Bromovalerylurea modulates GABAA receptor-mediated inhibitory neurotransmission while inducing sleep.

Bromovalerylurea (BU), an acyl urea derivative, was originally developed as a hypnotic/sedative. We recently reported that BU at a dose of 50 mg/kg ameliorates sepsis, Parkinson's disease, and traumatic brain injury in Wistar rat models through its anti-inflammatory actions on microglia and macrophages. However, since BU was developed more than 100 years ago, its hypnotic mechanism and characteristics are poorly understood. Herein, we conducted an electroencephalogram (EEG) study and found that BU, when administered at a dose of more than 125 mg/kg but not at a dose of 50 mg/kg in Wistar rats, significantly increased non-rapid eye movement (NREM) sleep duration and dose-dependently decreased rapid eye movement (REM) sleep duration. This characteristic of sleep induced by BU is similar to the effect of compounds such as barbiturate, benzodiazepine, and z-drugs, all of which require γ-aminobutyric acid A receptors (GABAAR) for hypnotic/sedative activity. To investigate whether BU could potentiate GABAAergic neurotransmission, we conducted a whole-cell patch-clamp recording from pyramidal neurons in rat cortical slices to detect spontaneous GABAAR-mediated inhibitory postsynaptic currents (IPSCs). We found that BU dose-dependently prolonged IPSCs. Importantly, the prolonged IPSCs were not attenuated by flumazenil, a benzodiazepine receptor antagonist, suggesting that modulation of IPSCs by BU is mediated by different mechanisms from that of benzodiazepine. Taken together, these data elucidate the basic characteristics of the hypnotic effects of BU and suggest that the enhancement of GABAAR-mediated Cl- flux may be a possible mechanism that contributes to its hypnotic/sedative activity.

Impact of Gestational Haloperidol Exposure on miR-137-3p and Nr3c1 mRNA Expression in Hippocampus of Offspring Mice.

BACKGROUND: Schizophrenia is a mental disorder caused by both environmental and genetic factors. Prenatal exposure to antipsychotics, an environmental factor for the fetal brain, induces apoptotic neurodegeneration and cognitive impairment of offspring similar to schizophrenia. The aim was to investigate molecular biological changes in the fetal hippocampus exposed to haloperidol (HAL) by RNA expression as a model of the disorder. METHODS: HAL (1 mg/kg/d) was administered to pregnant mice. Upregulated and downregulated gene expressions in the hippocampus of offspring were studied with RNA-sequencing and validated with the qPCR method, and micro-RNA (miR) regulating mRNA expressional changes was predicted by in silico analysis. An in vitro experiment was used to identify the miRNA using a dual-luciferase assay. RESULTS: There were significant gene expressional changes (1370 upregulated and 1260 downregulated genes) in the HAL group compared with the control group on RNA-sequencing analysis (P < .05 and q < 0.05). Of them, the increase of Nr3c1 mRNA expression was successfully validated, and in silico analysis predicted that microRNA-137-3p (miR-137-3p) possibly regulates that gene's expression. The expression of miR-137-3p in the hippocampus of offspring was significantly decreased in the first generation, but it increased in the second generation. In vitro experiments with Neuro2a cells showed that miR-137-3p inversely regulated Nr3c1 mRNA expression, which was upregulated in the HAL group. CONCLUSIONS: These findings will be key for understanding the impact of the molecular biological effects of antipsychotics on the fetal brain.

Eosinophilic enteritis accompanied by cytomegalovirus disease: a case report.

BACKGROUND: Eosinophilic enteritis is a chronic inflammatory disorder of the intestinal tract that is characterized by eosinophil infiltration. Cytomegalovirus (CMV), a common virus, has a broad infectivity range. CMV is retained in the host body after infection. Impairment of host immune defences may reactivate the latent CMV, leading to symptoms of overt disease. CASE PRESENTATION: A Japanese female in her 70 s was admitted to a hospital due to diarrhoea and then transferred to our hospital. Laboratory data showed hypoalbuminemia. Computed tomography (CT) revealed oedema of the small intestine. Lower gastrointestinal endoscopy revealed oedema of the submucosa, without any remarkable changes in the mucosa of the terminal ileum. Histological examination of the terminal ileum revealed infiltration of > 20 eosinophils per high-power field (HPF). These findings aided in diagnosing eosinophilic enteritis. We administered methylprednisolone (500 mg/day) for three days, followed by tapering prednisolone. However, the patient's general condition and hypoalbuminemia failed to improve. Immunoglobulin (Ig) G- CMV and IgM-CMV tests were positive. CMV antigenemia was extremely high. Therefore, we administered ganciclovir intravenously, which improved the patient's condition. Furthermore, azathioprine was administered to taper and discontinue prednisolone without relapse of eosinophilic enteritis. This treatment helped stabilize the patient's condition for approximately four years. CONCLUSION: We present a case of eosinophilic enteritis accompanied by CMV disease during prednisolone treatment. The patient's condition improved after administration of ganciclovir. Azathioprine aided in discontinuing prednisolone and stabilizing the patient's condition for approximately four years.

Quantitative measurement of peritumoral concentrations of glutamate, N-acetyl aspartate, and lactate on magnetic resonance spectroscopy predicts glioblastoma-related refractory epilepsy.

BACKGROUND: Increased extracellular glutamate is known to cause epileptic seizures in patients with glioblastoma (GBM). However, predicting whether the seizure will be refractory is difficult. The present study investigated whether evaluation of the levels of various metabolites, including glutamate, can predict the occurrence of refractory seizure in GBM by quantitative measurement of metabolite concentrations on magnetic resonance spectroscopy (MRS). METHODS: Forty patients were treated according to the same treatment protocol for primary GBM at Ehime University Hospital between April 2017 and July 2021. Of these patients, 23 underwent MRS to determine concentrations of metabolites, including glutamate, N-acetylaspartate, creatine, and lactate, in the tumor periphery by applying LC-Model. The concentration of each metabolite was expressed as a ratio to creatine concentration. Patients were divided into three groups: Type A, patients with no seizures; Type B, patients with seizures that disappeared after treatment; and Type C, patients with seizures that remained unrelieved or appeared after treatment (refractory seizures). Relationships between concentrations of metabolites and seizure types were investigated. RESULTS: In 23 GBMs, seizures were confirmed in 11 patients, including Type B in four and Type C in seven. Patients with epilepsy (Type B or C) showed significantly higher glutamate and N-acetylaspartate values than did non-epilepsy patients (Type A) (p < 0.05). No significant differences in glutamate or N-acetylaspartate levels were seen between Types B and C. Conversely, Type C showed significantly higher concentrations of lactate than did Type B (p = 0.001). Cutoff values of lactate-to-creatine, glutamate-to-creatine, and N-acetylaspartate-to-creatine ratios for refractory seizure were > 1.25, > 1.09, and > 0.88, respectively. CONCLUSIONS: Extracellular concentrations of glutamate, N-acetylaspartate, and lactate in the tumor periphery were significantly elevated in patients with GBM with refractory seizures. Measurement of these metabolites on MRS may predict refractory epilepsy in such patients and could be an indicator for continuing the use of antiepileptic drugs.

Microglial metabolic disturbances and neuroinflammation in cerebral infarction.

Cerebral ischemia/reperfusion injury activates microglia, resident immune cells in the brain, and allows the infiltration of circulating immune cells into the ischemic lesions. Microglia play both exacerbating and protective roles in pathological processes and are thus often referred to as "double-edged swords." In ischemic brains, blood-borne macrophages play a role that is distinct from that of resident activated microglia. Recently, the metabolic alteration of immune cells in the pathogenesis of inflammatory disorders including cerebral infarction has become a critical target for investigation. We begin this review by describing the multifaceted functions of microglia in cerebral infarction. Next, we focus on the metabolic alterations that occur in microglia during pathological processes. We also discuss morphological changes that take place in the mitochondria, leading to functional disturbances, accompanied by alterations in microglial function. Moreover, we describe the involvement of the reactive oxygen species that are produced during aberrant metabolic activity. Finally, we discuss therapeutic strategies to ameliorate aggravative changes in metabolism.

Dual Roles of Microglia in the Basal Ganglia in Parkinson's Disease.

With the increasing age of the population, the incidence of Parkinson's disease (PD) has increased exponentially. The development of novel therapeutic interventions requires an understanding of the involvement of senescent brain cells in the pathogenesis of PD. In this review, we highlight the roles played by microglia in the basal ganglia in the pathophysiological processes of PD. In PD, dopaminergic (DAergic) neuronal degeneration in the substantia nigra pars compacta (SNc) activates the microglia, which then promote DAergic neuronal degeneration by releasing potentially neurotoxic factors, including nitric oxide, cytokines, and reactive oxygen species. On the other hand, microglia are also activated in the basal ganglia outputs (the substantia nigra pars reticulata and the globus pallidus) in response to excess glutamate released from hyperactive subthalamic nuclei-derived synapses. The activated microglia then eliminate the hyperactive glutamatergic synapses. Synapse elimination may be the mechanism underlying the compensation that masks the appearance of PD symptoms despite substantial DAergic neuronal loss. Microglial senescence may correlate with their enhanced neurotoxicity in the SNc and the reduced compensatory actions in the basal ganglia outputs. The dual roles of microglia in different basal ganglia regions make it difficult to develop interventions targeting microglia for PD treatment.

Microglia and the Aging Brain: Are Geriatric Microglia Linked to Poor Sleep Quality?

Poor sleep quality and disrupted circadian behavior are a normal part of aging and include excessive daytime sleepiness, increased sleep fragmentation, and decreased total sleep time and sleep quality. Although the neuronal decline underlying the cellular mechanism of poor sleep has been extensively investigated, brain function is not fully dependent on neurons. A recent antemortem autographic study and postmortem RNA sequencing and immunohistochemical studies on aged human brain have investigated the relationship between sleep fragmentation and activation of the innate immune cells of the brain, microglia. In the process of aging, there are marked reductions in the number of brain microglial cells, and the depletion of microglial cells disrupts circadian rhythmicity of brain tissue. We also showed, in a previous study, that pharmacological suppression of microglial function induced sleep abnormalities. However, the mechanism underlying the contribution of microglial cells to sleep homeostasis is only beginning to be understood. This review revisits the impact of aging on the microglial population and activation, as well as microglial contribution to sleep maintenance and response to sleep loss. Most importantly, this review will answer questions such as whether there is any link between senescent microglia and age-related poor quality sleep and how this exacerbates neurodegenerative disease.

Phagocytic elimination of synapses by microglia during sleep.

Synaptic strength reduces during sleep, but the underlying mechanisms of this process are unclear. This study showed reduction of synaptic proteins in rat prefrontal cortex (PFC) at AM7 or Zeitgeber Time (ZT0), when the light phase or sleeping period for rats started. At this time point, microglia were weakly activated, displaying larger and more granular somata with increased CD11b expression compared with those at ZT12, as revealed by flow cytometry. Expression of opsonins, such as complements or MFG-E8, matrix metalloproteinases, and microglial markers at ZT0 were increased compared with that at ZT12. Microglia at ZT0 phagocytosed synapses, as revealed by immunohistochemical staining. Immunoblotting detected more synapsin I in the isolated microglia at ZT0 than at ZT12. Complement C3- or MFG-E8-bound synapses were the most abundant at ZT0, some of which were phagocytosed by microglia. Systemic administration of synthetic glucocorticoid dexamethasone reduced microglial size, granularity and CD11b expression at ZT0, resembling microglia at ZT12, and increased synaptic proteins and decreased the sleeping period. Noradrenaline (NA) suppressed glutamate-induced phagocytosis in primary cultured microglia. Systemic administration of the brain monoamine-depleting agent reserpine decreased NA content and synapsin I expression in PFC, and increased expression of microglia markers, C3 and MFG-E8, while increasing the sleeping period. A NA precursor l-threo-dihydroxyphenylserine abolished the reserpine-induced changes. These results suggest that microglia may eliminate presumably weak synapses during every sleep phase. The circadian changes in concentrations of circulating glucocorticoids and brain NA might be correlated with the circadian changes of microglial phenotypes and synaptic strength.

B lymphocytopenia and Bregs in a not-to-die murine sepsis model.

Sepsis is a leading cause of mortality in intensive care units due to multi-organ failure caused by dysregulated immune reactions. In this study, kinetic changes in the immune system were analyzed for 72 h in cecal ligation and puncture (CLP)-induced septic mice while preventing animal death by keeping body temperature. Increase of myeloid cells and decrease of B cells in circulation at 6 h after CLP were markedly observed. At the same time point, interleukin (IL)-10 expressing CD5+ regulatory B cells (Bregs) appeared. IL-10 and programmed death-ligand 1 (PD-L1) mRNA as well as IL-1ß, IL-6 and interferon γ (IFNγ) mRNA was increased in the spleen at 6 h. A gradual decrease in Bcl-2 and abrupt increase of Bim expression in the spleen at the late phase were also found. These results showed that B lymphocytopenia with the appearance of Bregs is the earliest event, likely leading to immunoparalysis in sepsis.

Taro koji of Amorphophallus konjac enabling hydrolysis of konjac polysaccharides to various biotechnological interest.

Due to the indigestibility, utilization of konjac taro, Amorphophallus konjac has been limited only to the Japanese traditional konjac food. Koji preparation with konjac taro was examined to utilize konjac taro as a source of utilizable carbohydrates. Aspergillus luchuensis AKU 3302 was selected as a favorable strain for koji preparation, while Aspergillus oryzae used extensively in sake brewing industry was not so effective. Asp. luchuensis grew well over steamed konjac taro by extending hyphae with least conidia formation. Koji preparation was completed after 3-day incubation at 30°C. D-Mannose and D-glucose were the major monosaccharides found in a hydrolyzate giving the total sugar yield of 50 g from 100 g of dried konjac taro. An apparent extent of konjac taro hydrolysis at 55°C for 24 h seemed to be completed. Since konjac taro is hydrolyzed into monosaccharides, utilization of konjac taro carbohydrates may become possible to various products of biotechnological interest.

Comparison of the detrimental features of microglia and infiltrated macrophages in traumatic brain injury: A study using a hypnotic bromovalerylurea.

Microglia and blood-borne macrophages in injured or diseased brains are difficult to distinguish because they share many common characteristics. However, the identification of microglia-specific markers and the use of flow cytometry have recently made it easy to discriminate these types of cells. In this study, we analyzed the features of blood-borne macrophages, and activated and resting microglia in a rat traumatic brain injury (TBI) model. Oxidative injury was indicated in macrophages and neurons in TBI lesions by the presence of 8-hydroxy-2'-deoxyguanosine (8-OHdG). Generation of mitochondrial reactive oxygen species (ROS) was markedly observed in granulocytes and macrophages, but not in activated or resting microglia. Dihydroethidium staining supported microglia not being the major source of ROS in TBI lesions. Furthermore, macrophages expressed NADPH oxidase 2, interleukin-1ß (IL-1ß), and CD68 at higher levels than microglia. In contrast, microglia expressed transforming growth factor ß1 (TGFß1), interleukin-6 (IL-6), and tumor necrosis factor α at higher levels than macrophages. A hypnotic, bromovalerylurea (BU), which has anti-inflammatory effects, reduced both glycolysis and mitochondrial oxygen consumption. BU administration inhibited chemokine CCL2 expression, accumulation of monocytes/macrophages, 8-OHdG generation, mitochondrial ROS generation, and proinflammatory cytokine expression, and markedly ameliorated the outcome of the TBI model. Yet, BU did not inhibit microglial activation or expression of TGFß1 and insulin-like growth factor 1 (IGF-1). These results indicate that macrophages are the major aggravating cell type in TBI lesions, in particular during the acute phase. Activated microglia may even play favorable roles. Reduction of cellular energy metabolism in macrophages and suppression of CCL2 expression in injured tissue may lead to amelioration of TBI.

Truncated CD200 stimulates tumor immunity leading to fewer lung metastases in a novel Wistar rat metastasis model.

CD200 mediates immunosuppression in immune cells that express its receptor, CD200R. There are two CD200 variants; truncated CD200 that lacks the part of N-terminal sequence necessary for CD200R binding (CD200S) and full-length CD200 (CD200L). We established a novel lung metastasis model by subcutaneously transplanting C6 glioma cells into the backs of neonatal Wistar rats. All transplanted rats developed large back tumors, nearly 90% of which bore lung metastases. To compare the effects of CD200S and CD200L on tumor immunity, CD200L (C6-L)- or CD200S (C6-S)-expressing C6 cells were similarly transplanted. The results showed that 100% of rats with C6-L tumors developed lung metastases, while metastases were found in only 44% of rats with C6-S tumors (n = 25). Tumors disappeared in approximately 20% of the C6-S-bearing rats, and these animals evaded death 180 d after transplantation, while all C6-L tumor-bearing rats died after 45 d. Next generation sequencing revealed that C6-S tumors expressed chemokines and granzyme B at much higher levels than C6-L tumors. Flow cytometry revealed that C6-S tumors contained more dead cells and more CD45+ cells, including natural killer cells and CD8+ lymphocytes. In particular, multiple subsets of dendritic cells expressing CD11c, MHC class II, CD8, and/or CD103 were more abundant in C6-S than in C6-L tumors. These results suggested that CD200S induced the accumulation of multiple dendritic cell subsets that activated cytotoxic T lymphocytes, leading to the elimination of metastasizing tumor cells.

Sustained anti-inflammatory effects of TGF-ß1 on microglia/macrophages.

Ischemic brain injuries caused release of damage-associated molecular patterns (DAMPs) that activate microglia/macrophages (MG/MPs) by binding to Toll-like receptors. Using middle cerebral artery transiently occluded rats, we confirmed that MG/MPs expressed inducible nitric oxide synthase (iNOS) on 3days after reperfusion (dpr) in ischemic rat brain. iNOS expression almost disappeared on 7dpr when transforming growth factor-ß1 (TGF-ß1) expression was robustly increased. After transient incubation with TGF-ß1 for 24h, rat primary microglial cells were incubated with lipopolysaccharide (LPS) and released NO level was measured. The NO release was persistently suppressed even 72h after removal of TGF-ß1. The sustained TGF-ß1 effects were not attributable to microglia-derived endogenous TGF-ß1, as revealed by TGF-ß1 knockdown and in vitro quantification studies. Then, boiled supernatants prepared from ischemic brain tissues showed the similar sustained inhibitory effects on LPS-treated microglial cells that were prevented by the TGF-ß1 receptor-selective blocker SB525334. After incubation with TGF-ß1 for 24h and its subsequent removal, LPS-induced phosphorylation of IκB kinases (IKKs), IκB degradation, and NFκB nuclear translocation were inhibited in a sustained manner. SB525334 abolished all these effects of TGF-ß1. In consistent with the in vitro results, phosphorylated IKK-immunoreactivity was abundant in MG/MPs in ischemic brain lesion on 3dpr, whereas it was almost disappeared on 7dpr. The findings suggest that abundantly produced TGF-ß1 in ischemic brain displays sustained anti-inflammatory effects on microglial cells by persistently inhibiting endogenous Toll-like receptor ligand-induced IκB degradation.

Modafinil alleviates levodopa-induced excessive nighttime sleepiness and restores monoaminergic systems in a nocturnal animal model of Parkinson's disease.

Treatment with dopaminergic agents result excessive daytime sleepiness (EDS) and some studies have shown the benefit of using modafinil for treating excessive daytime sleepiness of Parkinson's disease (PD) patient. We investigated whether modafinil have ameliorative properties against levodopa induced excessive nighttime sleepiness (ENS) in MPTP-treated murine nocturnal PD model. Our EEG analyses of whole day recordings revealed that modafinil reduce ENS of this nocturnal PD models with levodopa medications. Therefore, we investigated whether, modafinil post-treatment followed by MPTP shows any effect on monoamine contents of brain and found to robustly increased noradrenaline (NA) concentration of MPTP treated mice. Modafinil post-treatment, in neurorestorative context (5 days post-lesion) led to increased striatal dopamine (DA) concentrations of MPTP-treated mice. Here, we first confirmed that modafinil ameliorates levodopa induced excessive sleepiness and restores monoaminergic systems. The arousal and anti-parkinsonian effects displayed by modafinil indicate that in combination with dopaminergic agents, modafinil co-administration may be worthwhile in trying to suppress the excessive daytime sleepiness and progressive dopaminergic neuron loss in PD.

Microglia may compensate for dopaminergic neuron loss in experimental Parkinsonism through selective elimination of glutamatergic synapses from the subthalamic nucleus.

Parkinson's disease (PD) symptoms do not become apparent until most dopaminergic neurons in the substantia nigra pars compacta (SNc) degenerate, suggesting that compensatory mechanisms play a role. Here, we investigated the compensatory involvement of activated microglia in the SN pars reticulata (SNr) and the globus pallidus (GP) in a 6-hydroxydopamine-induced rat hemiparkinsonism model. Activated microglia accumulated more markedly in the SNr than in the SNc in the model. The cells had enlarged somata and expressed phagocytic markers CD68 and NG2 proteoglycan in a limited region of the SNr, where synapsin I- and postsynaptic density 95-immunoreactivities were reduced. The activated microglia engulfed pre- and post-synaptic elements, including NMDA receptors into their phagosomes. Cells in the SNr and GP engulfed red fluorescent DiI that was injected into the subthalamic nucleus (STN) as an anterograde tracer. Rat primary microglia increased their phagocytic activities in response to glutamate, with increased expression of mRNA encoding phagocytosis-related factors. The synthetic glucocorticoid dexamethasone overcame the stimulating effect of glutamate. Subcutaneous single administration of dexamethasone to the PD model rats suppressed microglial activation in the SNr, resulting in aggravated motor dysfunctions, while expression of mRNA encoding glutamatergic, but not GABAergic, synaptic elements increased. These findings suggest that microglia in the SNr and GP become activated and selectively eliminate glutamatergic synapses from the STN in response to increased glutamatergic activity. Thus, microglia may be involved in a negative feedback loop in the indirect pathway of the basal ganglia to compensate for the loss of dopaminergic neurons in PD brains.

Elevated Na+/H+ exchanger-1 expression enhances the metastatic collective migration of head and neck squamous cell carcinoma cells.

Cancer cells can migrate as collectives during invasion and/or metastasis; however, the precise molecular mechanisms of this form of migration are less clear compared with single cell migration following epithelial-mesenchymal transition. Elevated Na+/H+ exchanger1 (NHE1) expression has been suggested to have malignant roles in a number of cancer cell lines and in vivo tumor models. Furthermore, a metastatic human head and neck squamous cell carcinoma (HNSCC) cell line (SASL1m) that was isolated based on its increased metastatic potential also exhibited higher NHE1 expression than its parental line SAS. Time-lapse video recordings indicated that both cell lines migrate as collectives, although with different features, e.g., SASL1m was much more active and changed the direction of migration more frequently than SAS cells, whereas locomotive activities were comparable. SASL1m cells also exhibited higher invasive activity than SAS in Matrigel invasion assays. shRNA-mediated NHE1 knockdown in SASL1m led to reduced locomotive and invasive activities, suggesting a critical role for NHE1 in the collective migration of SASL1m cells. SASL1m cells also exhibited a higher metastatic rate than SAS cells in a mouse lymph node metastasis model, while NHE1 knockdown suppressed in vivo SASL1m metastasis. Finally, elevated NHE1 expression was observed in human HNSCC tissue, and Cariporide, a specific NHE1 inhibitor, reduced the invasive activity of SASL1m cells, implying NHE1 could be a target for anti-invasion/metastasis therapy.

Effects of hypnotic bromovalerylurea on microglial BV2 cells.

An old sedative and hypnotic bromovalerylurea (BU) has anti-inflammatory effects. BU suppressed nitric oxide (NO) release and proinflammatory cytokine expression by lipopolysaccharide (LPS)-treated BV2 cells, a murine microglial cell line. However, BU did not inhibit LPS-induced nuclear translocation of nuclear factor-κB and subsequent transcription. BU suppressed LPS-induced phosphorylation of signal transducer and activator of transcription 1 (STAT1) and expression of interferon regulatory factor 1 (IRF1). The Janus kinase 1 (JAK1) inhibitor filgotinib suppressed the NO release much more weakly than that of BU, although filgotinib almost completely prevented LPS-induced STAT1 phosphorylation. Knockdown of JAK1, STAT1, or IRF1 did not affect the suppressive effects of BU on LPS-induced NO release by BV2 cells. A combination of BU and filgotinib synergistically suppressed the NO release. The mitochondrial complex I inhibitor rotenone, which did not prevent STAT1 phosphorylation or IRF1 expression, suppressed proinflammatory mediator expression less significantly than BU. BU and rotenone reduced intracellular ATP (iATP) levels to a similar extent. A combination of rotenone and filgotinib suppressed NO release by LPS-treated BV2 cells as strongly as BU. These results suggest that anti-inflammatory actions of BU may be attributable to the synergism of inhibition of JAK1/STAT1-dependent pathways and reduction in iATP level.

High mobility group box 1 enhances hyperthermia-induced seizures and secondary epilepsy associated with prolonged hyperthermia-induced seizures in developing rats.

Levels of high mobility group box 1 (HMGB1), an important inflammatory mediator, are high in the serum of febrile seizure (FS) patients. However, its roles in FS and secondary epilepsy after prolonged FS are poorly understood. We demonstrate HMGB1's role in the pathogenesis of hyperthermia-induced seizures (HS) and secondary epilepsy after prolonged hyperthermia-induced seizures (pHS). In the first experiment, 14-15-day-old male rats were divided into four groups: high-dose HMGB1 (100 µg), moderate-dose (10 µg), low-dose (1 µg), and control. Each rat was administered HMGB1 intranasally 1 h before inducing HS. Temperature was measured at seizure onset with electroencephalography (EEG). In the second experiment, 10-11-day-old rats were divided into four groups: pHS + HMGB1 (10 µg), pHS, HMGB1, and control. HMGB1 was administered 24 h after pHS. Video-EEGs were recorded for 24 h at 90 and 120 days old; histological analysis was performed at 150 days old. In the first experiment, the temperature at seizure onset was significantly lower in the high- and moderate-dose HMGB1 groups than in the control group. In the second experiment, the incidence of spontaneous epileptic seizure was significantly higher in the pHS + HMGB1 group than in the other groups. Comparison between pHS + HMGB1 groups with and without epilepsy revealed that epileptic rats had significantly enhanced astrocytosis in the hippocampus and corpus callosum. In developing rats, HMGB1 enhanced HS and secondary epilepsy after pHS. Our findings suggest that HMGB1 contributes to FS pathogenesis and plays an important role in the acquired epileptogenesis of secondary epilepsy associated with prolonged FS.

Oct-3/4 modulates the drug-resistant phenotype of glioblastoma cells through expression of ATP binding cassette transporter G2.

BACKGROUND: Drug resistance is a major obstacle for the efficacy of chemotherapeutic treatment of tumors. Oct-3/4, a self-renewal regulator in stem cells, is expressed in various kinds of solid tumors including glioblastoma. Although Oct-3/4 expression has been implicated in the malignancy and prognosis of glioblastomas, little is known of its involvement in drug resistances of glioblastoma. METHODS: The involvement of Oct-3/4 in drug resistance of glioblastoma cells was assessed by lactate dehydrogenase assay, efflux assay of an anticancer drug, poly ADP-ribose polymerase cleavage, and in vivo xenograft experiments. Involvement of a drug efflux pump ATP binding cassette transporter G2 in Oct-3/4-induced drug resistance was evaluated by quantitative PCR analysis and knockdown by shRNA. RESULTS: Oct-3/4 decreased the susceptibility to chemotherapeutic drugs by enhancing excretion of drugs through a drug efflux pump gene, ATP binding cassette transporter G2. Moreover, the expression of Oct-3/4 was well correlated to ATP binding cassette transporter G2 expression in clinical GB tissues. CONCLUSION: Oct-3/4 elevated the ATP binding cassette transporter G2 expression, leading to acquisition of a drug-resistant phenotype by glioblastoma cells. GENERAL SIGNIFICANCE: If the drug-resistance of glioblastoma cells could be suppressed, it should be a highly ameliorative treatment for glioblastoma patients. Therefore, signaling pathways from Oct-3/4 to ATP binding cassette transporter G2 should be intensively elucidated to develop new therapeutic interventions for better efficacy of anti-cancer drugs.

Blood vessels expressing CD90 in human and rat brain tumors.

Blood vessels in brain tumors, particularly glioblastomas, have been shown to express CD90. CD90(+) cells in and around blood vessels in cancers including brain tumors have been identified as endothelial cells, cancer stem cells, fibroblasts or pericytes. In this study, we aimed to determine the nature or type(s) of cells that express CD90 in human brain tumors as well as an experimental rat glioma model by double immunofluorescence staining. The majority of CD90(+) cells in human glioblastoma tissue expressed CD31, CD34 and von Willebrand factor, suggesting that they were endothelial cells. Vasculatures in a metastatic brain tumor and meningioma also expressed CD90. CD90(+) cells often formed glomeruloid structures, typical of angiogenesis in malignant tumors, not only in glioblastoma but also in metastatic tumors. Some cells in the middle and outer layers of the vasculatures expressed CD90. Similar results were obtained in the rat glioma model. There were cells expressing both α-smooth muscle actin and CD90 in the middle layer of blood vessels, indicating that smooth muscle cells and/or pericytes may express CD90. CD90(+) vasculatures were surrounded by tumor-associated macrophages (TAMs). Thus, in addition to endothelial cells, some other types of cells, such as smooth muscle cells, pericytes and fibroblasts constituting the vasculature walls in brain tumors expressed CD90. Because CD90 has been shown to interact with integrins expressed by circulating monocytes, CD90 might be involved in angiogenesis through recruitment and functional regulation of TAMs in tumors. CD90(+) vasculatures may also interact with tumor cells through interactions with integrins. Because CD90 was not expressed by vasculatures in normal brain tissue, it might be a possible therapeutic target to suppress angiogenesis and tumor growth.