NRCMs in normal culture conditions. Under the 'safe dose' of APPJ treatment, we also found that the APPJ treatment could maintain NRCM viability under OGD conditions and reduce CK-MB and cTnI release from cardiomyocytes. Further studies revealed that the cytoprotective effect of APPJ may be related to NO production induced by APPJ treatment. Our results gave the first evidence of the cardiotoxicity and cytoprotective effect of APPJ on cardiomyocytes against OGD injury, and furthermore, contributed to new insights into the potential medical applications of plasma in cardiovascular diseases.

Objective To explore the effect of blood-brain barrier disruption on expression of AQP-4,through comparing the cell morphology and the expression of aquaporin-4(AQP-4)of cultured astrocytes in medium with and without fetal bovine serum(FBS). Methods Cerebral cortical astrocytes from female Wistar rats were cultured in serum free medium,DMEM supplement-ed with 10% FBS,and serum free medium supplemented with 10% FBS.Phase contrast microscope was used to detect the cell morphology and cell size. Immunofluorescence staining and reverse real-time quantitative poly-merase chain reaction(RT-qPCR)were used to examine the expression of glial fibrillary acidic protein(GFAP), AQP-4 and metabotropic glutamate receptor 5(mGluR5). Results Astrocytes in serum free medium showed extensive process bearing morphology,small body and nucleus,and high refractivity.In contrast,in two kinds of 10% FBS-containing medium,astrocytes were flat with large body and nucleus,weak refractivity,as well as short process.Analysis of immunofluorescence staining and RT-qPCR revealed a down-regulation of GFAP and AQP-4 protein and mRNA expression in two kinds of 10% FBS-con-taining medium, compared with that in serum free medium (P<0.001), however, there was no difference in mGluR5 protein and mRNA expression(P>0.05). Conclusion FBS changed astrocyte morphology and down-regulated the expression of GFAP and AQP-4.

Background/Aims: Circular RNAs (circRNAs) are a class of long noncoding RNAs with a closed loop structure that regulate gene expression as microRNA sponges. CircRNAs are more enriched in brain tissue, but knowledge of the role of circRNAs in temporal lobe epilepsy (TLE) has remained limited. This study is the first to identify the global expression profiles and characteristics of circRNAs in human temporal cortex tissue from TLE patients. Methods: Temporal cortices were collected from 17 TLE patients and 17 non-TLE patients. Total RNA was isolated, and high-throughput sequencing was used to profile the transcriptome of dysregulated circRNAs. Quantitative PCR was performed for the validation of changed circRNAs. Results: In total, 78983 circRNAs, including 15.29% known and 84.71% novel circRNAs, were detected in this study. Intriguingly, 442 circRNAs were differentially expressed between the TLE and non-TLE groups (fold change≥2.0 and FDR≤0.05). Of these circRNAs, 188 were up-regulated, and 254 were down-regulated in the TLE patient group. Eight circRNAs were validated by real-time PCR. Remarkably, circ-EFCAB2 was intensely up-regulated, while circ-DROSHA expression was significantly lower in the TLE group than in the non-TLE group (P<0.05). Bioinformatic analysis revealed that circ-EFCAB2 binds to miR-485-5p to increase the expression level of the ion channel CLCN6, while circ-DROSHA interacts with miR-1252-5p to decrease the expression level of ATP1A2. Conclusions: The dysregulations of circRNAs may reflect the pathogenesis of TLE and circ-EFCAB2 and circ-DROSHA might be potential therapeutic targets and biomarkers in TLE patients.

Objective To explore the methods of primary and passage cultures of astrocytes by using serum-free medium.Methods Cells from cerebral cortices of Wistar rats within 24 h of birth were mechanically dispersed and cultured in serum-free medium.Reaching the confluence of 80％-90％,cells were digested by EDTA (ethylenediaminetetraacetic acid)-trypsin or trypsin,followed by 0.2 mg/ml or 1 mg/ml soybean trypsin inhibitor (SBTI) treatment and centrifugation for 3 min (3 000 × g,4 ℃) to stop digestion,then cells were subcultured with the ratio of 1 ∶2 or 1 ∶3.The cell phenotype was identified by immunofluorescence staining for glial fibrillary acidic protein (GFAP).The cell proliferation was detected by MTT assay.Results Under the phase-contrast microscopy,primary cells in serum-free medium were prosperous,showed high refractivity and demonstrated small cell body and nucleus with extensive processes.About 95％ of the primary cells were positively stained for GFAP.The passage cells grew and proliferated well in the serum-free medium.The MTT results showed that the cell number in EDTA-trypsin group was higher than that in trypsin group (0.290 ± 0.007 vs.0.270 ± 0.006,P ＜ 0.010).There was no difference in cell count either between 0.2 mg/ml or 1 mg/ml SBTI groups (0.290 ± 0.013 vs.0.290 ± 0.017,P =0.820) or between 1∶2 and 1 ∶ 3 groups (0.340 ± 0.070 vs.0.320 ± 0.030,P =0.770).Conclusion The results showed that we have established the method of primary and passage cultures of rat astrocytes by using serum-free medium,which may provide a new cell model for better evaluation of astrocyte function in vitro without interference of serum.

Background: In vitro systems allowing maintenance and experimentation on primary astrocyte cultures have been used for decades. Astrocyte cultures are most maintained in serum-containing medium which has been found to alter the morphology and gene profiles of astrocytes. New method: Here, we reported a new serum-free medium for astrocyte culture, which consisted of DMEM and NB media supplemented with insulin and heparin-binding epidermal growth factor-like growth factor (HB-EGF) (SF-I-H medium). Meanwhile FBS-containing (FBS) medium composed of DMEM medium containing 10% FBS were used for comparison study. Cerebral cortex was harvested from postnatal day 1 Wistar rats and brain cells were isolated and seeded to poly-L-lysine coated culture dishes after 15 min differential velocity adherence. Results: Compared with FBS medium, astrocytes in SF-I-H medium were smaller and exhibited process bearing morphologies. MTT assays showed that cell density and proliferation rate were higher in SF-I-H medium than in FBS medium all the time, and flow cytometry analysis revealed that SF-I-H medium promoted cell mitosis in a manner comparable to FBS medium. Consistently, western blot analysis further revealed that insulin and HB-EGF synergistically activated the PI3K/AKT and MAPK/ERK1/2 signaling cascades as FBS. Comparison with existing method(s): Astrocytes cultured in SF-I-H medium grew faster than FBS medium. Conclusion: Taken together, our results indicated that SF-I-H medium, in which cell morphology was similar with astrocytes in brain, was more effective for astrocyte survival and proliferation than FBS medium, providing a new cell model to study astrocyte functions without the interference of serum.

Astrocytes are one of the most abundant cell types in the mammalian central nervous system (CNS), and astrocyte swelling is the primary event associated with brain edema. Glutamate, the principal excitatory amino acid neurotransmitter in the CNS, is released at high levels after brain injury including cerebral ischemia. This leads to astrocyte swelling, which we previously demonstrated is related to metabotropic glutamate receptor (mGluR) activation. Aquaporin 4 (AQP4), the predominant water channel in the brain, is expressed in astrocyte endfeet and plays an important role in brain edema following ischemia. Studies recently showed that mGluR5 is also expressed on astrocytes. Therefore, it is worth investigating whether AQP4 mediates the glutamate-induced swelling of astrocytes via mGluR5. In the present study, we found that 1 mM glutamate induced astrocyte swelling, quantified by the cell perimeter, but it had no effect on astrocyte viability measured by the cell counting kit-8 (CCK-8) and lactate dehydrogenase (LDH) assays. Quantitative reverse transcription polymerase chain reaction analyses revealed that AQP4, among AQP1, 4, 5, 9 and 11, was the main molecular expressed in cultured astrocytes. Glutamate-induced cell swelling was accompanied by a concentration-dependent change in AQP4 expression. Furthermore, RNAi technology revealed that AQP4 gene silencing inhibited glutamate-induced astrocyte swelling. Moreover, we found that mGluR5 expression was greatest among the mGluRs in cultured astrocytes and was co-expressed with AQP4. Activation of mGluR5 in cultured astrocytes using (S)-3,5-dihydroxyphenylglycine (DHPG), an mGluR5 agonist, mimicked the effect of glutamate. This effect was abolished by co-incubation with the mGluR5 antagonist fenobam but was not influenced by DL-threo-β-benzyloxyaspartic acid (DL-TBOA), a glutamate transporter inhibitor. Finally, experiments in a rat model of transient middle cerebral artery occlusion (tMCAO) revealed that co-expression of mGluR5 and AQP4 was increased in astrocyte endfeet around capillaries in the penumbra, and this was accompanied by brain edema. Collectively, these results suggest that glutamate induces cell swelling and alters AQP4 expression in astrocytes via mGluR5 activation, which may provide a novel approach for the treatment of edema following brain injury.